key: cord- -isw jeir authors: flori, laurence; gao, yu; laloë, denis; lemonnier, gaëtan; leplat, jean-jacques; teillaud, angélique; cossalter, anne-marie; laffitte, joëlle; pinton, philippe; de vaureix, christiane; bouffaud, marcel; mercat, marie-josé; lefèvre, françois; oswald, isabelle p.; bidanel, jean-pierre; rogel-gaillard, claire title: immunity traits in pigs: substantial genetic variation and limited covariation date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: isw jeir background: increasing robustness via improvement of resistance to pathogens is a major selection objective in livestock breeding. as resistance traits are difficult or impossible to measure directly, potential indirect criteria are measures of immune traits (its). our underlying hypothesis is that levels of its with no focus on specific pathogens define an individual's immunocompetence and thus predict response to pathogens in general. since variation in its depends on genetic, environmental and probably epigenetic factors, our aim was to estimate the relative importance of genetics. in this report, we present a large genetic survey of innate and adaptive its in pig families bred in the same environment. methodology/principal findings: fifty four its were studied on large white pigs vaccinated against mycoplasma hyopneumoniae and analyzed by combining a principal component analysis (pca) and genetic parameter estimation. its include specific and non specific antibodies, seric inflammatory proteins, cell subsets by hemogram and flow cytometry, ex vivo production of cytokines (ifnα, tnfα, il , il , il , ifnγ, il , il , il ), phagocytosis and lymphocyte proliferation. while six its had heritabilities that were weak or not significantly different from zero, and its had moderate ( . <h ≤ . ) or high (h > . ) heritability values, respectively. phenotypic and genetic correlations between its were weak except for a few traits that mostly include cell subsets. pca revealed no cluster of innate or adaptive its. conclusions/significance: our results demonstrate that variation in many innate and adaptive its is genetically controlled in swine, as already reported for a smaller number of traits by other laboratories. a limited redundancy of the traits was also observed confirming the high degree of complementarity between innate and adaptive its. our data provide a genetic framework for choosing its to be included as selection criteria in multitrait selection programmes that aim to improve both production and health traits. increasing robustness by improving resistance/tolerance to pathogens is an important selection objective in most livestock species, particularly in pigs. in the past years, selection for growth, carcass leanness, meat quality and prolificacy, combined with stringent sanitary rules, vaccination and use of antibiotics, has been highly effective in pigs [ ] . since the early 's, prophylactic use of antibiotics as growth promoters has been forbidden by european legislation. as a result, the health status of numerous farms has deteriorated, leading to an increase in the therapeutic use of antibiotics. indeed, animals highly selected for production traits may be more susceptible to pathogens or less able to maintain performance after infection. deterioration of the global health status may also be due to environmental trends. in this context, including health traits in existing breeding schemes using direct and/or indirect strategies is an emerging trend in pig breeding. direct strategies target animal resistance/tolerance to specific pathogens but may result in increased susceptibility to other diseases [ , ] . alternatively, an indirect and putatively more global approach focuses on immune traits (its) providing a measure of immune capacity (i.e. immunocompetence) and hopefully predicting the responses to pathogens in general [ ] . the choice of relevant its is further based on knowledge of the immune system. this highly interactive and cooperative system is classically separated into two arms referred to as innate and adaptive, which produce a combined response. innate immunity is the first line of defence. its activation is non pathogen-specific and depends on the recognition of evolutionarily conserved pathogenassociated molecular patterns such as lipopolysaccharides constituting bacterial cell walls [ ] . innate immunity involves physical barriers, innate immune cells such as dendritic cells (dcs), monocytes, natural killers (nk cells) or cd t lymphocytes, and inflammatory cytokines such as il b, il and tnf. adaptive immunity is antigen-specific and requires the recognition of specific ''non-self'' antigens via a process of antigen presentation and results in an immunological memory. adaptive immunity is divided into cell-and humoral-mediated immunity with different effector functions [ ] . in order to include its in a breeding plan to improve pig immunocompetence, the genetic and phenotypic parameters of the different its need first to be estimated. several studies in swine, mice, poultry and cattle demonstrated the possibility of selecting animals with high or low immune response (ir) as characterized by one or a few its [ , , , , ] . a study on yorkshire pigs selected for eight generations for high and low adaptive ir (hir and lir, respectively) on an index combining four standardized measures of specific antibodies and cellmediated ir, after stimulation with specific antigens (bacillus calmette-guérin and hen egg white lysozyme), has revealed that hir and lir animals differ in response to immunization and infection [ , , , , ] . other studies have also shown that various innate and adaptive its are genetically controlled. for example, variation in innate its, such as nk cells, monocytes, interferon a (ifna) production or phagocytosis [ , , ] is heritable and several adaptive its have moderate to high heritability values including total white blood cells (wbc), cd + t lymphocyte, cd a + t lymphocyte and b lymphocyte subsets [ , , ] , delayed-type hypersensitivity reaction [ , ] , lymphocyte proliferative response [ ] , interleukin- (il ) production by lymphocytes [ ] and antibody response [ , , , ] . clapperton and colleagues have also reported that variation in acute phase protein levels is heritable [ , ] . finally, several significant qtls for total leukocyte count ( [ , ] ; animal-qtldb, http://www.animalgenome.org/cgi-bin/qtldb/index), mitogen-induced proliferation [ ] , antibody response [ , ] , cytokine production (il and ifnc) [ ] , complement activity [ ] , and acute phase protein serum concentration [ ] have been detected and mapped to different pig chromosomes. taken together these data demonstrate that variation in some its is under genetic control. however, most of the results reported so far have targeted a limited number of traits and very few studies have combined innate and adaptive its. our global goal is to identify immunocompetence traits for inclusion in selection schemes aiming to improve both zootechnical performances and health traits in pigs. for this purpose, we have launched a genetic and genomic study of numerous its covering innate and adaptive ir [ ] . in this report, we present the results of a global genetic study, combining principal component analysis (pca), and genetic parameter estimation applied to a large number of innate and adaptive its in a pig population vaccinated against mycoplasma hyopneumoniae (m. hyopneumoniae). a set of its was measured on a population of pigs three weeks after vaccination against m. hyopneumoniae (tables and ; table s ; figure ). these its comprise either traits related to ir (phagocytosis, lymphocyte proliferation, cytokine production after in vitro stimulations, levels of total and specific antibodies, levels of acute phase proteins) or traits related to total leukocyte and leukocyte subpopulation counts. the various characteristics and descriptive statistics of the traits measured on each animal of the studied population (n = to ) are summarized in tables and . among the cell-mediated its evaluated after diverse stimulations, higher responses in cytokine levels were observed after phorbol myristate acetate (pma)-ionomycin (pmaiono) stimulation compared to lipopolysaccharide (lps) and concanavalin a (cona) stimulations, except for il production. conversely, a higher lymphocyte proliferation was detected after cona and pmaiono stimulations than after lps stimulation. ample phenotypic variation was observed for most traits. the coefficient of variation (cv) was equal to . on average and ranged from . to . (tables and ). limited dispersion (cv# . ) was observed for traits derived from hemograms, cell subsets characterized by fluorescence-activated cell sorting (facs), phagocytosis capacity and non-specific immunoglobulins. moderate dispersion ( . ,cv# . ) was observed for most cytokines produced in vitro except for tumour necrosis factor a (tnfa) and mitogen proliferation-related traits. finally, the cv for seric c reactive protein (crp) and haptoglobin (hapt) levels were close to . and , respectively. the highest cv ( . ) was obtained for the specific iggs directed against m. hyopneumoniae. these data clearly indicate that the seric inflammatory protein levels and the specific iggs had the greatest phenotypic variance in our study. in order to analyse the factors causing the variation, we performed a normed pca with traits (tables and ). for cellmediated adaptive ir, we included only those traits related to cytokine production and lymphocyte proliferation after pmaionomycin stimulation. for innate its, we included facscharacterized cell subtypes including the percentages of b lymphocytes (igm + ), cd t lymphocytes (tcrcd + ), three subsets of ab t lymphocytes (cd + cd + , cd -cd + and cd + cd -), nk cells (cd + cd + ) and three monocyte subsets (cd + cd a + , cd + mhcii + , mhcii + cd a + ). we excluded from the analysis four haematological traits not directly involved in immunity: red blood cell count (rbc), hematocrit (ht), red blood cell distribution width (rdw) and platelet count (plt). the percentage of variance (inertia) explained by the first five components was over % (figure a ). each of these five components explained more than % of the total variance and the first two components accounted for . and . % of the total variance, respectively. taking into account the components from pca, multivariate normal mixture modelling and modelbased clustering (see materials and methods) were used to identify clusters of its. the highest bayesian information criterion (bic) was obtained using the diagonal model with variable shape and variable variance (vvi in pink on figure b ) and k = (first factorial plan on figure c ). no parameter was located near the correlation circle indicating that the phenotypic correlations between its are globally weak. a first cluster (k in blue on figure c ) groups together four hemogram-derived cell counts: white blood cell count (wbc), lymphocyte count (lym), monocyte count (mon) and neutrophil count (neu). this cluster is representative of total cell number traits despite the eosinophil count (eos) not being included. a second cluster (k in green on figure c ) groups together all the traits related to facscharacterized leukocyte subpopulations (expressed as the percentage of cells with one or two surface antigens) except cd t lymphocytes (tcrcd + ), with a cell response parameter (il -pmaiono) and the seric level of haptoglobin (hapt). this cluster can be considered as representative of the leukocyte subsets. a third cluster (k in red on figure c ) includes all other cell response traits, one hemogram-derived cell count (eos) and one facs-characterized leukocyte subpopulation. note that k and k related traits, which mainly correspond to cell subsets and explain around % of the phenotypic variance, show clear clustering ( figure c ). these traits are grouped on the first pca axis and separated on the second axis. traits belonging to k , representative of cell activity (cytokine production, phagocytosis and antibody production), are more spread out on the other axes (data not shown). interestingly, cluster analysis did not highlight any cluster of innate or adaptive its. the estimation of phenotypic correlations (r r p ) with wombat confirmed that the its are weakly correlated, except i) among a few cell count traits (wbc, lym, mon, neu), and ii) between cell count traits and a few leukocyte subsets (wbc, lym, cd + cd + and cd -cd + ) for whichr r p greater than . were estimated (table s ; figure s ). weakr r p were mainly positive ( ) with negative. no strongly negativer r p (# . ) were found. taken together, pca and estimations of phenotypic correlations showed that the level of redundancy between the different immune parameters was limited. heritability estimates of the analyzed its was equal to . on average (se = . ; table for the large set of adaptive its, the mean heritability was . (se = . ). no significant difference in average heritability values was detected either between group of its qualifying the innate and adaptive immunity or the humoral and cellular adaptive immunity. in addition, an equivalent proportion of innate and adaptive its had significant heritability values. indeed, % ( / ), % ( / ) and among the traits involved in cell-mediated immunity, variation in ab t lymphocyte (cd -cd + , cd + cd + and cd + cd cells) counts was highly heritable. heritability estimates of the three cytokine (il , il , ifng) levels were moderate to high after pmaiono and cona stimulations and weak to moderate after lps stimulation. for those cytokines induced by cona or lps stimulation, confidence interval ( ci) for the heritabilities overlapped zero, except for il -cona. il production after pmaiono, cona and lps stimulations gave high estimates of heritability significantly different from zero for il -pmaiono and il -lps. proliferation measurements after various stimulations (prolif-cona, prolif-pmaiono, prolif-lps) provided moderate estimates of heritability not significantly different from zero. among the traits involved in humoralmediated adaptive immunity, heritabilities for total igg and iga antibody levels were higher than for total igm and specific antibodies, and weak h values were obtained for b lymphocyte count (igm + cells). heritability for innate its such as i) total cell number (eos and neu), ii) leukocyte subsets (cd + cd + cells, cd + cd acells, cd + mhcii + cells and tcrcd + lymphocytes), iii) cytokine production (ifna and il ), and iv) phagocytosis were high and significantly different from zero. in addition, several innate its showed weak to moderate heritability, including proinflammatory cytokines (il b, il , tnf and il ), mon, cd -cd + cells, cd + cd cells, mhcii -cd a + cells, mhcii + cd acells, cd -cd a + cells and cd + cd a + cells. variation in acute phase proteins was moderately (crp) to highly (hapt) heritable. among the four traits, which measured the total number (mon) or proportions (mhcii + cd a + , cd + cd a + and cd + mhcii + ) of monocytes, moderate to high h , but not significantly different from zero, were estimated, except for cd + mhcii + cells. other haematological traits (rbc, ht, rdw and plt) gave high h estimates, of which ht and plt were significant. pairwise genetic correlations are presented in table s and illustrated in figure . genetic correlation estimates among most its were generally weak but a few high genetic correlations were observed. the number of positive genetic correlations ( ) was higher than that of negative correlations ( ), as already observed for phenotypic correlations. positive genetic correlations were higher (in absolute values) than negative ones (table s , figures and s ). only ( . % of the total number of correlations) and five r ĝ ( . % of the total number of correlation estimates) were higher than . or lower than - . , respectively. the unsupervised hierarchical clustering distinguished two main clusters of traits ( figure ). the first cluster of traits (cluster a) could be divided into two groups: i) a group of four traits including one innate immunity cytokine (ifna), two antibody levels (total igg and specific igg-mh), and one facscharacterized leukocyte subpopulation (cd + cd a -) and ii) a group of traits with nine hemogram-based cell counts or facscharacterized leukocyte subpopulations (wbc, lym, mon, neu, eos, cd + mhcii + , cd + cd + , cd -cd + cells) and two cell activity traits (igm and il -pmaiono). the second cluster of traits (cluster b) is also subdivided into two groups of traits: i) a group of three different cell response traits (il , iga and ifng-pmaiono) and one facs-characterized leukocyte subpopulation (igm + cells), and ii) a group of nine cell response traits (il , il -pmaiono, il -pmaiono, tnf, prolif-pma, hapt, crp, il b, il and phag) and four facs-characterized leukocyte subpopulations (cd + cd -, cd + cd -, tcrcd + and mhcii + cd a + cells). in cluster a, the first group of traits showed moderately to highly positive genetic correlations with each other. indeed, r ĝ values greater than . were estimated between wbc, lym, mon, neu, eos, cd -cd + and cd + cd + (nk) cells ( figure , table s ). in cluster b, r ĝ values greater than . were estimated between i) tnf, il and phag, and ii) crp and hapt ( figure , table s ). strong negative r ĝ values (, . ) were found between a few traits from both clusters: i) tnf and three cell number traits (wbc, lym, mon), ii) il and cd + mhcii + , and iii) crp and iga. the measured its globally cover innate and adaptive immunity the large-scale study reported here allowed us to estimate the genetic and phenotypic parameters of numerous its measured on pigs bred in the same environment. innate immunity is represented by traits, humoral-mediated immunity by five traits and cell-mediated adaptive immunity by traits. we have also considered the total number of white blood cells and lymphocytes, and four other haematological traits (rbc, ht, rdw and plt). within cell-mediated immunity, we explored both th and th responses by measuring cytokine production. figure summarizes the traits that we selected to cover immunity globally. these traits include in vivo measures on blood such as quantification of cell populations by hemogram, dosage of circulating immunoglobulins and acute phase proteins, as well as ex vivo measures obtained after in vitro tests such as lymphocyte proliferation, phagocytosis capacity and cytokine production after blood stimulation. all these its have been widely studied in humans [ , ] . the trait typology we have used (table ) follows a model based on a clear distinction between innate and adaptive immunity, which may be over-simplistic since both immune systems are closely interconnected [ , ] . monocytes are involved in innate immunity but are also antigen-presenting cells required for adaptive immunity, and cytokines such as il are at the interface between innate and adaptive immunity. similarly, the cell-mediated and humoral adaptive immunity subdivision is artificial. for example, il is a cytokine produced by th lymphocytes that is usually classified as part of adaptive cellmediated immunity, whereas it is also involved in antibody production and thus adaptive humoral immunity. in addition, the conventional paradigm that cd + ab t lymphocytes differentiate into th and th lineages expressing specific cytokines is collapsing. indeed, recent studies have revealed that cytokine production by the different cd + t cell subsets (th , th , th, th and itreg) is highly flexible, providing new insight into the th cell plasticity [ ] . nevertheless, although schematic, the approach used in our report provides a comprehensive overview of genetic variation and co-variation across the entire immune spectrum in pigs (figure ). table illustrates various ranges of phenotypic variation in the measured immune traits, with most having a cv over . . such variability has already been reported in large panels of healthy humans [ ] , and in previous studies on pigs [ , , , ] . for instance, we substantiated the high level of variation of cytokine production previously reported for il production and virusinduced ifna production in a swedish yorkshire population [ ] . for innate immunity-related cytokines and il , stimulation was performed with a mixture of lps, pma and ionomycin. il was the cytokine produced with the highest levels followed by il b, tnf, il and lastly il . these four cytokines are not expected to be expressed at similar levels at all time points after stimulation and a kinetic study would help to improve comparison of cytokine production levels. weaker levels of adaptive ir cytokines are observed after lps stimulation compared to cona or pmaiono. these differences could be related to the distinct modes of action of these molecules. indeed, pma, a plant-derived functional analog of diacylglycerol, in conjunction with ionomycin, a calcium ionophore produced by streptomyces conglobatus, and cona, a lectin originally extracted from the jack-bean canavalia ensiformis, are known to be potent mitogens of blood lymphocytes [ , ] . lps, a major structural component of the outer membrane of gram-negative bacteria, which binds the cd / the five first components, which explain more than % of the total variance, are in red. b. plot of the bayesian information criterion (bic) calculated with different models according to number of clusters. six models are compared: eii (spherical with equal volume and equal shape), vii (spherical with variable volume and equal shape), eei (diagonal with equal shape and equal volume), vei (diagonal with variable shape and equal volume), evi (diagonal with equal shape and variable volume), vvi (diagonal with variable shape and variable volume). c. first factorial plan ( : first component, : second component) with three clusters identified by multivariate normal mixture modelling and model-based clustering taking into account the components (clusters k , k and k are in blue, green and red, respectively). doi: . /journal.pone. .g tlr /md receptor complex and promotes the secretion of proinflammatory cytokines, has been extensively used to study innate immune response [ ] . we have already shown that transcriptome modifications in peripheral blood mononuclear cells (pbmcs) differ between pmaiono and lps stimulation and that pmaiono and lps target different cells and cellular pathways [ ] . the combination of pma and ionomycin induces a stronger stimulation that may be related to a higher production of cytokines as detected in the present study. in addition, the lymphocyte proliferation induced by lps is weaker than that observed with other stimulants, as expected. our study provides the first heritability estimates for innate and adaptive cytokine production and for lymphocyte proliferation after pma-ionomycin and lps stimulations in pig. pro-inflammatory cytokines appear to show less heritable variation than adaptive system-related cytokines. among the adaptive system-related cytokines, estimated heritability was weakest for cytokines produced after lps stimulation, except for il production. heritability estimates for lymphocyte proliferation after cona, pma and lps stimulations were moderate and that of lymphocyte proliferation after cona stimulation was comparable to the value obtained by edfors-lilja and colleagues [ ] . moderate to high heritability estimates for cell count traits from hemogram or facs also confirmed those previously obtained for wbc, total lymphocytes, neutrophils, eosinophils and some leukocyte subsets (for cd + and cd + t lymphocytes, cd t lymphocytes, cd r + , cd r + cd a -, cd r + cd a + and cd + mhcii + ) [ , , , ] . likewise, our results confirmed the high heritability estimate for phagocytosis [ ] . conversly, a lower heritability estimate for crp than previously reported was observed [ , ] . overall, the heritability estimates for these traits appear robust regardless of populations, environments and protocols. some discrepancies exist between our heritability estimates and previous results for humoral-mediated adaptive its and some innate its. indeed, in our study, b lymphocyte levels (igm + cells) are not significantly heritable contrary to other results [ , ] and specific iggs (igg-mh) have lower heritability estimates ( . , se = . ) than previously reported (range from . to . ) for specific antibodies directed against other antigens [ ] . our estimated heritability for total igg is higher ( . , se = . ) than in published reports [ , , , ] . in addition, our estimated h for ifna production is moderate to high ( . , se = . ), contrary to previous results (range from to . ) [ ] , and for haptoglobin ( . , se = . ) is higher than previously published (range from . to . ) [ , ] . these discrepancies could be due to differences in the pig breeds and in environment factors but also to the absence of common standardised protocols between laboratories. in order to better qualify the phenotypes, protocol standardisation is needed. overall, we show that variation in both innate and adaptive its is under substantial genetic control (figure ; tables and ) . similar heritability estimates for innate and adaptive its and also between cell number and cell response parameters were observed. further, heritability estimates do not differ consistently between in vivo and ex vivo measures with no apparent bias due to phenotyping methods. these data also suggest candidate its for qtl mapping. indeed, mapping studies have already started for total leukocyte count ( [ , ] ; animalqtldb, http://www.animalgenome.org/cgi-bin/qtldb/index), mitogen-induced proliferation [ ] , antibody response [ , ] , cytokine production (il , il and ifnc) [ , ] , complement activity [ ] , and acute phase protein serum concentration [ ] . compared to the previously limited data on genetic correlations between its in pigs, our study provides a large-scale estimation of phenotypic and genetic correlations among its. pca results and correlation estimations highlight the weak phenotypic and genetic correlations between the different its, except mainly for cell subsets. no cluster of innate and adaptive its is revealed. these results illustrate that many of the its included in our study provide more or less independent potential clues for selecting for improved immunocompetence. such complementarity is expected since innate immunity is in place or ready for activation prior to infection or antigenic stimulation and collaborates with adaptive immunity, which is induced by infection or antigenic stimulation. nevertheless, a few highly positive genetic correlations have been detected between total number of white blood cells and some leukocytes subsets, and between some leukocyte subsets such as total number of lymphocytes, cd -cd + lymphocytes (which contains ab cd -cd + lymphocytes and nk), and nk cells (cd + cd + cells). phagocytosis, production of il and tnf, two pro-inflammatory cytokines produced by monocytes and macrophages, were positively correlated with acute inflammatory phase proteins produced by hepatocytes i.e. crp and hapt. a high correlation was also found between crp and hapt. clapperton and colleagues [ ] have shown that phenotypic correlations between leukocyte subsets and acute phase proteins are weak (, . ) and not significantly different from zero in agreement with our results. in contrast to our study, clapperton and collaborators have not detected any significant genetic and phenotypic correlations between different leukocyte subsets except when one subset was nested in another [ ] . our results provide a framework for including its in multitrait selection for immunocompetence in pigs. criteria for inclusion should take into account heritability, biological relevance, biological sensitivity and feasibility of measurement [ ] . the weak genetic correlations between most its suggest that it will be difficult to choose only a few its to select for immunocompetence, and that a combination of many traits may be required [ ] . the moderate to high heritabilities estimated for many traits together with the selection study on pigs carried out by wilkie and colleagues a decade ago support the feasibility of selecting for immunocompetence [ , ] . chickens have also been successfully divergently selected for carbon clearance (phagocytic activity), high antibody response to newcastle disease virus three weeks after vaccination (adaptive humoral ir) and wing web response to pha (high cell-mediated immune response) for more than twelve generations [ , ] . in order to include immunocompetence in selection for improved health, a major challenge will be to correlate variation in heritable its in healthy animals with inter-individual variability in response to various pathogens. testing this hypothesis will be a key point for further use of its as indirect selection criteria in multitrait selection to improve resistance to disease. some results on genetic and phenotypic relationships between immunocompetence and susceptibility to specific pathogens have already been reported in the literature for pigs. among pigs selected for eight generations for high (hir) or low (lir) response based on an index of four cell and humoral-mediated immunity traits, an increased specific antibody response and lower polyserositis were observed in the hir pigs compared to the lir pigs after challenge with a novel pathogen, mycoplasma hyorhinis [ , ] . thus, animals with a high ir level to unrelated challenges, as defined by wilkie and colleagues [ , ] , have a better response to infection with mycoplasma hyorhinis. however, hir pigs develop more severe arthritis than lir pigs. indeed, the levels of humoral and cell-mediated adaptive its included in the wilkie et al index induce the formation of immune complexes and/or the development of inflammatory responses, central to the pathogenesis of mycoplasma hyorhinis-induced arthritis. other correlation tests between its and response to various infections are needed. however, it is important to remain cautious with high responder animals, which could develop autoimmune pathologies or pathological iummne responses. all the studies on immunocompetence and resistance to disease will have to be completed by estimation of genetic correlations with economically important traits already under selection. negative genetic correlations have been reported between some its (monocytes, cd r + cells) and average daily gain [ ] . however a larger correlation study considering a higher number of its and pig performances is needed and is ongoing in our population. in the future, a more sustainable production system may require a compromise with a slight decrease in performance traded off for a gain in animal robustness. more studies are required to better understand the correlations between its and production traits and it is not established which levels of its would be good predictors for resistance to pathogens if any. in conclusion, our results show that variation in many its is under significant genetic control in pigs and these findings may provide insights in other species. moreover, based on heritability and correlation estimations, some of the its that we have studied might be incorporated into selection schemes, provided they are associated with improved global health and do not exhibit strong antagonisms with other economically important traits. our experiment was conducted in accordance with the french national regulations for humane care and use of animals in research. no ethics approval was required for the vaccination and the collection of blood samples under the then current regulations. experiments were performed under the individual license numbers - assigned to marcel bouffaud who was responsible for experiments in the test farm, and - assigned to a veterinarian, dr silvia vincent-naulleau. the experimentation agreement number for the test farm at le rheu was a - - . a total of large white pigs (castrated males, dam line) tested for performance traits in a pig test station (ue , inra, le rheu, france) was included in the study. the pigs were distributed in seven contemporary groups and belonged to nuclear families obtained from boars, with an average of . (+/ . ) piglets per boar. animals were born and weaned in different selection herds and arrived in the test station at five weeks of age with no prior vaccination. they were placed into pens of piglets in a post weaning unit and vaccinated against m. hyopneumoniae (stellamune, pfizer, one injection) one day after their arrival in the test station, when . days old in average. all pigs were apparently healthy with no clinical sign of infection. all animals were sampled three weeks after vaccination. blood samples were collected via the external jugular vein into tubes with or without anti-coagulants, according to further use. blood collected in ml tubes with no anti-coagulant was centrifuged at g for min at uc. the serum was collected and stored at uc until use. plasma was collected from blood sampled in heparinised tubes and stored at uc before use. hemograms were measured with an ms - counter (eli-techgroup, france) with blood sampled in edta tubes. among a set of traits, nine were included in the genetic analyses: total number of leukocytes, lymphocytes, monocytes, neutrophils, eosinophils, erythrocytes, platelets and hematocrit (tables and ). total concentrations of immunoglobulin subsets were measured by elisa as previously described [ ] . plasma samples were diluted : , : and : , to detect igm, iga and igg, respectively, in tris-buffered saline and added to plates coated with immunoglobulin class specific pig antibody (bethyl laboratories inc., interchim, france). the different subsets were detected with the appropriate peroxidase anti-pig igm, iga or igg (bethyl laboratories inc.) and were quantified by reference to standard curves constructed with known amounts of pig immunoglobulin subsets. anti-m. hyopneumoniae igg titers were also measured by elisa using a commercial kit (elisa id screenh m. hyopneumoniae indirect, idvet, france). absorbance was read at nm using an elisa plate reader (spectra thermo, tecan, nc, usa) and the biolise . data management software. haptoglobin and c reactive protein levels were measured in pig serum by colorimetric tests (phase haptoglobin assay, abcys biologie, france) and elisa assays (porcine c reactive protein assay, abcys biologie, france), respectively. absorbance was read at nm using an elisa plate reader (mrx revelation, dynex). pbmcs were purified by density gradient centrifugation. a volume of ml heparinised blood was added to leucosept tubes (greiner bio-one, france) pre-filled with ml ficoll (lymphocytes separation medium, eurobio, france) and centrifuged at rpm for min. pbmcs were collected at the ficoll interface and washed in ml d-pbs without mgcl and cacl (gibco, invitrogen, france). cells were then incubated in ml bd pharmlyse x (bd biosciences, france) at room temperature. purified pbmcs were washed in ml d-pbs without mgcl and cacl , incubated with ml pig serum at uc for min, washed again in ml d-pbs without mgcl and cacl and then washed in ml s/w buffer ( g/l nan , g/l bovine serum albumin in pbs, ph . ) at a final concentration of . cells/ml. cells were used for each antibody labelling. single, double or triple staining was performed using monoclonal antibodies (mabs) directed against i) cd (msa , isotype igg a, vmrd) and cd (mca , isotype igg , serotec), ii) cd (pt a, isotype igg a, vmrd) and cd a (pt b, isotype igg b, vmrd) iii) tcrcd (mac , isotype igg a, bd biosciences pharmingen), iv) igm (pig a, isotype igg b, vmrd) and v) mhcii (msa , isotype igg a, vmrd), cd (mca , isotype igg , serotec) and cd a ( - - a, isotype igg b, bd biosciences pharmingen). briefly, pbmcs were stained with primary mabs for min at uc, washed in s/w buffer and stained with allophycocyanin-conjugated anti-mouse igg (bd biosciences, france), phycoerythrin-conjugated antimouse igg a (southern biotech, france), fitc-conjugated antimouse igg b (southern biotech, france), apc-conjugated antimouse igg (bd biosciences, france), phycoerythrin-conjugated anti-rat igg a (bd pharmingen, france), or phycoerythrinconjugated anti-mouse igg b (southern biotech, france). after washing in s/w buffer, cells were fixed in bd cellfix solution (becton dickinson, germany). data acquisition and analysis were carried out with the facscan and cellquest software (becton dickinson, uk). synthesis of ifna by leukocytes of pigs was tested in vitro by incubating diluted total blood in the presence of pseudorabies virus (prv, suid herpesvirus )-infected, glutaraldehyde-fixed, pk cell monolayers, according to a protocol previously described for transmissible gastroenteritis virus [ ] . confluent pk cell monolayers grown in well plates were infected by prv at a multiplicity of infection of , fixed with . % glutaraldehyde h post-infection and washed with d-pbs and rpmi before the addition of blood samples. for each animal, monolayers were incubated with diluted heparinized blood ( ml of blood diluted : in dmem supplemented with antibiotics) for h at uc. plates were then centrifuged at x g for min at uc, and supernatants were collected and stored at - uc. ifna was assayed in the supernatants using a classical sandwich elisa test as previously described [ ] . production and dosage of il b, il , il , tnfa and il heparinized blood samples ( ml) were fivefold diluted in well plates in . ml rpmi medium (biowhittaker, belgium) supplemented with % heat-inactivated fetal bovine serum (qb perbio, uk), mmol/l l-glutamine, u/ml penicillin and mg/ml streptomycin. for stimulation, a mixture of ng/ml pma (sigma, france), mg/ml ionomycin (sigma, france) and mg/ml lps from escherichia coli o :b (sigma, france) was added to the diluted blood. for mock stimulation, a volume of pbs equal to the volume of stimulation reagents was added to the diluted blood. after incubation at uc for h, culture supernatants were collected by centrifugation at g for min and stored at uc before use. the cytokines il b, il , il , tnf and il were quantified using commercial elisa tests (duoset elisa development kits, r&d systems, usa). for quantification of basal levels of cytokines in supernatants from mock-stimulated cells, the samples were not diluted for quantification. supernatants collected from stimulated cells were diluted ( : for il b and il , : for il , : for tnf and : for il ). all samples were tested in duplicates. absorbance was read at nm using an elisa plate reader (mrx revelation, dynex). results were expressed as pg of cytokine/ml. heparinized blood diluted : in complete culture medium consisting of dmem (dulbecco's modified eagle medium, eurobio, france) supplemented with % fetal calf serum (hyclone, perbio, france), mm l-glutamine, u/ml penicillin and mg/ml streptomycin (eurobio, france) was stimulated with mg/ml cona (sigma, france), or with ng/ml of pma (sigma, france) and mg/ml of ionomycin (sigma, france) or mg/ml lps from escherichia coli (sigma, france). cytokine content was measured in supernatants using elisa tests as already described [ ] . briefly, purified fractions of anti-swine il- , il- , ifnc (clones a d f , a b f and a d b respectively, biosource, france) and il (clone , r and d system, france) were used as capture antibodies, in conjunction with the biotinylated anti-swine il- , il- , il and ifnc monoclonal antibodies (clones a d h , a b c and a d c , respectively, biosource, clinisciences, france) or anti-swine polyclonal antibody (goat anti-porcine il- , r and d system, france). streptavidin-horseradish peroxidase (biosource) and tmb (fermentas, md, usa) were used for detection. absorbance was read at nm using an elisa plate reader (spectra thermo, tecan, nc, usa) and the biolise . data management software. recombinant pig il- , il- , il and ifnc were used as standards. the detection limits were pg/ ml, pg/ml, pg/ml and pg/ml for il- , il- , il and ifnc, respectively. results were expressed as pg of cytokine/ ml. lymphocyte proliferation was performed in well plates as already described [ ] . briefly, heparinized blood samples were diluted : in complete culture medium consisting of dmem (dulbecco's modified eagle medium, eurobio, france) supplemented with % fetal calf serum (hyclone, perbio, france), mm l-glutamine, u/ml penicillin and mg/ml streptomycin (eurobio, france). for detection of unspecific lymphocyte proliferation, the diluted blood samples were seeded in well plates ( ml/well) and mock-stimulated for h by incubation in culture medium (control wells), or stimulated for h by incubation with the culture medium supplemented with either mg/ml cona (sigma, france), or ng/ml of pma (sigma, france) and mg/ml of ionomycin (sigma, france), or mg/ml lps (sigma, france). control wells remained unstimulated. after h of incubation at uc, . mci of h-methylthymidine (icn, france) was added to each well. after another h incubation period, the cells were harvested through glassfiber filters (whatman, united kingdom) by means of an automatic harvester (titerteck-skatron, molecular devices, france). incorporation of tritiated thymidine was measured with a liquid scintillation beta counter (kontron instruments, france). results were expressed as a stimulation index of lymphocyte proliferation calculated as mean counts per min (cpm) of the triplicate cultures in stimulated culture/mean cpm in control non-stimulated culture. preliminary statistical analyses were performed using r software [ ] . in order to test if trait distributions deviated from gaussian, a d'agostino normality test was used (p = . ). since most traits were not sampled from a gaussian distribution, they were all normalized using a box-cox transformation except for phenotypes reaching the value zero, which were normalized using ln( +x) transformation. significant effects of age at the time of vaccination, of time of vaccination, of breeding unit and of time of experiment were detected for most traits by variance analysis taking into account these effects. normed principal component analysis (pca, [ ] , dudi.pca function, ade package [ ] , r software [ ] ) on a subset of its adjusted for age at the time of vaccination, time of vaccination, breeding unit and experiment (table ) were performed using a linear model. clusters of its were detected using the r package mclust for normal mixture modelling and model-based clustering [ ] . it combines model-based agglomerative hierarchical classification, based on the classification likelihood, and the expectation-maximization (e-m) algorithm for maximum likelihood estimation of multivariate mixture models. variance components, genetic parameters and their standard errors were estimated by the restricted maximum likelihood (reml) method [ ] , using the wombat software [ ] . this is the reference method to estimate genetic parameters with a mixed model. univariate and bivariate mixed linear animal models were employed to estimate heritability and genetic correlations, respectively. for the univariate analyses, the fixed part of the model included experiment time, age at the time of vaccination, vaccination time and herd of origin effects and the random part included a common litter environmental effect and direct genetic effects. in matrix notation y~x b zw a azw c cze where y = the vector of observations; x b , w a and w c are known incidence matrix relating observations to fixed and random effects; ß = a vector of fixed effects and covariates; a = the vector of direct genetic effects; c = the vector of common litter environmental effects; and e = the vector of random residual effects. all random effects were assumed to follow a normal distribution with zero mean. for bivariate analyses, the same effects as for univariate analyses were taken into account in the fixed part of the model and a direct genetic effect was included in the random part of the model. % confidence intervals ( ci) were calculated for heritability (h ) estimates (h ). heritability estimates have been classified: high (h . . ), moderate ( . ,h , . ) or weak (h # . ). a graphical representation of the genetic correlations combined with a hierarchical clustering (euclidian distance, average link) was obtained with the heatmap function from the bioconductor software [ ] . comparisons of heritability average between subsets of traits were tested using the wilcoxon mann-whitney test (significance threshold p-value = . ). figure s heatmap of the phenotypic correlations between its. the correspondence between colour scale and genetic correlation levels are presented on the right-hand side of the heatmap. (tif) estimation of genetic trends in french large white pigs from to for growth and carcass traits using frozen semen selection for high immune response: an alternative approach to animal health maintenance? genetic aspects of health and disease resistance in pigs relationships between genetic change and infectious disease in domestic livestock toll-like receptors and innate immunity kuby immunology toward genetic dissection of high and low antibody responsiveness in biozzi mice genetic parameters for antibody response of chickens to sheep red blood cells based on a selection experiment correlated effects of selection for immunity in white leghorn chicken lines on natural antibodies and specific antibody responses to klh and m. butyricum evaluation of immune responses of cattle as a means to identify high or low responders and use of a human microarray to differentiate gene expression use of estimated breeding values in a selection index to breed yorkshire pigs for high and low immune and innate resistance factors immune responsiveness in swine: eight generations of selection for high and low immune response in yorkshire pigs multi-trait selection for immune response; a possible alternative strategy for enhanced livestock health and productivity cytokines in mycoplasma hyorhinis-induced arthritis in pigs bred selectively for high and low immune responses genetic variation in parameters reflecting immune competence of swine pig peripheral blood mononuclear leucocyte subsets are heritable and genetically correlated with performance traits associated with innate and adaptive immunity in pigs: heritability and associations with performance under different health status conditions an evaluation of immune competence in different swine breeds immunological traits have the potential to improve selection of pigs for resistance to clinical and subclinical disease mapping quantitative trait loci for immune capacity in the pig confirmation of qtl on porcine chromosomes and influencing leukocyte numbers, haematological parameters and leukocyte function qtl for traits related to humoral immune response estimated from data of a porcine f resource population mapping quantitative trait loci for cytokines in the pig deciphering the genetic control of innate and adaptive immune responses in pig: a combined genetic and genomic study markers to measure immunomodulation in human nutrition intervention studies immunological parameters: what do they mean? innate immune recognition and control of adaptive immune responses innate immunity: impact on the adaptive immune response mechanisms underlying lineage commitment and plasticity of helper cd + t cells the association between plasma levels of acute phase proteins, haptoglobin, alpha- acid glycoprotein (agp), pig-map, transthyretin and serum amyloid a (saa) in large white and meishan pigs mitogenic activity of -o-tetradecanoyl phorbol- -acetate on peripheral blood lymphocytes from young and aged adults characterization of the response of human thymocytes and blood lymphocytes to the synergistic mitogenicity of -otetradecanoylphorbol- -acetate (tpa)-ionomycin lps/tlr signal transduction pathway transcriptome analysis of porcine pbmcs after in vitro stimulation by lps or pma/ionomycin using an expression array targeting the pig immune response mapping quantitative trait loci for innate immune response in the pig immune modulation: the genetic approach mycoplasma hyorhinis infection of pigs selectively bred for high and low immune response ingestion of deoxynivalenol (don) contaminated feed alters the pig vaccinal immune responses induction of alpha interferon by transmissible gastroenteritis coronavirus: role of transmembrane glycoprotein e a sensitive immunoassay for porcine interferon-alpha fumonisin b alters cell cycle progression and interleukin- synthesis in swine peripheral blood mononuclear cells ingestion of low doses of deoxynivalenol does not affect hematological, biochemical, or immune responses of piglets a language and environment for statistical computing. vienna: r foundation for statistical computing on lines and planes of closest fit to systems of points in space the ade package. i. one-table methods model-based methods of classification: using the mclust software in chemometrics the significance of the difference between two means when the population variances are unequal recovery of interblock information when block sizes are unequal wombat: a tool for mixed model analyses in quantitative genetics by restricted maximum likelihood (reml) bioconductor: open software development for computational biology and bioinformatics sincere thanks are due to mathieu gautier (inra, umr gabi, jouy-en-josas, france) for his computational help and to thierry tribout (inra, umr gabi, jouy-en-josas, france) for providing the pig genealogy. we thank fabrice andreoletti and stephan bouet (inra, umr gabi, jouyen-josas, france) for their contribution to animal experimentation. we are also grateful to hélène hayes (inra, umr gabi, jouy-en-josas, france) for helping in preparing the manuscript. we warmly thank christopher moran (faculty of veterinary science, university of sydney) for helpful comments and suggestions for the final submission of the manuscript. key: cord- -jgtsl q authors: harkouk, hakim; jacob, chantal; fletcher, dominique title: urgent development of an anaesthesiology-based intensive care unit for critical covid- infected patients date: - - journal: anaesth crit care pain med doi: . /j.accpm. . . sha: doc_id: cord_uid: jgtsl q nan the global covid- pandemic requires anaesthesiologists to adapt themselves to this unprecedented situation . beyond this first adaptation, the major influx of patients imposes to rapidly manage critical patients outside the usual intensive care structures, in addition to required care of surgical patients. in france, the first cases are diagnosed on january th , . on march th , all unessential institutions are shut down. since march th at noon, the population is confined at home with strict rules. on april st , patients are hospitalised in intensive care units while national maximal admissions are estimated to be patients. we shortly describe the use of professional skills and existing structures in a french anaesthesia department to deal with this covid- crisis. french anaesthesiologists have years of training with mixed skills in anaesthesia ( years) and resuscitation ( years). our beds university hospital is part of the assistance publique -hôpitaux de paris, the first hospitals group in ile de france, a region that is severely affected by the covid- pandemic. our structures include operating theatres and beds of recovery room (rr), performing an average of . scheduled or urgent surgical interventions a year, in trauma, visceral and vascular surgeries. the medical team includes anaesthesia consultants and residents; the paramedic team includes nurse anaesthetists and rr nurses. the intensive care unit (icu), managed by intensivists, has a capacity of resuscitation beds and continuous care beds. a regulatory team headed by an anaesthesiologist with the help of surgeons, usually meeting once a week, decides a new organisation evaluating rapidly both management of critical negative and positive covid- patients and surgical activity; chronological details are listed in table the man-power includes anaesthetist nurses and rr nurses and anaesthesiologists ( anaesthesiologists present hours a day). all these professionals work in -hour shifts, hours a day, days a week. this radical reorganisation within weeks of an operating theatre and a rr relies on the professional, structural and material resources of an anaesthesia department to create an icu with beds dedicated to critical covid- infected patients while maintaining the management of selected scheduled and emergency surgery. problems to overcome are numerous, covering both patient care, professional protection and urgency of management; we only discuss here three of them: . the isolation of covid or non-covid patients by restructuring the circulation areas, using the advantages of an operating theatre (clean and contaminated circuit, closed operating theatre under negative pressure) and identifying two separates care team selected both on professional skills and risk of viral exposition (age over years old and/or comorbidities). all successive decisions were validated with hospital hygiene team; . making the best of existing structures for patient care and professional protection: the rr allows easy centralised monitoring of patients but exposes to aerosolised virus, especially with high oxygen flow and requires enhanced protection for nursing professionals (ffp mask changed every hours, dressing and take-off procedures, gown) but also an adaptation of the rr , . negative pressure was installed on day after admission of first critical patients and rr was equipped with air extractors plasmair© (dalkia) which allow treating . m of air per hour (i.e. volumes per hour for a m rr) ; . the medical and paramedical anaesthesia teams had to upgrade rapidly their skills to be able to use high and very high oxygen flow therapy, ventilation of the patient with severe adult respiratory distress syndrome and to be kept informed of additional therapeutic solutions specific to these patients in collaboration with intensivists. the target physician/patient ratio was set to / ( anaesthesiologist hours a day) and / for nurses in the acute phase and / . in the steady phase. page of j o u r n a l p r e -p r o o f after days of functioning as icu for critical covid- infected patients, patients were admitted with patients with mechanical ventilation. patients start to be discharged from icu and hospital (respectively and ) and patient is deceased. we report our experience with mobilisation of an anaesthesia team and use of existing structures for urgent creation of an icu managing critical covid- patients in a pandemic which exceeds the usual resources of resuscitation structures. j o u r n a l p r e -p r o o f funding statement: support was provided solely from institutional and/or departmental sources conflicts of interest: the authors declare no competing interests service d'anesthésie, hôpital ambroise paré, assistance publique hôpitaux de paris france; for icu organisation and validation of the manuscript service d'anesthésie, hôpital ambroise paré, assistance publique hôpitaux de paris france; for icu organisation and validation of the manuscript service d'anesthésie, hôpital ambroise paré, assistance publique hôpitaux de paris france; for icu organisation and validation of the manuscript service d'anesthésie, hôpital ambroise paré, assistance publique hôpitaux de paris france; for icu organisation and validation of the manuscript service d'anesthésie, hôpital ambroise paré, assistance publique hôpitaux de paris france; for icu organisation and validation of the manuscript service d'anesthésie, hôpital ambroise paré, assistance publique hôpitaux de paris france; for icu organisation and validation of the manuscript service d'anesthésie, hôpital ambroise paré, assistance publique hôpitaux de paris france; for icu organisation and validation of the manuscript service d'anesthésie, hôpital ambroise paré, assistance publique hôpitaux de paris france; for icu organisation and validation of the manuscript service d'anesthésie, hôpital ambroise paré, assistance publique hôpitaux de paris france; for icu organisation and validation of the manuscript service d'anesthésie, hôpital ambroise paré, assistance publique hôpitaux de paris france; for icu organisation and validation of the manuscript references covid- infection: implications for perioperative and critical care physicians aerosol and surface stability of sars-cov- as compared with sars-cov- supplemental treatment of air in airborne infection isolation rooms using high-throughput in-room air decontamination units key: cord- -bb ekgdm authors: unlu, e.; leger, h.; motornyi, o.; rukubayihunga, a.; ishacian, t.; chouiten, m. title: epidemic analysis of covid- outbreak and counter-measures in france date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: bb ekgdm covid- pandemic has triggered world-wide attention among data scientists and epidemiologists to analyze and predict the outcomes, by using previous statistical epidemic models. we propose to use a variant of the well known seir model to analyze the spread of covid- in france, by taking in to account the national lockdown declared in march , . particle swarm optimisation (pso) is used to find optimal parameters for the model in the case of france. we propose to fit the model based only on the number of daily fatalities, where an r score based error metric is used. as number of confirmed cases shall not be fully representative due to low testing especially in the first phases of the outbreak, we present that basing the model optimisation on the fatalities can provide legitimate results. in december , novel coronavirus-sourced atypical pneumonia cases were reported in wuhan, china. rapidly, it evolved into an epidemic in its city of origin [ ] . despite taken counter-measures, the outbreak has gradually spread out globally. the world health organisation (who) declared it a pandemic in march , and called for augmented enforcing policies to all governments [ ] . countries have responded to outbreak with varying degrees of containment and other preventive actions; also with varying latency [ ] [ ] [ ] [ ] . the pandemic is ongoing by the date april , when this paper is written, with total confirmed cases more than . million and , deaths. this work focuses on france, where the first confirmed case was reported on january , . the situation then rapidly deteriorated, leading authorities to execute more draconian policies [ ] [ ] . a nation-wide, strict lockdown has been initiated on march and was announced to continue till may . by april , there are around , confirmed cases and , deaths caused by covid- . french government has announced that [ ] [ ] lockdown will be lifted starting on may , with strict measures. the exact details of the lockdown lifting policy are not publicized yet, however it is known that the allowed proportion of population and the commercial and public establishments will be gradually increased week by week. the reopening date of high risk establishments such as restaurants or schools is still unknown. it would not be speculative to state that the reproduction number r will also increase gradually over this period, however we expect that it will be much lower compared to pre-lockdown values due to increased awareness and public health measures. considering this setting, we aim to analyze the current situation and its future evolution using a variation of a widely employed mathematical epidemiology model: the seir (susceptible, exposed, infected, recovered) model [ ] [ ] . we have made the assumption to use two different reproduction numbers: r b and r q . r b is used to describe reproduction rate between the first confirmed death until the start of the lockdown and r q from the start to the end of the lockdown. the parameters of the model are fitted using particle swarm optimization (pso) [ ] [ ] . however, unlike other approaches we have decided to base our optimization on the number of fatalities only. in addition, rather than mean squared error (mse) or mean squared logarithmic error (msle); we minimize an r score based metric [ ] for daily deaths. the rationale behind this approach is that due to largely unknown dynamics of the novel coronavirus such as degree of infectiousness, length of incubation period and limited testing capability, using confirmed cases with such limited information may highly disrupt the validity of the model. however, the number of deaths caused by covid- is much more definitive, especially in the first days of the outbreak and thus expected to increase the accuracy. note that, with this approach the initial state values are also defined in terms of proportion of fatalities, hence they also are parameters of the pso optimization. according to our initial results, we estimate the reproduction number at around . before quarantine and . after total lockdown, in agreement with various recent studies around the globe for different scenarii [ ] [ ] . using the developed model we predict that if lockdown continues with strict measures, the total number of covid- fatalities should topple below , (which is currently around , ) by late august, ; where the effects of the epidemic start to significantly diminish. as it is not possible to predict the reproduction number for the forthcoming lockdown lift, we propose two scenarii with a reproduction number increase of respectively % and % per week during consecutive weeks. for these two scenarii, it is estimated that total number of fatalities may reach up to , - , , and that an epidemic situation could continue till november, . the seir model has been one of the keystone components of statistical epidemiology for a long time, and has proven its validity also for relatively recent regional or global epidemics such as mers, sars and ebola [ ] [ ] [ ] . the seir model is classified as a closed dynamical epidemiological model, as it divides the total population into four distinct categories, where the proportion of individuals belonging to each category evolves over time, subjects passing from one state to other. the temporal transitions between states are defined by several differential equations [ ] . initially, the entire population is considered as susceptible except very few infected individuals. gradually over time, according to base differential equations, susceptible individuals are exposed to disease, proportional to the reproduction number r . exposed subjects get infected, recover or die also based on the scalar parameters used in base differential equations. generally, these parameters are calculated for the considered case using optimization algorithms [ ] . in the past, researchers have also developed noteworthy number of extended seir/sir models, where additional number of states are added, considering the special circumstances of the evaluated epidemic [ ] [ ] . we further explain the statistical formulation of an extended seir model in the next section. covid- pandemic has immediately gained attention among the research community and numerous different approaches using seir model have been proposed. first studies were published in january, , for the initial epicenter wuhan, less than a month after the novel coronavirus was identified [ ] [ ] . one particular work aims to extend the model according to characteristics of covid- , by a research group from the university of basel [ ] [ ] . authors identify new states: h (hospitalized), c (critical) and d (dead). we base our methodology by following this guideline, as it appears to fit the dynamics of the on going pandemic. as mentionned in the previous section, we developed a seir-hcd model as in [ ] . the state transition diagram is shown in fig. . infected individuals may be hospitalized after a certain period. a proportion of the infected agents turn into critical cases, requiring intensive care; whilst the rest recovers. among the critical cases, a certain proportion of individuals eventually dies. these proportion constants are among the parameters of the model to be optimized. initially there are only a few infected agents, i(t ), while rest of the population is susceptible s(t ) = n − i(t ); where n is the population of france ( millions). also note that, at any time the sum of all states must be equal to n . one of the innovative proposal we make is to base our model completely on the number of deaths. as mentioned previously, the most definitive data for covid- case is the number of fatalities, due to current lack of pathological and epidemiological information about the disease and the low number of tests. especially, in the first phase of dissemination of the virus in france, the number of tests was much lower, further decreasing the validity of using confirmed cases as a model initiator. therefore, we propose to estimate the initial number of infected people, i(t ) from the initial number of fatalities d(t ), by simply reverse tracing the state transition diagram, using the proportion of hospitalized h(t ) and critical c(t ) cases. it is important to note that these proportion constants are parameters of the seir-hcd model we aim to optimize along a temporal axis. in other words, one novel outcome of our proposed algorithm is to be able to intrinsically calculate the initial number of infected citizens. it is highly reasonable to indicate that the number of infected people at the time where the first confirmed cases are announced shall be exponentially higher, due to very high proportion of asymptomatic cases [ ] . the transitions between states are explained by this set of differential equations in terms of proportion of the total population [ ] [ ] : all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . where t inc is the incubation period of the coronavirus, t inf is the infectiousness period of an infected agent, t hsp is the duration it takes for an infected agent to check in to a health facility and finally t crt is the duration it takes for an hospitalized person to turn into a critical case since the initial check-in. p a , p c , p f respectively refer to the proportion of asymptomatic infected individuals, the hospitalized agents who switched to a critical case and the critical cases resulting in death. r is the basic reproduction number for the coronavirus. we have decided to use the reported data for france, starting from february , when the first fatality was confirmed. without loss of generalization, we assume a binary reproduction number; r b for the interval between february , and march , for the pre-quarantine period and r q for lockdown period, which is still ongoing. finally, we need to optimize the model to find these parameters : r q , r b , p a , p c , p f , t inc , t inf , t hsp , t crt . we employ a particle swarm optimization (pso) for this task, which is a powerful evolutionary algorithm, well suited for this setting [ ] . as we propose to set our model solely on the initially reported covid- related fatalities, the the initial states of each dynamic component can be denoted as : other than suggesting a model optimization based on fatalities, we have also observed that the most adapted error metric is the r score of differentials of number of deaths (i. e. series of daily fatalities). this avoids overfitting, while preserving the parameter optimization in plausible ranges. the r score is a metric ranging from [−∞, ], where denotes the full accuracy, so we aimed to minimize the following value: the gradual lockdown lift, starting on may , is modeled as a % to % increase of quarantine time reproduction number for each week for next weeks (assuming reproduction number does nor grow after weeks), presenting two different scenarii for france. with pso optimization based on the defined error metric, the optimal parameters for france are found in table . we have estimated the reproduction number at . prior to lockdown and . after lockdown. in an independent research for france, these numbers are reported as . and . , where their method is not exposed; confirming integrity of our approach from a parallel perspective [ ] . we also report an approximate average reproduction number before lockdown that is consistent with the initial various studies on the issue [ ] [ ] , where the average of the studies suggests a value around . . as shown in table , all other parameters of the suggested model are within the range of other published research on covid- [ ] . for instance, asymptomatic ratio of . , incubation period of . days, infectiousness period of . days, hospitalisation period of . days, transition to critical state period of . , ratio of . for infected becoming critical and fatality ratio of . for critical patients are all close to the medians of the other reported work [ ] . these results are particularly interesting, since our seir model optimization approach with suggested error metric for number of fatalities is able to converge to the optimal point, and directly coronavirus related metrics such as incubation period, hospitalisation period etc. (assuming demographics etc. of a country does not influence considerably) are close to the average of the previous works. this asserts the validity of binary reproduction number approach for lockdown and its calculated values. note that, in complex dynamics of a seir model, slight variance of parameters may have a drastic impact on the outputs, such as the estimated number of deaths. we have calculated the mortality ratio of infected people ( − p a )p c p f as % . , where [ ] proposes % . for france. by the date april , our model estimates the proportion of already infected population as % . ( fig. - ) , while [ ] also reports as % . it is quite surprising to observe independent works show similar results, while our seir model optimization only takes fatalities into account for optimization. based on our model, we estimate that if lockdown is maintained, the number of fatalities for france might never pass the limit of , . if reproduction number grows by % each week during weeks following the lockdown lift; this upper limit might reach , (fig. ) . we observe that, if strict measures are respected till september, the epidemic almost terminates around this date. in case this growth rate all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. becomes % per week, the upper death toll limit might reach , (fig. ) ; where epidemic situation lasts till november, . considering the fact that, french government has already prepared a versatile and extensive plan for lifting, by limiting social gatherings, augmented surveillence and nationwide distribution face masks, these scenarii with % -% per week increase of quarantine time reproduction number seem legitimate. in this paper, we have proposed a seir model for covid- epidemic in france, similar to [ ] . unlike other similar attempts, we have used only confirmed number of deaths as an optimization metric. rationale behind this proposal is that deficiency in testing coverage, especially in the early phases of the outbreak, greatly underestimates the number of confirmed cases. however, the number of confirmed fatalities is a much more solid evidence in this setting. an error metric based on the daily death toll is presented and model parameters are all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may , . . optimized using pso algorithm. also note that, the initial state of the model (initial values of each state) is also defined based on the proportion of fatalities, hence all initial state values are also parameters for the pso optimization. we believe that, for epidemics at this scale starting a seir models with a hypothetical single infected individual may greatly lower the accuracy; especially considering their potential drastic impact. coronavirus disease (covid- ): situation report world health organization declares global emergency: a review of the how will country-based mitigation measures influence the course of the covid- epidemic? modeling the control of covid- : impact of policy interventions and meteorological factors covid- : extending or relaxing distancing control measures the effect of travel restrictions on the spread of the novel coronavirus analysis and forecast of covid- spreading in china, italy and france first cases of coronavirus disease (covid- ) in france: surveillance, investigations and control measures lifting lockdown: france looks ahead to options for easing coronavirus restrictions france to unveil end-of-lockdown plan within weeks global dynamics of a seir model with varying total population size a fractional order seir model with vertical transmission pyswarm: particle swarm optimization (pso) with constraint support particle swarm optimization pseudo-r measures for some common limited dependent variable models the reproductive number of covid- is higher compared to sars coronavirus novel coronavirus -ncov: early estimation of epidemiological parameters and epidemic predictions a modified seir model for the spread of ebola in western africa and metrics for resource allocation epidemic modelling using sars as a case study dynamical transmission model of mers-cov in two areas a comparison of delayed sir and seir epidemic models survey of models, methods and techniques for computational epidemiology nowcasting and forecasting the potential domestic and international spread of the -ncov outbreak originating in wuhan, china: a modelling study epidemic analysis of covid- in china by dynamical modeling about covid- scenarios seir-hcd model quantifying undetected covid- cases and effects of containment measures in italy covid- : seuls % des français infectés par le coronavirus le mai key: cord- - h tjs r authors: kuchenbuch, mathieu; d’onofrio, gianluca; wirrell, elaine; jiang, yuwu; dupont, sophie; grinspan, zachary m.; auvin, stephane; wilmshurst, jo m.; arzimanoglou, alexis; cross, j. helen; specchio, nicola; nabbout, rima title: an accelerated shift in the use of remote systems in epilepsy due to the covid- pandemic date: - - journal: epilepsy behav doi: . /j.yebeh. . sha: doc_id: cord_uid: h tjs r purpose: the purpose of the study was to describe epileptologists' opinion on the increased use of remote systems implemented during the covid- pandemic across clinics, education, and scientific meetings activities. methods: between april and may , we conducted a cross-sectional, electronic survey on remote systems use before and during the covid- pandemic through the european reference center for rare and complex epilepsies (epicare) network, the international and the french leagues against epilepsy, and the international and the french child neurology associations. after descriptive statistical analysis, we compared the results of france, china, and italy. results: one hundred and seventy-two respondents from countries completed the survey. prior to the covid- pandemic, . % had experienced remote systems for clinical care. during the pandemic, the use of remote clinics, either institutional or personal, significantly increased (p < (− )). eighty-three percent used remote systems with video, either institutional ( %) or personal ( %). during the pandemic, . % of respondents involved in academic activities transformed their courses to online teaching. from february to july , few scientific meetings relevant to epileptologists and routinely attended was adapted to virtual meeting (median: [ th– th percentile: – ]). responders were quite satisfied with remote systems in all three activity domains. interestingly, before the covid- pandemic, remote systems were significantly more frequently used in china for clinical activity compared with france or italy. this difference became less marked during the pandemic. conclusion: the covid- pandemic has dramatically altered how academic epileptologists carry out their core missions of clinical care, medical education, and scientific discovery and dissemination. close attention to the impact of these changes is merited. pandemics can lead to government regulations that limit social contact, decreased access to healthcare resources, and increased anxiety and fearall can disrupt the care path of patients with chronic diseases and decrease of face-to-face visits. in , a study on the outbreak of severe acute respiratory syndrome (sars) in china showed that the loss of contact with medical care providers led to an increase in the withdrawal of antiseizure medications resulting in an increase in seizure frequency [ ] . the current covid- pandemic is an important challenge for the management of patients with epilepsy worldwide. remote patient management systems [ ] (in use since the s and now integral to several national digital health strategies [ ] [ ] [ ] [ ] ) are a valuable tool during a pandemic to continue medical follow-up. they include different types of communications such as phone calls, one-way video links, and on live interactive communication. in addition, the epilepsy medical community is involved in educational activity and promoting knowledge dissemination through courses and scientific congresses. these activities also rely on face-to-face interactions and are also likely affected by the covid- pandemic. the aim of this study was to assess the impact of the covid- pandemic on the acute use of remote systems in clinics, education, and scientific meetings in the field of epilepsy and to explore the users' satisfaction and the perspectives of future use. we conducted a cross-sectional, electronic survey of epileptologists, neurologists, and pediatric neurologists mainly involved in the epilepsy field to determine the use of remote work during the covid- pandemic (supplementary data). to reach a wider public, this survey was addressed to adult and child neurologists specialized in epilepsy care through the european reference network for rare and complex epilepsies (epicare), international league against epilepsy (ilae), international child neurology association (icna), the french league against epilepsy (lfce), and the french society of child neurology (sfnp). the survey was comprised of questions divided into four sections: demographic and general information followed by remote work for clinical practice, education, and scientific meetings and symposia (for details, see supplementary data). items assessed practice before and during the covid- pandemic. the first two sections were mandatory (demographics and clinical practice). we used different types of questions: closed (n = ), semiopen (n = ) , and open (n = ). some questions used semiquantitative scales such as the likert scale. descriptive statistics included mean ± standard deviation for normal data, and median [ th- th percentile] for non-normal data. in the event of missing data, percentages were calculated per number of responses obtained, item by item. frequency of use of remote system was scored as follows: never = , used it once = . , few = , monthly = , weekly = , and daily = . wilcoxon signed-rank test were used to compare the frequency of the institutional and personal remote system use before and during the covid- pandemic and the frequency of use of these two systems during the same period. open-ended questions on free text allowed us to obtain qualitative data to illustrate respondents' feelings about their satisfaction with remote systems. we constructed a coding frame to analyze free-text data about satisfaction of remote clinic, online teaching, and virtual meeting. we subdivided into level categories to evaluate positive and negative aspects with some subcategory: cost, time, interaction, and target public. two authors (mk and rn) discussed the coding and interpretation of results. finally, we compared findings among the three countries with the highest number of respondents (france, china, and italy). quantitative or semiquantitative data were compared using kruskal-wallis h test followed, in case of significance (p b . ), by a dwass-steel-critchlow-fligner procedure. for qualitative data, we used chi tests. a p-value b . was considered as statistically significant, and a p-value b . as a tendency. the statistical analyses were performed using r software [ ] . this study was approved by the ethics committee of our institution necker hospital, aphp. participants were entirely free to participate and their consent was implicit. between april and may , , respondents in countries from continents completed the survey from all over the word (table , fig. ). responders were involved in caring for children with epilepsy (n = , . %), adults (n = , . %), or both (n = , . %). one hundred and fifty ( . %) worked in a public hospital. all had a clinical practice, were involved in clinical research ( . %), and in basic research activities ( . %). most of the participants were from europe (n = , . %). a containment policy due to the covid- pandemic was decreed in the countries of participants ( . %). indeed, participants ( %) belonged to the most impacted countries of the world in this period [ ] . the sections concerning remote work for education and scientific meetings were completed by participants ( % of all respondents). the questionnaire completion rate was % ( / , ). prior to the covid- pandemic, responders ( . %) had already experienced using a personal ( / , . %) or an institutional ( / , . %) remote system: / for patient direct care ( . %), for education of trainees ( . %), for clinical case discussions within other institutions ( . %), for research ( . %), and for clinical case discussions within their own institutions ( . %). for on the responders ( . %), this experience was at least monthly using institutional (n = / , . %) or personal (n = / , . %) remote systems ( fig. a) . there was no statistical difference between the frequency of use of institutional versus personal remote system (p = . ). the three main personal systems used were skype® (n = of the using personal remote system, . %), zoom® (n = , . %), and webex® (n = , . %). the means frequently used to contact remote respondents in an emergency were telephone calls (n = , %) and e-mails from families ( , %). other means (letters from families ( , . %) and letters ( , . %), telephone calls ( , %), and e-mails ( , . %) from the attending physician) were less frequently used. during the covid- pandemic, the use of remote systems increased, both institutional systems ( to ) and personal systems fig. b ). however, contrary to the pre-pandemic period, the use of institutional remote systems was significantly higher than that of personal systems (p = . ). only one respondent from china did not have to reschedule any face-to-face clinics compared with who rescheduled most or all their clinics ( . %). one hundred and sixty-two respondents ( . %) replaced faceto-face visits by various ways of remote connections with the families or the patients. this involved all clinics for ( . %), most of them for ( . %) and only a few for / respondents ( . %). sixty-eight ( %) used phone calls without any remote specific connection with or without video, and a remote system with video connection ( . %). this system was institutional for ( . %) either regularly available ( / , . %) or developed for this pandemic ( , . %). a personal remote system was used by ( . %). the duration of remote clinics was considered as identical as face-to-face ones for respondents ( . %), shorter for ( . %, including much shorter for ), and longer for ( . %, including much longer for ). regarding antiseizure medication changes, . % of respondents tended to make fewer amendments (n = ), . % same (n = ), and . % more (n = ). electroencephalogram (eeg) were less frequently requested for . % (n = ), without changing the frequency requested for . % (n = ), and more frequently requested for . % (n = ). blood test were less frequently requested for . % (n = ), without changing the frequency requested for . % (n = ), and more frequently requested for . % (n = ). respondents reported an increase in email and phone contacts by patients and their families (for , . % and , . % of respondents, respectively) but also by primary care physicians (for , . % and , . %, respectively for email and phone). one hundred and thirty-four respondents ( / , . %) were involved in educational activities. before the covid- pandemic, ( . %) had face-to-face lectures or small group teaching courses, and ( . %) had been involved in online teaching. educational activities were impacted by the covid- pandemic for respondents ( . %). eighty-two percent had at least a part of their activities canceled (n = / , %), postponed (n = , . %), or transformed to online teaching ( , . %). respondents' courses were either interactive ( / , . %), video recorded ( / , . %), or both ( / , . %). for / respondents, all courses were transformed to online teaching ( . %). before the covid- pandemic, . % ( / ) of the respondents had participated in remote scientific meetings, . %, ( / ) in workshops, . % ( / ) in clinical studies meetings, and . % ( / ) in research symposia. few had such experience with national . % ( / ) or international . % ( / ) congresses. during the period from february to july , responders had planned [ ] [ ] [ ] [ ] [ ] meetings. only a few were transformed to remote (median: [ - . ]) giving the opportunity to eighty-nine responders ( . %) who participated in at least one meeting transformed to remote. the rest of the meetings were canceled or postponed. sixty-one percent of respondents were satisfied by their remote clinics ( / , including very satisfied), . % by their online teaching ( / , including very satisfied), and . % by remote meetings ( / ) (fig. ) . feelings regarding family and patient satisfaction with the remote clinic were positive for . % of the respondents ( / , of which were very positive) and . % regarding students and online teaching ( / , of which were very positive). almost onequarter of responders reported dissatisfaction with remote work, mostly for remote education ( . %, n = / ), remote meetings ( %, / ), and remote clinics ( / , . %). respondents indicated they would likely continue greater use of remote work for remote clinics, education, and meetings after the covid- pandemic, in . % ( / ), . % ( / ), and . % ( / ), respectively (fig. ) . free text allowed us to have more qualitative data on the reasons to maintain remote working in their different activity axis. indeed, in their opinion, remote clinics had the advantage of decreasing time and cost for families and patients travel and consequently of work absenteeism. this was highlighted for follow-up visits but not for new patients having their first evaluation. for first visits, respondents declared a clear need for a face-to-face visit. saving time, adapting to the availability of students, and increasing the target audience due to the absence of the need to travel to attend the course seemed to be the positive factors identified by respondents regarding remote education. however, they identified several negative factors including a decrease in interactions, especially the immediate students' feedback reactions. workshops with a small number of participants was reported as particularly adapted to remote systems allowing a gain in term regarding travel, time, and cost. however, respondents agreed that national and international meetings are more adapted to in-person meetings as their major goal in addition to disseminate knowledge is to favor personal interactions and consolidate personal friendships and contacts to enhance collaboration and exchange of ideas. the pandemic began in december in china, late february in italy, and early march in france. these countries were all placed under quarantine (from january to april in china, from march to may in italy and from march to may in france). the peak of pandemic-related deaths occurred between february , [ ] . comparison of data from france (n = ), italy (n = ), and china (n = ) showed no significant differences in terms of age, gender, and practice (pediatric, adult, or both, public or private, epilepsy center or not). belonging to a healthcare network was statistically different between countries (p b − ). indeed, only four chinese respondents ( . %) belonged to a patient care network, whereas there were % in france ( responders) and . % in italy ( ) . before the covid- pandemic, the rate of respondents who had experienced remote working systems was higher in china than in the two other countries ( . % versus . % for france and % for italy, p = − ). in the same way, the number of respondents with an institutional remote work system was higher in china ( . % versus % for france and . % for italy, p b − ). however, the rate of respondents who had a personal remote work system was not statistically different (china: . %, france: % and italy: . %, p = ns). the frequency (scored from : never to : daily) of use of institutional remote systems was significantly different between the three countries the proportion of respondents using official remote systems or phone calls without video for remote clinics was not statistically different between france, italy, and china (official remote system: china: . %, france: . %, italy: . %, p = ns and phone call without video: china: . %, france: %, and italy: %, p = ns). however, in china, remote personal systems were more often used to manage patients than in other countries ( . %, italy: %, france: . %, p b − ). concerning educational activities, the proportion of respondents involved was not statistically different (china: . %, france: . %, and italy: . %, p = ns). before the covid- pandemic, the proportion of chinese respondents who had already experience online teaching was significantly higher ( . % versus . % for france and . % for italy, p = . ). the respondents who had their teaching activities impacted by covid- pandemic were % for china, . % for france, and . % for italy (p = ns). during the pandemic, all respondents in china replaced at least part of their course with online teaching ( / ) compared with % in france and % in italy (p = . ) in particular using interactive online teaching (china: . %, france: %, and italy: %, p = . ). concerning remote meetings, a large majority of respondents had already used this system without any statistical difference between countries (china: %, france: . %, and italy: . %, p = ns). for satisfaction scores (from very unsatisfied: − to very satisfied: + with a neutral position: ), only the impression on families' and patients' satisfaction for remote clinics had a tendency to be higher in france compared with china ( for france and [ - ] for china, p = . , italy: [ . ). all other satisfaction scores showed no significant difference. the covid- pandemic blockage has significantly strengthened the use of remote access technologies in medicine. our study showed that pandemic has increased the shift from classical to remote communication for epilepsy practitioners in all the fields of their activity, namely clinical activity, teaching, and scientific meetings. the satisfaction was acceptable, and almost all responders agreed on a possible future use of remote systems for some of the scientific and educational meetings or for occasional remote clinics excluding first visit. our study demonstrated that during the covid- pandemic, there has been a reduction of face-to-face visits with a replacement for most by remote clinics. in similar situations, remote systems had already been identified as a possible alternative to face-to-face visits, for example, during ebola or sars epidemics [ ] . in the same way, our study showed an increase of remote clinic frequency use during the pandemic compared with the pre-pandemic period. prior work on remote systems in epilepsy has shown notable benefits. a pilot study in canada compared remote systems to face-to-face clinics showing a decrease of costs of . % ($ . versus $ ) with a satisfaction for patients of % and only % preferring a face-to-face next visit in both groups [ ] . the main barriers to remote clinics are the need for clinical examination, technical support, and reimbursement [ ] . in our survey, respondents identified the same advantages and barriers, the first visit being the most challenging. in another study comparing the impression of new patients on remote visits with face-to-face visits, patients' perceptions of the neurologist's understanding, their ability to say what they wanted, their confidence in the neurologist, and the usefulness of the visit were similar [ ] . however, they stated more difficulties in describing their symptoms and concerns about confidentiality. in our study, respondents used personal remote system for remote clinics. this raises concerns about privacy and protection. of note is that the explosion of remote working systems due to covid- attracted hackers [ ] . one attack, called "zoom bombing", consists of an unwanted intrusion, causing disruption and possibly disclosure of medical confidentiality. in order to regulate the security of health data during remote clinics, countries have established strict rules such as the health insurance portability and accountability act (hipaa) in the usa [ ] and general data protection regulation in eu [ ] . most of the free-access personal remote systems in our study are not hipaa compliant. this point should be better addressed by health authorities in future development of remote clinics. until , attendance in medical classes was correlated with passing the examination [ ] [ ] [ ] . since , however, some studies have found no clear correlation [ , ] . for example, th year medical school students have more absences than nd year students due to conferences, meetings, and residency interviews, but unlike personal absences, this type of absence is not significantly associated with lower academic test scores [ ] . this is likely due to the improvement of means of communication that have enabled the students to fill in the gaps. in a recent study using a combined approach between online teaching and face-to-face interactive medical course, online teaching attendance was higher than face-to-face, and the exam score was correlated to online teaching attendance. ninety-eight percent were satisfied with this teaching, and % wished to extend it to the entire second cycle [ ] . this is a good illustration of the change of perspective that is taking place in undergraduate and postgraduate university education. factors associated with a good adherence to online teaching are mainly the quality of the technical system, support system, learner and instructor, and the perceived usefulness [ , ] . the advantages and disadvantages identified by the providers in our study were in line with the literature, i.e., on the one hand, a greater flexibility, an increase of the dissemination of knowledge, a decrease of travel cost and time, and better accessibility, on the other hand, less peer-to-peer exchange and feedback difficulties, including nonverbal communication [ ] . in the symposia and meetings, the same advantages and disadvantages as with teaching were identified, but the proportion of respondents who recommended this method for the future were lower than for clinics and teaching. the use of remote systems seemed to be more adapted for research networks and workshops than congresses. but during the covid- pandemic, the european academy of neurology replaced its congress by a virtual meeting free-of-charge. with more than , participants, they claimed this to be "the biggest neurology meeting ever" [ ] . a virtual congress allows for lower prices, time savings, and a greater dissemination of knowledge both to and from all over the world. however, the respondents interviewed stressed the importance of face-to-face for the development of collaborative projects. our questionnaire highlighted, before the covid- epidemic, a stronger experience of remote systems in china compared with france and italy. this may be due to previous epidemic crisis in china, a larger geographic area of china compared with france and italy, and a lower density of neurologists and child neurologists ( . and . per , persons for neurologists and child neurologists, respectively in south-east asia region versus . and . per , persons in europe [ ] ). indeed, prior to the covid- pandemic, some studies and reviews identified remote clinics in the field of epilepsy as an opportunity in rural regions and in resource-poor setting where the access to a specialist is an important barrier to epilepsy diagnosis and treatment [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, the covid- pandemic seemed to have accelerated the shift towards the implementation of remote clinics and had enabled france and italy to fill the gap with a strong development of remote patient management tools. the sample of this study was small, but respondents completed the survey just after the covid- in china and during the covid- pandemic and lock down in other countries, giving to this survey a value of "almost" real-time responses. responses were from many countries around the world thanks to the involvement of international societies. we cannot rule out the presence of a selection bias since this questionnaire was sent online. however, we believe that this study can present a picture about practitioners' opinion on remote work in epilepsy and help to develop future perspectives. in addition, a significant proportion of respondents in our sample focus on pediatric care. the use of remote clinics in this population is probably easier than in adults. indeed, parents may be able to successfully complete a visit on their child's behalf but adults with cognitive impairment or other limitations may have more difficulty negotiating the technical requirements of such a visit. finally, we did not request a detailed description of the applied methods of online teaching. the survey aimed to have answers on the three activity fields of the respondents without adding much details relatively long survey. the covid- pandemic has increased the shift from classical to remote communication for epilepsy practitioners in all the fields of their activity, namely clinical activity, teaching, and scientific meetings. the advances of these methods of communication have allowed a rapid adaptation to confinement policies using their flexibility and their accessibility. this allowed a maintained link between practitioners and patients, professors and students, and between groups and colleagues. the satisfaction was acceptable, and almost all responders agreed on a possible future for remote work, for some of the scientific and educational meetings or for occasional teleconsultations. in addition, the positive ecological impact of such approaches might be interesting in addition to the economic impact on health and academic costs. it is likely that in "the world after covid", the shift process to the implementation of these new modes of communication is moving forward, although the balance between face-to-face and remote work has yet to be determined in our different fields of activities, and the long-term benefit of such shift to virtual interaction should be evaluated. m. kuchenbuch, g d'onofrio, y. jiang, zm grinspan, j wilmshurst and r nabbout have any conflict of interest to disclose. s dupont has received honoria from eisai, ucb, gw, novartis, advicenne and shire. e wirrell has acted as an investigator for gw pharma and zogenix and has received consulting fees from biocodex and biomarin. s auvin has served as consultant or received honoraria for lectures from arvelle therapeutics, biocodex, eisai, gw pharma, novartis, nutricia, ucb pharma, zogenyx. he has been investigator for clinical trials for advicenne pharma, eisai, ucb pharma and zogenyx. a arzimanoglou has served as consultant, received honoraria for lectures from arvelle therapeutics, eisai, gw pharma, ucb pharma and zogenix. on behalf of his instiitution he has been investigator for clinical trials sponsored by eisai, gw, ucb pharma and zogenix. jh cross has acted as an investigator for studies with gw pharma, zogenix, vitaflo and marinius. she has been a speaker and on advisory boards for gw pharma, zogenix, and nutricia; all remuneration has been paid to her department. her research is supported by the national institute of health research (nihr) biomedical research centre at great ormond street hospital, nihr, epsrc, gosh charity, eruk, the waterloo foundation. n specchio has acted as an investigator for studies with zogenix, marinus, biomarin, and livanova, and has received consulting fees from zogenix, biomarin, arvelle, livanova. the impact of sars on epilepsy: the experience of drug withdrawal in epileptic patients advantages and limitations of teleneurology scotland ' s digital health & care strategy n.d danish ministry of health south african national department of health. national digital health strategy for south africa rdct. a language and environment for statistical computing. r found stat comput world health organization. coronavirus disease (covid- ) situation report- 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application and telephone telemedicine: safe and effective epilepsy care in rural nepal telemedicine for epilepsy: a useful contribution telemedicine for epilepsy support in resource-poor settings. front public heal managing epilepsy by telemedicine in resource-poor settings telemedicine in epilepsy: how can we improve care, teaching, and awareness? epilepsy behav the authors wish to thank the practitioners who participated in this study and the networks without whom this study could not have been carried out, namely the international league against epilepsy, the international child neurology association, the french league against epilepsy, the french society of pediatric neurology, and the ern epicare network. rima nabbout was supported by state funding from the agence nationale de la recherche under "investissements d'avenir" program (anr- -iahu- ) and the "fondation bettencourt schueller". this research was supported by the agence nationale de la recherche under "investissements d'avenir" program (anr- -iahu- ) and the "fondation bettencourt schueller", paris, france. supplementary data to this article can be found online at https://doi. org/ . /j.yebeh. . . key: cord- - dnz yaa authors: coldefy, magali; curtis, sarah e. title: the geography of institutional psychiatric care in france – : historical analysis of the spatial diffusion of specialised facilities for institutional care of mental illness date: - - journal: soc sci med doi: . /j.socscimed. . . sha: doc_id: cord_uid: dnz yaa as in other european countries, specialised psychiatric hospitals were established throughout france during the th century. the construction of these hospitals can be considered as the concrete expression of a therapeutic innovation which recognized insanity as an illness that could be treated in such specialised institutions. the spatial diffusion of these innovative institutions through th and th century france is analysed and we explore how far this can be understood through theories of diffusion of innovations including geographical models of hierarchical and expansion diffusion (or whether other conceptual models are more appropriate). the research reported here particularly focuses on the period – . it involved the construction of an original historical database of both psychiatric hospitals and information on the cities where these institutions were located. this was used to examine and interpret the different phases of development of psychiatric institutions and the parts of the country and types of geographical setting where they were concentrated. a multiple correspondence analysis was then performed to examine the connections between different aspects of the diffusion process. the study shows the limitations of classical models of spatial diffusion, which are found to be consistent with some, but not all aspects of the development of psychiatric institutions in france. an alternative political ecology approach seems more appropriate to conceptualise the various processes involved; national policies, social representations, medicalisation of care of mental illness, and urban and economic growth all seem to be associated with the emergence of a variable and complex pattern. this paper also opens a large field of research. compared with other western countries, the geography of french psychiatric care is relatively under-researched, although there has been a strong spatial dimension to mental health policy in the country. this analysis provides a context for studies of more contemporary processes of french deinstitutionalisation, which is strongly structured by the past heritage of these large asylum facilities. this paper critically explores the relevance of innovation diffusion theories to the geographical development of psychiatric asylums in th and th century france. since hagerstrand's pathbreaking work in the s, geographers have emphasized the role of spatial structures in processes of innovation diffusion. from various case studies, hagerstand highlighted temporal and spatial regularities in diffusion processes (hägerstrand, ) . spatial diffusion of an innovation expresses both the conservation and transformation of geographical spatial structures (saint julien, ) . innovation spread is driven by dynamic spatial interaction. two models are classically presented: the hierarchical diffusion model and the contagious diffusion model. the first takes into account the functional hierarchy of settlements. innovation spreads between densely populated urban areas with a high level of interaction and subsequently filters down to smaller, less influential areas. size and rank in the urban spatial system are therefore determining criteria of the hierarchical diffusion process. the second model is based on effects of 'distance decay' and contiguity in the spatial diffusion process and involves 'contagious' spread to areas in close proximity. an innovation will tend to spread within neighbourhoods close to its point of adoption (daudé, ) . hagerstrand showed that in most cases, diffusion is achieved through a combination of 'hierarchical transmission' and 'neighbourhood contagion'. empirical observations of innovation diffusion processes have systematically demonstrated that vertical diffusion down through the urban hierarchy has been dominant in a large number of diffusion processes, accompanied by 'horizontal', contagious diffusion around the larger centres (pumain & saint julien, ) . innovations first appear in large cities before spreading into the whole urban system. however, despite the rather general relevance of these diffusion models, not every diffusion process can be described solely using these concepts. according to saint julien ( ) , other factors can interact with diffusion flows, such as: chance events, market characteristics independent of the urban hierarchy, effects of the existence of a centralised or decentralised management of the diffusion process or the competitive or noncompetitive nature of the system. in health geography, research on spatial diffusion has mainly focused on the diffusion of infectious diseases, especially nonvectored infectious diseases giving rise to epidemics through human contact (meade & earickson, ) . there is a long history of research to describe and predict how epidemics spread geographically, providing information for action to anticipate, treat and perhaps prevent epidemics. since pioneering work in the th and th centuries (currie, ; currie, ; snow, ; webster, ) , the emergence of new infectious diseases at the end of the th century has given a new impetus to research in this field. hiv/ aids, for example, was largely studied in the late s (amat-roze & remy, ; bastos & barcellos, ; dias & nobre, ; gould, ; kearns, ; shannon, ; wallace & wallace, ; wood et al., ) . recent research has also focused on ancient epidemics, like the plague (especially the second pandemic) or influenza (spanish influenza for instance) (anatra, ; hunter & young, ; lemey, suchard, & rambaut, ; merler & ajelli, ; sabatini, ; smallman-raynor, johnson, & cliff, ; tuckel, sassler, maisel, & leykam, ) . more recently, numerous studies have focused on the international diffusion of severe acute respiratory syndrome (sars) and h n avian influenza (souris, gonzalez, shanmugasundaram, corvest, & kittayapong, ) , which are examples of diseases presenting new challenges to public health in this era of more pronounced globalisation (affonso, andrews, & jeffs, ; bowen & laroe, ; meng, wang, liu, wu, & zhong, ; shannon & willoughby, ; smallman-raynor & cliff, ; wang, christakos, han, & meng, ) . less attention has been paid to the diffusion of medical innovations and new types of care structure within health care delivery systems. however, research of this type can be helpful in formulating and evaluating policies aiming to improve the provision of care, suggesting what factors may help or hinder the dissemination of good practice and how effectively new initiatives are introduced throughout a health system. for example, complex systems such as health services rely on large and expensive infrastructures and on the provision of trained staff that are difficult to move once they are in place, and considerable effort is often required to achieve universal changes in professional practice. services therefore develop in a way that is 'path dependent'; the history of development of a service can influence the potential for new development in the future. investigations of this type include studies of the diffusion of: tomography scanners in the us (baker, ) ; abortion facilities in the north-eastern us (henry, ) ; the administration of antipsychotic olonzapine to urban and rural children in michigan (penfold & kelleher, ) ; alternative chiropractic and naturopathic practices in canada (williams, ) , and the international diffusion of yoga (hoyez, ) . diffusion of innovations in health policy and health promotion has also been studied from a geographical perspective. shannon, bashshur and metzner ( ) analysed the spatial diffusion of a prepaid group practice health plan and nykiforuk, eyles, and campbell ( ) studied the diffusion of smoke-free spaces in canada using roger's ( ) framework for the diffusion of innovations and classic geographical diffusion models. some geographers have also studied the spatial diffusion of hospitals and (most pertinent here) the evolution of national systems of psychiatric hospitals from an historical perspective. these are interesting for the way that they demonstrate the growth of medical power and influence as well as changes in access to care. they also provide the context for studies of more contemporary processes of deinstitutionalisation that have often retained vestiges of the older health care system, still influencing the way care is provided today. jones ( ) compares implementation and spatial aspects of mental health policy reforms in united kingdom and italy since the s, noting that in italy, the diffusion of reform was spatially uneven. it was more advanced in the industrialised and urbanised north of the country while in the poorer, more rural south, development was retarded and mainly left to the management of voluntary and religious sector organizations (galzigna & terzian, ) . jones suggests that in the uk, psychiatric hospitals developing through the larger urban centres eventually led to a more equitable distribution in the national space. for jones, this was due to the strong intervention of the british government in the implementation of a national system of institutions, but the dynamic process of diffusion is not detailed in her paper. in a particularly comprehensive discussion, philo ( ) gives an account of the development of mental asylums in england and wales up to , which suggests that various forces came into play. debate and rivalry among medical professionals were important in the early phases of development. philo also points to developments from the late th to the middle of the th centuries, when initiatives to locate asylums in what were thought to be more humane and therapeutic settings outside major cities became increasingly influential. it seems that trends depend on national context since a rather contrasting american study (hunter, shannon, & sambrook, ) , reports the emergence and diffusion of public 'lunatic asylums' in the united states during the th century, demonstrating how over time the establishment of these facilities spread from the north-east to the west of the country. further research conducted by bretagnolle, giraud, and mathian ( ) on american urbanisation allows us to draw a parallel with the diffusion of the railway network, suggesting that in america, the diffusion of institutions for mental health care (as well as other services) followed geographical processes of colonization and social and economic development taking place at the time. the role of railways and transport networks on the spread of disease and health care has been examined by hogbin in south africa during the first part of the th century (hogbin, ) . it is thus clear that a good deal can be learned from studies of diffusion of mental health care institutions in the th, th and early th centuries. it shows the interdependencies between socio-economic development and health care developments across national spaces. this diffusion of institutional structures is interesting in that it also represents the concrete implementation of ideas about appropriate models of psychiatric care. the emergence and dissemination of an idea concerning psychiatric care is not necessarily perfectly matched by the implementation of the idea through construction of the specialised psychiatric hospitals that are of interest here. in this study we are particularly concerned with the diffusion of this concrete expression of a new care model through the modification of the psychiatric infrastructure, since it is at the point of construction of these new facilities that changes in provision of psychiatric care will have started to have an impact on the care environment for people with mental illness. the innovation diffusion model also raises some interesting issues concerning whether or not there is a specific 'tipping point' in time and space at which an innovation begins to spread, or whether change is influenced by more continuous processes of path-dependency whereby past actions and thought influence present patterns of change. in this paper we contribute to the international discussion concerning the importance of national context in the history of psychiatric care provision by considering the development of psychiatric institutions in france during the study period. the analysis aims to determine the relevance of 'classic' diffusion models in this process (which might suggest psychiatric care development was part of socio-economic growth and development in france, as in america). following philos' and jones' european examples, we also seek to identify other key processes, associated with professional medical influence and governmental health care policy at the time, that also appear to have driven the growth of the system. the lunatic asylum as an innovation in th and th century france: from the 'alienist' perspective on government policy here we briefly summarise the processes that influenced the diffusion of the 'lunatic asylum' as a model of psychiatric care in france during the th and th centuries. prior to these developments, no specific medical or health care response was proposed for people with mental disorders. they were placed in institutions for the indigent and criminals. hitherto 'insanity' had not been understood as a treatable illness, so the aim was to restrain people identified as 'mad' and prevent them from disturbing public order, not to try to cure them. foucault ( , chapter ii) suggests that in france, and particularly in paris, this approach was clearly illustrated in institutions called hôpitaux généraux created in (imbert, ) to implement this policy described by foucault ( edition, p. e ) as 'the great confinement'. the lamentable conditions of their confinement were already being identified in the th century (colombier & doublet, ) . at around the start of the th century, in france as in other countries, we begin to see the seeds of innovation: insanity began to be interpreted as an illness that could be cared for in specialised institutional settings. this therapeutic innovation was rooted in the emergence of the philanthropic and humanist ideals of the th century. these were associated with a shift away from demonological interpretations of madness and the growing pre-eminence of naturalistic explanations. the idea of the curability of mental illness and the legitimacy of the physician's role in the social management and treatment of madness also contributed to the emergence of this innovation gauchet & swain, ) . these ideas were promulgated through the 'alienist' school of thought, calling for the separation of 'mad' people into specialised, therapeutic settings as recommended by pinel ( ) in france, and tuke ( ) and browne ( ) in the united kingdom. they were developing the concept of mental 'alienation' (mental illness viewed as a person's inability to integrate in society), arguing that a mental disorder inhibited the sufferer's feelings to such an extent that eventually, both the self and the external world seemed unreal. for the french alienist pinel ( ) , the asylum was the only suitable place for 'moral treatment' requiring the patient's isolation from society as a whole, as well as from other groups who were seen as 'deviant' and dangerous to society. the lunatic asylum thus became the preferred therapeutic instrument of this moral treatment, secluding mentally ill people from the stresses of mainstream society and family life and incarcerating them in a secluded place, ideally situated in tranquil countryside where a strict moral framework was imposed. foucault ( edition, ) argues that '.the asylum becomes, in pinel's hands, an instrument of moral uniformity and social denunciation.'. 'place' has considerable significance in this model; physicians aimed to put the mentally sick in a new situation, removed from places, objects, people and circumstances that shaped their usual relationships and behaviour. at this period, well before the introduction of psychotropic drugs, 'moral treatment', acting on intellect and feelings, also marked a move away from physical treatment by traditional methods of blood-letting and purges applied to the patient's body (goldstein, ) . pressure of opinion was building in france in favour of extensive reform and was beginning to be felt by both the government and the medical profession. in a report on institutions for the 'insane' presented to the french interior minister in , the alienist physican esquirol wrote: 'these unfortunate people are treated worse than criminals and reduced to a worse condition than animals'. it was at around this period that esquirol introduced the term 'asylum' to distinguish psychiatric care institutions from both the 'hôpital général' carceral regime and 'hôtel-dieu' hospitals for paupers, since these earlier types of institution were considered oppressive, arbitrary in their treatment of mentally ill people, and likely to exacerbate their condition (lantéri-laura, ). esquirol wrote: 'i would like us to give these facilities a specific name which does not bring to mind a painful image; i propose we name them asylums' authors' translation from (esquirol, , p. ) . the term 'lunatic asylum' was still used as late as when it was replaced by 'psychiatric hospital'. by then, psychiatry had become an established practice within the medical profession and psychiatric institutions had entered into the clinical domain. the later phase of our study period thus arguably represents the shift to a different model of care associated with a new phase in the diffusion of changing ideas about psychiatric treatment that were expressed in the new facilities built most recently. the governmental response to the 'alienist' model, promoting the asylum as an institutional model, was the lunacy act. this required that every french département (representing the local administrative tier of national government in france) provide a 'facility dedicated to host and care for lunatics'. promulgated under the july monarchy, the lunacy act continued to influence the provision of care for mental disorders, for over years as it was only revised on june th with the 'act relative to the rights and protection of people hospitalised because of mental disorders and to their hospitalisation conditions'. the lunacy act of instituted the mandatory provision of mental health care in each administrative département either by the creation of at least one asylum or by contracting with an authorized voluntary hospital to do so. this legislation could therefore be expected to have had a significant impact on the geographical diffusion of this type of institutional structure, albeit that the 'lunatic asylum' was not specified as the model on which these facilities were to be built. individuals that were to be housed in these new facilities were nevertheless described as 'lunatics' rather than 'insane' or 'agitated' which suggests that the law makers were influenced by the 'alienation' paradigm proposed by pinel. while the lunacy act did not include direct guidelines on the type of site that should be preferred for asylum facilities, psychiatric ideas on the subject had already been clearly expressed in france. the esquirol ( ) thus specified that asylums should be built outside cities for economic and therapeutic reasons. the following quote illustrates alienist ideas that dictated th century views of what might constitute (or undermine) a therapeutic setting for care of mental illness: "most lunatic asylums are located in cities, a few in the countryside, in the plains or on the heights. in cities, space is lacking, the sick are excited by the hubbub and the noise of the population; visits are more numerous and more frequent; nurses are more distracted, more inclined to leave, while in countryside, there is more space, the sick enjoy more peace and quiet, can go out for a walk in tranquil surroundings or engage in gardening; they have fewer visitors and finally, there are economic advantages. buildings on a high plateau are more favourably situated but when the plateau is not sufficiently extensive, buildings cannot develop on the same level or be sufficiently spaced out; terraces and steps are then required because of the uneven ground." he specifically cites the example of antiquaille hospital in lyon: "located at mid-altitude on the fourviere mountain, it is built on the ruins of an ancient roman construction. this choice of location was unfortunate. it was impossible to design suitable buildings: yards are too narrow, promenade galleries are missing, the ground is arid, and vegetation cannot improve the view or refresh the air. water is not very abundant whereas it is required in such a house. views are certainly very extensive, but the insane can constantly see their fellow citizens coming and going on the banks of the saone river and in the neighbouring streets. they hear the hubbub of the city; is that not sufficient to provoke feelings of irritation likely to increase and to maintain delirium?" (translated by the authors from esquirol, ( , p. )). in the following analysis we shall treat the establishment of 'asylum' facilities as a 'proxy marker' for the implementation of a major innovation in the care of people with mental disorders in france. these asylums constituted a new type of clinical and therapeutic environment for care of mental illness as conceived by pinel. in the following discussion we use the term 'asylum' to refer to state sponsored, specialised psychiatric hospitals in france that were either established following the lunacy act, or preexisting facilities, including voluntary or religious institutions, recognized by the government as meeting the requirements of the act. other institutions providing mental health care (in multispecialty general hospitals or independent institutions not recognized by the state) are not included in this category, although their contribution in the general context of care provision is taken into consideration in our analysis. in this study, 'asylum' therefore refers to an administrative category of residential institution. these institutions did not all systematically incorporate every aspect of pinel or tuke's asylum model of care, and it is likely that the care provided over the period covered, varied from one institution to the next. however, one aspect of asylum design does become apparent in this analysis; the preference for a rural or semi-rural location as an ideal site. our analysis indicates that this had a significant and lasting influence on the geographical development of psychiatric care in france and contributed to the specific geographical pattern of diffusion of asylum facilities around the country, as will be discussed below. our focus on the establishment of institutions corresponds to the schumpeterian definition (schumpeter, (schumpeter, , of an innovation, which is distinct from an invention as it describes the process by which a new idea is effectively adopted by society (dortier, ) . the lunatic asylum can also be considered as an 'institutional innovation' according to the pederson's ( ) definition, because it does not directly apply to individuals or households (as in 'individualistic' or 'domestic' innovation), but involves the introduction of a collective service. this is underlined by the way the innovation was not left to develop randomly, or under the sole influence of the medical profession. government legislation was introduced as a means of organizing and centrally coordinating the even spread of asylums to every part of the country. using the functionalist perspective proposed by brown ( ), we consider to what extent the diffusion of 'lunatic asylums' in france corresponded to a 'decentralised process' (spreading autonomously throughout the national space) or a 'centralised process' propagated under the control of a national agency or policy, which determines diffusion conditions (daudé, ) . centrally managed innovation diffusion may follow different time space paths than individualistic or decentralised processes. in this case, the adopter of the innovation was central government, aiming to influence the process of innovation through local administrative and geographical levels of government throughout the country. the government of the day was keen to demonstrate the effectiveness of this recently created government structure, inspired by the egalitarian and republican goals of the french revolution ( ). in , legally assigned with new powers in terms of resources, broader responsibilities and greater facilities, french départements provided a conduit for central power to all parts of france, ensuring the management of national space in line with central government policy (burguière & revel, ) . this was paralleled by increasing spatial accessibility of most parts of the country, particularly in the first part of the th century with the expansion of the railway network (suggesting interesting potential parallels with bretagnolle's study mentioned in the introduction). these processes might have been expected to encourage homogenization and evenness in social and economic development across all french départements, though they might also have tended to encourage early adoption of the new model of psychiatric hospital in the geographical centres of central governmental control in paris, the capital city, and in provincial centres of government. this review of the processes influencing asylum diffusion through french national space suggests it can be viewed as an example of an innovation diffusion process in which the original innovation took place through an informal network of reformers, (which might have produced rather randomly distributed sites for the very first asylums), but that after , the leading adopter was a collective (state) agent, operating through a highly structured geographical and administrative hierarchy. the following analysis explores how these processes influenced the diffusion of asylums in th and th century france. the state hierarchy was strongly centred in the capital city and its regional seats of government, and had the potential to control the pattern of spreading the innovation through the national space. this could lead one to expect an even, more or less simultaneous geographical diffusion of asylum facilities designed to ensure provision in each département. in many other cases of innovation diffusion, the largest urban centres are most likely to be the sites for early adoption. however, in this case, the diffusion phase dominated by the 'alienist' model of care could be expected to result in the early establishment of asylums in rural or semi-rural settings close to major towns and more particularly, in the proximity of regional administrative centres. to investigate spatial diffusion of psychiatric hospitals in france from the th century to the present day, the initial task involved building an original historical database of psychiatric hospitals, their location and date of establishment, indicating the points at which, in different parts of france, asylum facilities were first adopted as innovative care institutions for the mentally ill. this was achieved using a number of different data sources. these data were then analysed and interpreted in the light of the conceptual frameworks and the historical context discussed above. data from the national register of health and social facilities (fichier national des etablissements sanitaires et sociaux, finess) were employed. this is based on information provided by local agencies of the ministry of health and social affairs. created in , the finess inventory made it possible to precisely locate existing facilities and the date on which establishments set up since became operational. however, it does not allow us to reconstitute the history of hospital development prior to ; hospitals which closed before do not appear in the register, and the date of establishment for older facilities is not included. this inventory is therefore not sufficient for our purpose but is useful to supplement and consolidate historical information from other sources. archival data were used for the earlier period. the french national statistics service (la statistique générale en france sgf) published data on asylums from to . in the introduction to the volume covering the period e , the minister of agriculture, commerce and public works indicates to 'his majesty the emperor' that 'this work not only allows us to appreciate the administrative situation of our asylums and its degree of development; it also contains a certain amount of strictly medical information, which appears to be helpful for the very delicate and difficult study of one the cruellest human infirmities' (translated from statistique de la france, , p. ). the format of this publication was modified over time. while finess was produced as a register in list format, the sgf provided more comprehensive statistical data on hospitals presented by département and by year. these data allow us to correctly date the creation of asylums established between and . complementary information was sought in historical studies on french psychiatry. two main archival sources were used. the first was the website created by dr caire on the french history of psychiatry (http://psychiatrie.histoire.free.fr/). this site constitutes a rich documentary database on psychiatric hospitals. hospitals are presented by département with the date of creation when known. in addition, personal communication with the author made it possible to enhance the information available from this source. the other useful source was found in the paper by longin ( ) , which presents historical periodisation of the construction of psychiatric hospitals. institutions can be dated and located within départements. most of the data were taken from official reports (constans, lunier & dumesnil, ; esquirol, ) . to analyse the spatial diffusion of psychiatric institutions at departmental level, a temporal and geographical database was constructed showing french departmental boundaries and the associated resident populations for each period. first drawn up in , the boundaries of french départements were modified throughout the th and th centuries, partly because of modifications to national borders (such as germany's annexation of the alsace and moselle regions during e ) and partly because of changes within the national space due to demographic and urban growth during the th century. rapid and spatially uneven population growth since the th century led to increasing disparities in population size between départements. various base maps were constituted for different years from a historical database of french towns and their attribution to départements (http:// cassini.ehess.fr). demographic data used to assess the scale of urban development were collected from different sources: ined-insee census demographic tables (croze, ) for the period to , and the royal almanach for the years and (http://sref.free.fr, http://splaf.free.fr/). for local analyses, another database comprised of historical data on french cities initially produced by pumain (pumain & riandey, ) and completed by guerin and paulus (guérin-pace, ; guerois & paulus, ; pumain & riandey, ) was used. this database contains city population figures for the period to , and a classification of cities distinguishing between: urban centres (most populated parts of urban agglomerations), isolated cities (urban areas bounded within a single urban space), suburban areas and rural areas. this morphological definition of cities, taking into account both population size and continuity of built up areas, combined with information on the dates urban areas first developed, reflects the structure of the french urban system at different time periods. more detailed information was compiled for each asylum analysed and for locations in which they were located (table ) . the analysis was designed to explore whether the geographical pattern of the diffusion of asylums in france seemed consistent with the processes thought to be influencing this diffusion, as reviewed above. the analysis proceeded by first trying to establish whether the legislation provided a major impetus to the development of asylums throughout the country, which would be consistent with the idea of a centralised institutional innovation. then, at different historical phases of development of asylums, the analysis investigated the parts of the country where they were set up and the types of geographical setting where they were concentrated. in order to model the neighbourhood diffusion process, a contiguity matrix of french départements was created and in each département, euclidian distance was calculated between asylum locations and the city where the departmental administrative centre, (representing the local seat of government power), was located. finally, to bring all this information together, a multiple correspondence analysis (mca) was performed on the dataset to examine the connections between different aspects of the diffusion process. at this point we were also able to explore the possible significance of independent and religious institutions that were not recognized by the state as psychiatric 'asylums', but which may have influenced the spread of alienist ideas. descriptive analysis was carried out using the sas statistical package, mca was carried out with spad software (morineau & aluja-banet, ; morineau & morin, ) . mca is a useful tool to identify the main dimensions of a spatiotemporal diffusion process (saint julien, ) . it allows us to highlight key components of the diffusion process and to analyse their interactions. of the various techniques for multivariate analysis available, mca (or 'homogeneity analysis') (everitt & dunn, ) was selected because it can include categorical variables (lebart, morineau & piron, ; volle, ) . alternative methods also considered were multiple factorial analysis (mfa) (escofier & pagès, ) or mixed data factor analysis (mdfa) (pagès, ) . however, mca was preferred since it is widely used and understood, as well as being the most likely to offer statistically robust results. to carry out this mca, quantitative variables were converted into nominal categories, choosing a classification which would generate similar numbers of categories as were present in the qualitative variables. if the variables in mca differ significantly in the number of categories, this will tend to distort their impact on the analysis. this is because variables with a large number of categories will carry disproportionate weight in the resulting dimensions. table variables characterising the asylum facilities and the places where they were located, incorporated in the mca. the units of analysis for the mca are the lunatic asylum locations created in french départements since the th century. variables used in the mca are listed in table . the full range of variables are only included in the mca for asylum institutions of interest here (state-sponsored psychiatric hospitals that were recognized by, or were established in response to, the lunacy act). however some information relating to private and non-specialised institutions that were not in this category are also projected on the mca plots as illustrative individual cases, which may influence the pattern of relationships in the rest of the analysis. for example, a psychiatric ward in a general hospital, could have constituted an 'acceptable' way of caring for people with mental health problems in a département and such provision may have resulted in a delay in the establishment of a dedicated asylum facility in that area, or progressive independent institutions may have played a role in the dissemination of alienist ideas in psychiatry. the mca includes information on the position of départements within each of statistical regions in france, (using the territorial units for statistics nomenclature defined for the member states of the european union (nuts )). this gives an indication of the geographical position of the département where innovation took place at different time points, and the category of settlement in which the asylum was located. components of the mca were then used to build a classification of different types of lunatic asylum locations. this cluster analysis was based on a hierarchical ascendant classification using 'ward criteria' aimed at both maximising inter-group inertia and minimising intra-group inertia. to optimize cluster homogeneity, we used the 'dynamic nodes' method, a consolidation procedure involving aggregation around moving centroïds. temporal-spatial trends in the adoption of lunatic asylum facilities in french départements from to fig. shows the time trend in the proportion of départements adopting the asylum model of care (i.e. for each year, the proportion of départements that had established at least one asylum). the diffusion of these psychiatric institutions through france lasted almost years, from to . as shown on fig. , the diffusion process is still incomplete, because eight départements out of have never had a specialised, public sector psychiatric hospital, whereas the 'deinstitutionalisation' of psychiatric care began in the s with the introduction of acute psychiatric units for the provision of care within general hospital structures. four of these eight départements had been accommodating people with mental disorders in specialised wards in general hospitals since the th century. the other four départements had never previously provided a specialised public hospital service for mentally ill patients but currently provide acute psychiatric beds in multi-specialty hospitals. the general form of the curve is consistent with the typical pattern of development of innovation diffusion processes. the curve is similar to an 's-shaped' logistic form, apart from a perturbation caused by the resumption of new adoptions after the s, as registration of new hospitals recommenced in france after a hiatus during world war ii. particularly notable is the absence of any change in the trend associated with the introduction of the act. the rate of innovation had started to progress most rapidly well before this date, and the rate of diffusion of the asylum model across départements in france actually slowed down shortly after the act was passed. therefore, it seems that at most the act only confirmed a pre-existing trend of introducing the process, but there is no evidence that it led to its acceleration. as is typical of diffusion processes, four main phases in the introduction of french asylums can be identified, similar to the stages of 'emergence', 'expansion', 'consolidation' and 'new expansion' proposed by hägerstrand ( ) . these are marked on fig. and the départements involved in each phase are plotted on the maps in fig. . details of the type of locality in which the new institutions were set up are also given in table . phase : emergence of asylum institutions ( th and th centuries) initial innovation during the th and th centuries commenced in certain geographically dispersed centres around the emergence of the innovation expansion consolidation new expansion country (in some départements in the north and west of france and in dispersed locations in the south and west e see fig. ). in , only out of the existing départements at that time had a public or voluntary lunatic asylum. the voluntary sector, rather than the state, was the predominant early adopter ( % of the new establishments). as shown in fig. , earlier adopters of the innovation appeared in diverse regions of france. most of the earliest adopters (before ) were départements located in northern france. in the th and th centuries, the north of france had higher level of education and industrialisation than the south (furet & ozouf, ; pumain, saint julien, & ferras, ) . these wealthier northern départements were also privileged areas for exchange and production (pumain et al., ) . it is interesting that paris, as the governmental and cultural centre of france, was not among the first to establish asylum facilities. although two hospitals with psychiatric wards and one private asylum existed during the th century, no state lunatic asylum was established in paris during the first part of the period. it was not until that it opened its first lunatic asylum 'sainte-anne'. asylums for the curable and incurable would be built outside the city at a later date (lamarche-vadel & préli, ) . this may have been because of rigidities in the system of institutional provision that already existed in paris, where the hôpital général had become firmly established. it would also be consistent with the preference for locating 'lunatic asylum' facilities in less urban settings. although pinel developed the 'lunatic asylum' concept through his observation and critique of conditions in the hôpital général setting, his ideas were initially concretised in new institutional facilities elsewhere in the country. with the exception of two départements, the early adopters were also generally more populated than non-adopters ( , inhabitants on average for this group of early adopters vs. , on average for non-adopters). apart from the striking absence of the parisian capital at the emergent phase of the process, this gives the impression of a hierarchical diffusion process, with the innovation spreading initially in the more populated and economically advanced areas and later reaching the more sparsely populated and economically 'backward' regions (saint julien, ) . this may have been a simple effect of the pressure of potential demand (which would be greatest in populated areas). however there may have been qualitative differences in the propensity for innovation and the availability of resources for new developments so that areas that were socially and economically more dynamic (pumain, ) led the way in adopting the new style of asylum. this was a period of very rapid industrial and economic growth in the north associated with the exploitation of coal and the industrial revolution, so that the region saw rapid urbanization and population growth and was relatively wealthy at this time with sufficient community resources for new health care development. the diffusion process advanced rapidly throughout most of the th century. an increasing rhythm of change is observed after e , and well before the lunacy act. this may have been due to state intervention preceding legislation, and was probably also strongly influenced by the alienist network of reformists. during the years preceding the act, the question of care for the 'insane' was on the government agenda. the french alienists pinel and esquirol, both parisian doctors, were disseminating their ideas about treatment for the insane. the influence of pinel's report entitled medico-psychological treatise for mental alienation published in and reprinted in , reached beyond the medical and bureaucratic fields (goldstein, ) . it is very likely that this original paradigm shift in psychiatry provoked by pinel (and by his colleagues in other countries such as william battie and william tuke in england) initiated the lunatic asylum diffusion process rather than national government policy (see philo, , and foucauld, ) . with his theory on mental alienation and moral treatment, pinel laid the foundations of french psychiatry through the diffusion of his ideas. on the eve of the act, départements out of had already developed asylums to implement the innovations he proposed. voluntary initiatives remained numerous during this phase. if there was a 'tipping point' at which innovation started to escalate, it occurred prior to . the legislation appears to have simply taken up and officially endorsed a previously established movement by encouraging the diffusion of the innovation throughout the national space. the political and economic context may also have played a role in these developments. the french government, under the imperial regime ( e ) and the following restoration (of monarchical sovereignty) until the s, brought a degree of political stability and economic expansion favouring hospital development (longin, ) . psychiatric establishments created by the state became predominant after the act. between and , % of the new asylums were the result of public initiatives (as opposed to institutions set up by voluntary bodies and recognized by the state after their inception). at the eve of the th century, out of the départements in existence by that time had adopted the innovation by constructing an asylum facility. the maps for and in fig. show that more central and southern parts of the country had begun to establish asylums. the geographical pattern of asylum development also suggests that the 'contagion model' of diffusion is also relevant throughout the th century. this is confirmed by the finding that a département was more likely to be an adopter when a neighbouring area had already established asylum facilities. among the neighbouring départements bordering earlier (pre- ) adopters, % had adopted the innovation during the period e versus % of départements not neighbouring previous adopters. this contagious diffusion model seems to be more relevant in the north of france. this might be associated with the more advanced development of communication networks in the north of france at this time. in the south, the innovation seems to have been taken up more spontaneously and randomly in space (fig. ) . the rate of the new establishment of asylum facilities slowed in the th century, as most départements that had not already done so adopted this type of mental health care facility. the apparent acceleration in is the result of a bias in the data, noted above; some psychiatric hospitals established by could not be precisely attributed to the preceding years. then we see a period of relative stabilisation until the s when most of the 'late adopters' made some provision of this type. during the first part of the th century, the drive to expand provision seemed to focus particularly on rural and less populated départements. the north-east and south-west of france constitute areas with high levels of adoption during this period (see fig. ). classic models of the diffusion of innovations would predict a slowing down in the third phase of a diffusion process, but longin ( ) suggests a further explanation of this relative stabilisation at the beginning of the th century, linked with the development of secularism. the act on separation of church and state prevented any denominational private enterprise. furthermore, damage during the first world war strongly affected some asylums. closures and transformations were considered in some cases. this is a period of rehabilitation and repair rather than of new construction of asylums. the second world war resulted in less destruction of hospitals but more than , patients died in french psychiatric hospitals during this period. concern over conditions in asylum facilities, the discovery of neuroleptics, together with changes in the economic and political situation after wwii, subsequently led to a new mental health strategy: the 'sectorisation' policy. sectorisation interrupted the classical diffusion process proposed by hagerstrand, so that the final phase of complete saturation predicted by his model did not occur. instead, sectorisation introduces a new paradigm in psychiatric care, with a shift from large residential institutions to community-based services. in france, this process of deinstitutionalisation was initially planned on the basis of a territorial strategic framework. the 'psychiatric sector' was defined as a geodemographic area of around , inhabitants, for which a specialised team and a range of community-based services were dedicated. at this point the incentive to provide psychiatric hospital facilities was no longer driven by the aim of providing institutions for long term care, but by the need to convert and redevelop the service infrastructure to provide acute inpatient services as part of a deinstitutionalised model of care. to be able to implement this new policy, départements who had not yet followed the trend to build asylums had to create such acute facilities. the last départements to build inpatient psychiatric services often set up psychiatric hospitals or psychiatric wards in the multi-specialty hospitals serving the area, resulting in a more rapid period of growth in the number of psychiatric facilities after the s. arguably, these were part of a new phase of innovation in psychiatric care, rather than the last stages of the diffusion of asylums. however, they can also be seen as a continuation of psychiatric service infrastructure development that had been set in motion though the asylum development process, since in the french case the original asylums have often been retained and converted to the community care model. choice of location within départements: distancing the 'mad' from the city by shifting the scale of analysis to a more local one, one can also discern what seem to be the effects of changing ideas about what constituted both a therapeutic setting and an appropriate location for a 'lunatic asylum'. the communes where new asylums were located were examined in terms of their population size, their general position relative to urban areas and their distance from the administrative centre for the département where the prefecture (government headquarters for the département) was located. as discussed above, the lunacy act gave départements no precise guidelines as to the ideal setting for such facilities but alienist theories suggested that a rural location was preferable to an urban setting. at present, more than half of the public or integrated psychiatric hospitals created throughout the period are located in what are now urban centres; % in suburban areas, % in isolated towns and % in urban areas (french population census, ) . however, it must be borne in mind that the urban geography of france has developed over time so that a third of the asylums that were initially established in rural settings were later absorbed by urban sprawl and are now suburban areas, while some previously rural settings have become urban centres or small towns. table thus shows that historically, % of the psychiatric hospitals were initially located in urban centres, % in rural areas, % in isolated towns, and % were initially located on the outskirts of cities. table also shows how, from the early s to the first part of the th century, the distribution of new asylum locations shifts over time from predominantly urban to more rural and semi-rural settings. a different perspective on the geographical position of these asylum buildings is provided in table , which shows their average distance from the administrative centre of the département and the proportion of the asylum buildings that were located within the city centre where the prefecture (local seat of state government) was located. the pioneering asylum facilities set up before were on average located km from the main city centre ( % were within the main administrative city centre). this is consistent with the theory that dominant centres in the urban hierarchy adopt the innovation before smaller centres. the mean distance to the prefecture is greater for the asylums set up during the th and early th centuries ( e km from the main urban centre of the département). this may be a reflection of the diffusion of alienist ideas concerning the appropriate setting for an asylum. after the average distance to the main city centre declined to km indicating a growing proportion of more urban locations for more recently established facilities. however, by this phase the geographical pattern of diffusion became quite complex. table also shows that in the second part of the th century, the positions of new asylums were more widely distributed in suburban settings and in more isolated urban centres. while rural locations were still often selected, we can also observe a larger number of psychiatric hospitals being set up in large cities (table ) . while urban areas with populations of over , adopting this innovation slowly decreased until the s, the trend reversed in the last part of the th century. this should be considered in the 's context of deinstitutionalisation associated with a psychiatric paradigm shift. the aim in this most recent period was no longer to isolate and distance people with mental health problems, but to integrate them into the community and to bring the health care facilities closer to population centres. multiple correspondence analysis: relationships between different temporal-spatial trends these trends are summarised in a multiple correspondence analysis (mca) to explore the complex associations between the attributes of asylum facilities, listed in table . a scree plot analysis of eigenvalues showed that the first four dimensions from the factorial analysis account for % of the variability. fig. presents the first two dimensions (which together explain about % of the variability) and the size of the data points indicate their influence on the pattern of correspondence between the different variables. the first dimension on the horizontal axis is strongly structured by characteristics of the places where hospitals were sited. it clearly opposes hospitals located in middle to large-sized urban centres or main administrative centres (to the left of the diagram) to sparsely populated rural areas or semi-rural settlements (less than inhabitants) more distant from the main cities (to the right). by projecting the temporal dimension on this factorial component (the phase in the diffusion process when the hospital was established, marked as a jagged line in fig. ) , a path emerges demonstrating a strong relationship between location and time. thus middle to large-sized urban centres are more likely to be the settings of pioneer lunatic asylums, created before the lunacy act, while less central and more rural locations are more often sites table mean distance of asylum locations from the city where the prefecture (regional government office for the département), was located, according to phase of diffusion. for th century establishments before the s. this seems consistent with the idea of a hierarchical diffusion trend, with early adoption of the asylum model in larger centres and later adoption in smaller settlements. the second dimension, on the vertical axis, is essentially structured by the relationship between the locations of public sector institutions of special interest here and the presence of facilities provided by independent charitable organizations. this dimension opposes (at the bottom of the diagram) voluntary hospitals (frequently of religious origin, often located in places with no preexisting provision) to places with existing, 'embryonic' provision in a public and secular hospital (at the top of the plan). this is consistent with a theory of path-dependency in service development, later hospital developments being associated with earlier patterns of development. the most densely populated départements also appear in the upper part of the diagram, suggesting a longer history of provision of public facilities in these départements. the third and fourth axes are not illustrated. the third axis opposes suburban asylum locations, often close to the main urban centre of a département, to hospitals located in isolated places, further from the main cities. the former group was more likely to be in the voluntary sector, whereas the latter group was more often public sector facilities. the projection of the temporal dimension of the diffusion process provides a clear pattern; suburban locations (which were on the city fringes when the asylum was established) were more common for pioneer establishments while isolated locations were later developments. the fourth axis showed suburban locations were more common in rather sparsely populated départements while very rural locations were more often chosen in more populated départements. this may suggest that in rather urbanised and industrialised settings the move toward tranquil rural settings promoted by the alienist movement was particularly strong. our study was faced with several challenges, so that the conclusions are subject to several caveats. the first of these was the question of how to analyse a hierarchical diffusion hypothesis when the urban hierarchy was changing significantly through the period studied. the dramatic modification of the french national urban hierarchy during the th century due to the unprecedented urban growth biases the hierarchical diffusion model. the départements' mean population density increased by a factor of . during the th century with a good deal of local variability, which radically altered their demographic ranking. the urban hierarchy also became more differentiated: in , for the least and the most populated départements, the population density ratio was around one to seven; by , the ratio was one to . this unstable urban hierarchy makes the hierarchical hypothesis difficult to apply, even if we hypothesise that the greatest population growth was associated with a greater probability of adopting the lunatic asylum innovation. similar challenges also face other studies of diffusion over extended time periods. we also note that percentages in the tables and the results of the mca need to be interpreted with caution due to the relatively small numbers of data points. this makes it especially difficult to assess the later stages of the diffusion of the asylum model. it would be interesting to have more information on the capacity of the institutions and the numbers of patients using them, in order to assess the extent to which provision was related to likely demand in départements of varying population size. bearing in mind these limitations, this analysis of spatial diffusion of asylum facilities as an innovation has shown the limits of the relevance of classical models of spatial diffusion. the contagious diffusion model, arguing for diffusion governed by geographical proximity, does not seem very appropriate for our case study, except during the early expansion phase in the th century. in addition, its applicability remains limited to the northern part of france, perhaps because of the stronger economic, industrial and demographic development of this french region at that period. elsewhere in france, it is difficult to distinguish the effect of contagious diffusion specific to psychiatric hospitals. while the hierarchical diffusion model seems to be more relevant in our study, it nevertheless proves to be inadequate in explaining the whole process of diffusion and location of lunatic asylums. some large urban centres, and namely the capital city, paris, delayed in the construction of asylum facilities despite the fact that it had been the centre of emergence of the clinical and therapeutic ideas about the moral treatment and the need for such asylums. this delay in the creation of a lunatic asylum in paris was criticized at the time and interpreted as an administrative failure. for example, semelaigne wrote in : 'in france, several large cities already have model establishments, and rival improvements are developing in foreign countries. in paris, however, through a regrettable anomaly, the bicetre and salpetriere hospitals are not affected by this trend, as indicated by both their imperfections and gaps in their scientific progress and actual achievements. this immobility, in a centre from which fruitful initiatives usually emanate, cannot continue. the capital city is embarrassing itself. a special commission has been established to consider the changes required in this situation'. (translated from a quote from daumézon ( ) ). this phenomenon reflects both the social rejection of people with mental illness and the facilities to treat them, and processes operating in landscapes of power as defined by dear and wolch ( ) . while paris was a centre of psychiatric knowledge, it may have been slow to establish asylums because of the effort required to reorganize the existing provision in 'hôpital général' facilities, and there may also have been motives to distance people with mental illness from the capital by devolving provision for 'the insane' to the provincial départements. this analysis therefore differentiates between the site of innovation in the sense of development of a new model of care (the alienist approach) and the diffusion of the concrete expression of this model: i.e., special purpose residential care facilities designed to deliver this psychiatric care. while diffusion of alienist ideas may have followed the classical hierarchical diffusion model from paris to other large urban centres (and other countries), and then to smaller urban centres, the diffusion of asylum facilities was influenced by other factors which will have favoured or impeded their establishment. among these we can include the local influence of organizations prepared to create unconventional facilities, (these were apparently often not-for-profit private associations or religious institutions), and the economic and social dynamism of communities within some départements. of course, there exist alternative interpretations of pinel's ideas and taking them into consideration highlights how change in health care systems is multi-faceted and complex. foucault, for example, interprets the diffusion of the therapeutic benefits of the asylum model in terms of the diffusion of growing power and discipline exerted by the medical profession in psychiatry (foucault, , . other authors have interpreted the development of psychiatric medicine less in terms of repression and punitive action towards mentally ill people and more in terms of innovative knowledge of the social and psychological determinants of mental illness progressively leading to a long term shift towards new models of care and risk governance (gauchet & swain, ; quétel, ; swain, ) . either interpretation is particularly interesting in the french context in that it is, arguably, rather unusual in france for professional associations, rather than the state, to determine national policies and welfare strategies. apart from the situation in paris, the hierarchical diffusion model seems to apply to our case study quite well, particularly before the act. except for some rural départements where religious communities initiated asylum development, pioneer départements were usually densely populated and tended to be relatively advanced both socially and economically. the average size of new adopters (absolute and relative to the period) tended to decrease until , which suggests that the innovation was filtering down the urban hierarchy. having said this, there were some inconsistencies in the general trends; certain départements with small populations established asylums quite early, while some more populated areas were slower to set up asylums. to some extent, the act, requiring each french département to have an asylum, disrupted the hierarchical diffusion process, imposing a more universal diffusion of asylum development. although the act did not proactively initiate the trend to set up asylums, it nevertheless framed the later stages of the process and may have influenced its course of development. likewise the 'sectorisation' policy introduced in , in the wake of deinstitutionalisation of psychiatric care, also 'interrupted' the final stages of the diffusion process, as discussed above. our findings therefore raise questions concerning the relevance of classical diffusion models for the interpretation of this example of health system development and argue for an approach based on more complex models. a more relevant conceptual framework might be political ecology, involving the exploration of large-scale social, economic and political influences that shape the local context (e.g. described by mayer ( ) , richmond,c., elliott,s., matthews,r., & elliott,b. ( ) ), as well as locally specific factors that influence the trajectory of development of health care systems. such a perspective would also place more emphasis on mental health system development in its wider social, economic and political context, including the evolution of the social representations and medical knowledge of mental illness, the political management of the diffusion of these innovations, and also the profound changes in the urban hierarchy of the country through the th century. a conceptual framework based on political ecology would also make it possible to consider that decisions regarding the development of psychiatric care were being made simultaneously at different geographical scales, from local to national level. furthermore, it would allow us to emphasize the importance of a historical perspective, stressing the path-dependency that helps us to understand how historical conditions influenced the dynamic processes of innovation considered here. ideas of path-dependency also continue to be particularly relevant for french mental health provision because of the continuity between past and present in the geography of the provision of services. contemporary processes of french deinstitutionalisation are strongly structured by the past heritage of asylum institutions. unlike the situation in the united kingdom or the united states, french deinstitutionalisation has not led to the mass closure of psychiatric hospitals. to date in , no psychiatric hospital closure has been registered in france following the deinstitutionalisation principles. the present psychiatric sectorisation policy therefore has to adapt to this pre-existing asylum geography. this creates issues of accessibility and problems of rehabilitation and transformation of parts of disused buildings, often costly to maintain and difficult to convert to other uses, especially when buildings are classified as historical monuments. this paper also opens up a large field of research, since the geography of french mental health care has not been previously studied, despite the strong spatial dimensions of mental health care planning, as enshrined in the act, in the policy for geographical division of the country into psychiatric sectors, and, more recently, in the 'area health plans' (projets médicaux de territoire) aiming to facilitate and coordinate primary and hospital care, social and health services. the urban geography of sars: paradoxes and dilemmas in toronto's health care la géographie du sida en afrique. cahiers géos e the plague of e in the western mediterranean: the italian side diffusion of high technology medical innovation -computedtomography scanner example the social geography of aids in brazil airline networks and the international diffusion of severe acute respiratory syndrome (sars) la mesure de l'urbanisation aux etats-unis, des premiers comptoirs coloniaux aux metropolitan areas ( e ). cybergeo, systèmes, modélisation, géostatistiques what asylums were, are and ought to be histoire de la france. l'espace français instruction sur la manière de gouverner les insensés, et de travailler à leur guérison dans les asyles qui leur sont destinés rapport sur le service des aliénés en tableaux démographiques. la population en france: histoire et géographie historical account of the climates and diseases of the united states of america a view of the diseases most prevalent in the united states of america analyse de processus centralisés de diffusion spatiale: le cas des établissements des réseaux de services aux entreprises rapport présenté à la séance du novembre de la commission de santé mentale landscapes of despair: from deinstitutionalization to homelessness analysis of spatial diffusion patterns for aids cases in some brazilian states dictionnaire des sciences humaines analyses factorielles simples et multiples. dunod des établissements consacrés aux aliénés en france et les moyens de les améliorer des maladies mentales considérées sous les rapports médical, hygiénique et médico-légal applied multivariate data analysis histoire de la folie à l'âge classique surveiller et punir. naissance de la prison new york: virgin books. translation to english by richard howard of foucault lire et écrire: l'alphabétisation des français de calvin à jules ferry l'archivio della follia la pratique de l'esprit humain. l'institution asilaire et la révolution démocratique consoler et classifier: l'essor de la psychiatrie française. les empêcheurs de penser en rond la dynamique d'un système de peuplement: évolution de la population des villes françaises de à commune centre, agglomération, aire urbaine: quelle pertinence pour l'étude des villes? 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the authors would like to thank pr. denise pumain for her support and advice during the research process and dr. michel caire for his valuable assistance in the building of the database. this study was funded by an industrial agreement for training through research (cifre contract), through the national agency for technical research. key: cord- -eiphxwmn authors: trouillet-assant, sophie; viel, sebastien; gaymard, alexandre; pons, sylvie; richard, jean-christophe; perret, magali; villard, marine; brengel-pesce, karen; lina, bruno; mezidi, mehdi; bitker, laurent; belot, alexandre title: type i ifn immunoprofiling in covid- patients date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: eiphxwmn covid patients in icu present a high mortality rate and immunoprofiling reveals heterogeneous ifn-α production with about % of critically-ill patients unable to produce ifn-α , highlighting the immune response heterogeneity and opening avenues for targeted therapies. sophie trouillet-assant , * , phd, sebastien viel , , , * , pharmd, phd, alexandre gaymard merazga for their excellent work. we thank fabien subtil for his helpful advice for statistical analysis. we also thank the life (lyon immunopathology federation) community for fruitful discussion. capsule summary: covid patients in icu present a high mortality rate and immunoprofiling reveals heterogeneous α production with about % of critically-ill patients unable to produce ifn-α , highlighting the immune response heterogeneity and opening avenues for targeted therapies. to the editor, severe acute respiratory syndrome coronavirus (sars-cov- ) infection (covid- ) is characterized by a wide spectrum of disease encompassing asymptomatic carriage, mild to severe upper respiratory tract illness that can evolve into respiratory failure or rapidly progressing severe viral pneumonia with acute respiratory distress syndrome (ards). disease severity depends on viral strain and host risk factors have been identified such as age and male gender. in addition, an excessive immune response has been identified in patients showing a cytokine storm associated with ards . various immunosuppressive drugs, including il- blockers or jak-stat signaling inhibitors have been suggested for the treatment of sars-cov- infection whereas additional clinical trials are evaluating the use of recombinant interferon to foster host antiviral response. (clinicaltrials nct , nct ). type i interferons (ifn-i) are major components of the innate immune system and represent critical antiviral molecules . to date, ifn-i response has not been evaluated in covid- patients and its contribution to the viral control and inflammation is unknown. in this study, we assessed the kinetics of plasma ifn-i in covid- patients with a spectrum of severity degree. this study was approved by an ethical committee for biomedical research (comité de protection des personnes hcl). (supplemental material and method of this article online repository). firstly, we explored three patients issued from the first covid cluster diagnosed in france (les contamines, haute savoie, france) in february . we took advantage of the new digital elisa technology single-molecule arrays (simoa) and analyzed the kinetics of plasma inflammatory cytokines. interleukin (il)- , c-reactive protein (crp) and interferon γ-induced protein (ip- ) were elevated in the two symptomatic patients (pt , ) (supplementary figure in the online repository). strikingly, no ifn-α was detectable in these two patients. in contrast, il- , crp and ip- elevation of plasmatic ifn-α was observed. viral loads were low with no obvious quantitative difference between all three patients. we further explored a larger cohort of critically ill covid patients from one of the intensive care unit (icu) at hospices civils de lyon (lyon, france). of note, all the patients were treated with standard of care and none received antiviral or immunotherapies. considering the first days of infection, more than half of critically ill patients required invasive mechanical ventilation ( / ). we observed that patients demonstrated a peak in ifn-α at day - of symptoms onset corresponding to the viral replication phase, that decreased overtime to low but still detectable ifn-α the timing of interferon exposition may be critical to control the virus and avoid immunopathogenesis. channappavanar et al. have shown that delayed ifn-i expression can be detrimental in mice in the context of sars-cov- infection . our data suggests that screening patients for ifn production is instrumental to select those who could benefit from early intervention with ifn. following day , il- remains increased while ifn-α tapered. this kinetics highlight that cytokine inhibitors could be helpful at the second phase of the disease following ifn-i decrease. viral characteristic or individual genetic susceptibility should be explored to understand the defect of ifn- α production in some covid patients. some ifn-α positive patients also experienced fatal outcome highlighting the multifactorial causes of disease severity. we acknowledge limitations of this study, related to the small number of included patients and the technical limitation for the measurement of ifn-β and ifn-λ, in this proof of concept study. here, we provide new argues for an early intervention with recombinant ifn-α and we also highlight the window of opportunity for immunosuppressors at the second phase of the disease, delay between symptom onset and icu admission (days) [ - ] [ - ] . bacterial co-infection during icu stay (n (%)) ( %) ( %) diabetes (n (%)) ( %) ( %) chronic obstructive pulmonary disease (n (%)) ( %) ( %) cardiovascular disease (n (%)) ( %) ( %) hypertension (n (%)) ( %) ( %) cancer (n (%)) ( %) ( %) active smokers (n (%)) ( %) ( %) mortality at d after symptom onset(n(%)) ( %) ( %) . crp -c-reactive protein, icu -intensive care unit, bmi -body mass index table -clinical characteristics of covid- patients in intensive care unit p-value are calculated using mann-whitney test for quantitative values and using fisher-exact test for qualitative ones. a. plasma ifn-α concentrations (fg/ml) were determined by single molecule array (simoa) b.c.d. il- , crp and ip- concentrations were measured using a multiplexed assay with the ella platform. e. viral load is represented as cycle threshold of ip rt-qpcr using assay designed by pasteur institut in paris. ifn-interferon ; il- -interleukin ; crp -c-reactive protein ; ip- -interferon γ-induced protein a. ifn score is a transcriptionnal signature defined by interferon-stimulated gene (isg) quantified using nanostring technology and obtained from paxgene tubes in covid- patients. b-d. normal values for healthy volunteers was indicated by grey area. vertical bar indicates median delay between symptom onset and icu admission. concentrations of ifn-γ were quantified in only / patients because of lack of material. clinical features of patients infected with novel coronavirus in wuhan, china covid- : consider cytokine storm syndromes and immunosuppression type i interferons (α/β) in immunity and autoimmunity / and directive / /ec) and the french data protection law (law n° - on / / and décret n° - on / / ), we obtained consent from each patient or his next of kin usa) on plasma samples of covid- patients. the assay was based on a -step protocol using an hd- analyzer (quanterix). il- , crp and interferon γ-induced protein (ip- ) concentrations were measured using a multiplexed assay with the ella platform (protein simple© ca, usa), according to manufacturer's instructions. plasma il a/b and il- (type iii interferon) have been quantified by elisa (pbl laboratories rna integrity was then evaluated by agilent rna microarray (agilent technologies© data standardization was obtained using the geometric mean of internal control and housekeeping genes count number. interferon score was calculated as previously described . virus quantification load viral load was quantified from nasopharyngeal swabs or endotracheal aspirates. rna extraction was performed by the automated nuclisens® easymag® (biomérieux, marcy l'etoile, france) using manufacturer's instructions. a μl reaction contained μl of rna p-value were calculated using mann-whitney test for quantitative values and using fisher-exact test for qualitative ones comparison of rt-qpcr and nanostring in the measurement of blood interferon response for the diagnosis of type i interferonopathies walzer international center of research in infectiology, lyon university, inserm u , cnrs umr , ens, ucbl, lyon, france we explored the first three sars-cov- positive patients diagnosed in france (les contamines, france) in february . patient : a high risk contact (a -year-old man) initially negative for sars-cov- developed fever and cough with respiratory crackles at auscultation on the fifth day of hospital isolation. a bilateral interstitial syndrome at the ct-scan with bilateral ground-glass opacification predominant on the left. sars-cov was detected from endotracheal aspirates (eta), all nasopharyngeal swabs were always negative. the daily follow-up revealed a short-lasting excretion with only two successive eta for these three patients, no other respiratory pathogens were detected. these patients did not need oxygenation, nor antibiotics, steroids or antiviral agents. plasma samples and paxgene® tubes were collected from covid- patients hospitalized in the university hospital of lyon (hospices civils de lyon), france. diagnosis of covid- was confirmed in all patients by rt-pcr.all critically ill patients, admitted to icu, were included in the mir-covid study. this study was registered to the french national data protection agency under the number - and was approved by an ethical committee for biomedical research (comité de protection des personnes hcl) under the number n° - . in agreement with the general data protection regulation (regulation key: cord- - mqc mqd authors: roques, lionel; klein, etienne k.; papaïx, julien; sar, antoine; soubeyrand, samuel title: impact of lockdown on the epidemic dynamics of covid- in france date: - - journal: front med (lausanne) doi: . /fmed. . sha: doc_id: cord_uid: mqc mqd the covid- epidemic was reported in the hubei province in china in december and then spread around the world reaching the pandemic stage at the beginning of march . since then, several countries went into lockdown. using a mechanistic-statistical formalism, we estimate the effect of the lockdown in france on the contact rate and the effective reproduction number r(e) of the covid- . we obtain a reduction by a factor (r(e) = . , %-ci: . – . ), compared to the estimates carried out in france at the early stage of the epidemic. we also estimate the fraction of the population that would be infected by the beginning of may, at the official date at which the lockdown should be relaxed. we find a fraction of . % ( %-ci: . – . %) of the total french population, without taking into account the number of recovered individuals before april st, which is not known. this proportion is seemingly too low to reach herd immunity. thus, even if the lockdown strongly mitigated the first epidemic wave, keeping a low value of r(e) is crucial to avoid an uncontrolled second wave (initiated with much more infectious cases than the first wave) and to hence avoid the saturation of hospital facilities. covid- epidemic was reported in the hubei province in china in december and then spread around the world reaching the pandemic stage at the beginning of march ( ) . to slow down the epidemic, several countries went into lockdown with different levels of restrictions. in the hubei province, where the lockdown has been set long before the other countries (on january ), the epidemic has reached a plateau, with only sporadic new cases by april [from the data of johns hopkins university center for systems science and engineering ( ) ]. in france, the first cases of covid- were detected on january , and the lockdown has been set on march . this national lockdown means important restrictions on movement, with a mandatory home confinement except for essential journeys including food shopping, care, h individual sporting activity and work when teleworking is not possible, and closing of the borders of the schengen area. it also includes closures of schools and universities as well as all non-essential public places, including shops (except for food shopping), restaurants, cafés, cinemas, and nightclubs. the basic reproduction number r corresponds to the expected number of new cases generated by a single infectious case in a fully susceptible population ( ) . several studies, mostly based on chinese data, aimed at estimating the r associated with the covid- epidemic, leading to values from . to . , with an average of . ( ) . as the value of r can be interpreted as the product of the contact rate and of the duration of the infectious period, and since the objective of the lockdown and associated restriction strategies are precisely to decrease the contact rate, an important effect on the number r e of secondary cases generated by an infectious individual is to be expected. this value r e is often referred to as "effective reproduction number, " and corresponds to the counterpart of r in a population that is not fully susceptible ( ) . if r e > , the number of infectious cases in the population follows an increasing trend, and the larger r e , the faster this trend. on the contrary, if r e < , the epidemic will gradually die out. the control measures in china have been shown to have a significant effect on the covid- epidemic, with growth rates that shifted from positive to negative values (corresponding to r e < ) within weeks ( ). the study ( ) showed that containment policies in hubei province also led to a subexponential growth in the number of cases, consistent with a decrease in the effective reproduction number r e . fitting a seir epidemic model to time series of reported cases from provinces in china, tian et al. ( ) found a basic reproductive number r = . before the implementation of the emergency response in china, a value that was divided by more than once the control measures were fully effective. using contact surveys data for wuhan and shanghai it was estimated in zhang et al. ( ) that the effective reproduction number was divided by a factor in wuhan and . in shanghai. standard epidemiological models generally rely on sir (susceptible-infected-removed) systems of ordinary differential equations and their extensions [for examples of application to the covid- epidemic, see ( , ) ]. with these models, and more generally for most deterministic models based on differential equations, when the loss of information due to the observation process is heavy, specific approaches have to be used to bridge the gap between the models and the data. one of these approaches is based on the mechanistic-statistical formalism, which uses a probabilistic model to connect the data collection process and the latent variable described by the ode model. milestone articles and textbook have been written about this approach or related approaches ( ) , which is becoming standard in ecology ( , ) . the application of this approach to human epidemiological data is still rare. in a previous study ( ) , we applied this framework to the data corresponding to the beginning of the epidemic in france (from february to march ), with a sir model. our primary objective was to assess the infection fatality ratio (ifr), defined as the number of deaths divided by the number of infected cases. as the number of people that have been infected is not known, this quantity cannot be directly measured, even now (on april ). the mechanistic-statistical framework allowed us to compute an ifr of . % ( %-ci: . - . %), which was consistent with previous findings in china ( . %) and in the uk ( . %) ( ) and lower than the value previously computed on the diamond princess cruse ship data ( . %) ( ) . in this previous study, we also computed the r in france, and we found a value of . ( %-ci: . - . ). although the number of tests at that stage was low, an advantage of working with the data from the beginning of the epidemic was that the initial state of the epidemic was known. here, we develop a new mechanistic-statistical approach, based on a sird model (d being the dead cases compartment), in the aim of • estimating the effect of the lockdown in france on the contact rate and the effective reproduction number r e ; • estimating the number of infectious individuals and the fraction of the population that has been infected by the beginning of may (at the official date at which the lockdown should be relaxed). we obtained the number of positive cases and deaths in france, day by day from santé publique france ( ) , from march to april . we obtained weekly data on the number of individuals tested (in private laboratories and hospitals) from the same source. we assumed that during each of these weeks the number of tests per day was constant. this assumption is consistent with the small variations between the number of tests during the first week ( , ) and the second week of observation ( , the mechanistic-statistical framework consists in the combination of a mechanistic model that describes the epidemiological process, a probabilistic observation model and an inference procedure. the dynamics of the epidemic are described by the following sird compartmental model: with s the susceptible population, i the infectious population, r the recovered population, d the number of deaths due to the epidemic and n the total population. for simplicity, we assume that n is constant, equal to the current french population, thereby neglecting the effect of the small variations of the population on the coefficient α/n. the parameter α is the contact rate (to be estimated) and /β is the mean time until an infectious becomes recovered. based on the results in zhou et al. ( ) , the median period of viral shedding is days, but the infectiousness tends to decay before the end of this period: the results in he et al. ( ) indicate that infectiousness starts - days before symptom onset and declines significantly days after symptom onset. based on these observations we assume here that the mean duration of the infectiousness period is /β = days. in li et al. ( ) , the duration of the incubation period was estimated to have a mean of . days. thus, the mean duration of the non-infectious exposed period is relatively short (about - days), and can be neglected without much differences on the results, as shown in liu et al. ( ) . inclusion of an exposed compartment (as in seir models) is particularly relevant when exposed individuals can indirectly transmit the disease e.g., through insect vectors [e.g., ( ) ], which is seemingly not the case for coronaviruses. the parameter γ corresponds to the death rate of the infectious (to be estimated). the model is started at a date t corresponding to april st. the initial number of infectious i(t ) = i is not known and will be estimated. the total number of recovered at time t is also not known. however, as the compartment r has no feedback on the other compartments, we may assume without loss of generality that r(t ) = , thereby considering only the new recovered individuals, starting from the date t . we fixed d(t ) = , the number of deaths at hospital by march . the initial s population at the beginning of the period, should still be close to the total french population: by march only , cases had been observed in france, corresponding to . % of the total population. a factor had been estimated in roques et al. ( ) between the cumulated number of observed cases and the actual number of cases at the beginning of the epidemic. even though this factor may have changed, this means that the proportion of the total population that has been infected by march is still small. we can get an upper bound for the cumulated number of cases by march by dividing the number of hospital deaths at the end of the observation period ( , by april ) by the hospital ifr [ . %, as estimated in ( )] leading to about million cases. this means that the value of s(t ) is between and million cases. for our computation, we assumed that s(t ) = · , corresponding to about . % of the french population. as shown in figure s , our results are not much sensitive to the value of s(t ) (at least when s/n remains close to ). the ode system ( ) was solved thanks to a standard numerical algorithm, using matlab r ode solver. the number of cases tested positive on day t, denoted byδ t , is modeled by independent binomial laws, conditionally on the number of tests n t carried out on day t, and on p t the probability of being tested positive in this sample: the tested population consists of a fraction of the infectious cases and a fraction of the susceptibles: n t = τ (t) i(t)+τ (t) s(t). thus, with κ t : = τ (t)/τ (t), the relative probability of undergoing a screening test for an individual of type s vs an individual of type i. we assumed that the ratio κ was independent of t over the observation period. the coefficient σ corresponds to the sensitivity of the test. in most cases, rt-pcr tests have been used and existing data indicate that the sensitivity of this test using pharyngeal and nasal swabs is about − % ( ). we assumed here σ = . ( % sensitivity). each day, the number of new observed deaths (excluding nursing homes), denoted byμ t , is modeled by independent poisson distributions conditionally on the process d(t), with mean value d(t) − d(t − ) (which measures the daily increment in the number of deaths): note that the time t in ( ) is a continuous variable, while the observationsδ t andμ t are reported at discrete times. for the sake of simplicity, we used the same notation t for the days in both the discrete and continuous cases. in the formulas ( ) and ( ) i(t), s(t), and d(t) are computed at the end of day t. the unknown parameters are α, γ , κ, and i . we used a bayesian method ( ) to estimate the posterior distribution of these parameters. the likelihood l is defined as the probability of the observations (here, the increments {δ t ,μ t }) conditionally on the parameters. using the observation models ( ) and ( ), and using the assumption that the incrementsδ t andμ t are independent conditionally on the underlying sird process and that the number of tests n t is known, we get: with t i the date of the first observation and t f the date of the last observation. in this expression l(α, γ , κ, i ) depends on α, γ , κ, i through p t and d(t). the posterior distribution corresponds to the distribution of the parameters conditionally on the observations: p(α, γ , κ, i |{δ t ,μ t }) = l(α, γ , κ, i ) π(α, γ , κ, i ) c , where π(α, γ , κ, i ) corresponds to the prior distribution of the parameters (detailed below) and c is a normalization constant independent of the parameters. regarding the contact rate α, the initial number of infectious cases i and the probability κ, we used independent noninformative uniform prior distributions in the intervals α ∈ ( , ), i ∈ ( , ) and κ ∈ ( , ). to overcome identifiability issues, we used an informative prior distribution for γ . this distribution, say f g , was obtained in roques et al. ( ) during the early stage of the epidemic (f g is depicted in figure s ). in roques et al. ( ) , the number of infectious cases i at the beginning of the epidemic was known (equal to ), and did not need to be estimated. thus, we estimated in roques et al. ( ) the distribution of the parameter γ by computing the distribution of the infectious class and using the formula d ′ (t) = γ i(t) together with mortality data (which were not used for the estimation of the other parameters, unlike in the present study). finally, the prior distribution is defined as follows: π(α, γ , κ, i ) = (α,κ,i )∈( , )×( , )×( , ) f g (γ ). the numerical computation of the posterior distribution is performed with a metropolis-hastings (mcmc) algorithm, using independent chains, each of which with iterations, starting from the posterior mode. to find the posterior mode we used the bfgs constrained minimization algorithm, applied to − ln(l) − ln(π), via the matlab r function fmincon. in order to find a global minimum, we applied this method starting from , random initial values. the matlab r codes are available as supplementary material. denote by (α * , γ * , κ * , i * ) the posterior mode, and s * (t), i * (t), r * (t), d * (t) the solutions of the system ( ) associated with these parameter values. the observation model ( ) implies that the associated expected number of cases tested positive on day t is n t p * t (expectation of a binomial) with the observation model ( ) implies that the expected cumulated number of deaths on day t is d * (t). to assess model fit, we compared these expectations and the observations, i.e., the cumulated number of cases tested positive, t : = c + {s=t ,...,t + }δs with c the number of cases tested positive by march (c = , ) and the cumulated number of deaths m t : = m + {s=t ,...,t + }μs , with m the number of reported deaths (at hospital) by march (m = ). the results are presented in figure . we observe a good match with the data. the pairwise posterior distributions of the parameters (α, i ), (α, γ ), (α, κ), (γ , i ), (γ , κ), (κ, i ) are depicted as figure s . with the exception of the parameter γ (figure s ), for which we chose an informative prior, the posterior distribution is clearly different from the prior distribution, showing that new information was indeed contained in the data. the effective reproduction number can be simply derived from the relation r e = α/(β + γ ) when s is close to n ( ). the distribution of r e is therefore easily derived from the marginal frontiers in medicine | www.frontiersin.org posterior distribution of the contact rate α (since we assumed β = / ; see section . ). it is depicted in figure . we observe a mean value of r e of . ( %-ci: . - . ). the marginal posterior distribution of i indicates that the number of infectious individuals at the beginning of the considered period (i.e., april st) is . · ( %-ci: . · − . · ). the computation of the solution of ( ) with the posterior distribution of the parameters leads to a number of infectious i(t f ) = . · and a total number of infected cases (including recovered) (i + r)(t f ) = . · at the end of the observation period (april ). by may , if the restriction policies remain unchanged, we get a forecast of i(t) = . · infectious cases ( %-ci: . · − . · ) and (i + r)(t) = . · infected cases including recovered ( %-ci: . · − . · ). the dynamics of the distributions of i and i + r are depicted in figure . by may , the total number of infected cases (including recovered) therefore corresponds to a fraction of . % of the total french population. this value does not include the recovered cases before april st. many studies focused on the estimation of the basic reproductive number r of the covid- epidemic, based on data-driven methods and mathematical models [e.g., ( , ) ] describing the epidemic from its beginning. in average, the estimated value of r was about . . we focused here on an observation period that began after the lockdown was set in france. we obtained an effective reproduction number that was divided by a factor , compared to the estimate of the r carried out in france at the early stage of the epidemic, before the country went into lockdown [a value r = . was obtained in ( ) ]. this indicates that the restriction policies were very efficient in decreasing the contact rate and therefore the number of infectious cases. in particular, the value r e = . is significantly below the threshold value were the epidemic starts dying out. the decay in the number of infectious cases can also be observed from our simulations. it has to be noted that, although the number of infectious cases is a latent, or "unobserved" process, the mechanistic-statistical framework allowed us to estimate its value (figure ) . the cumulated number of infected cases that we obtained by may (i +r) corresponds to a fraction of . % ( %-ci: . - . %) of the total french population, without taking into account the number of recovered individuals before april st, which is not known. based on a value r = . , the herd immunity threshold, corresponding to the minimum fraction of the population that must have immunity to stop the epidemic, would be − /r ≈ % [a threshold of % was proposed in ( ) ]. this proportion will probably not be reached by may . as emphasized by angot ( ) , a too fast relaxation of the lockdown-related restrictions before herd immunity is reached or efficient prophylaxis is developed), would expose the population to an uncontrolled second wave of infection. in the worst-case scenario, the effective reproduction number r e would approach the initially estimated value of r , and the second wave would start with about . · infectious individuals (in comparison with the few cases that initiated the first wave in france) and about · susceptible individuals. keeping a low value of r e is therefore crucial to avoid the saturation of hospital facilities. we deliberately chose a parsimonious mechanistic model with a few parameters to avoid identifiability issues. possible extensions include stage-structured models, where the infectious class i and the contact rate α would depend on another variable: i = i(t, τ ) and α = α(t, τ ) with τ the time since infection, to take into account the dynamics of the viral load on the infectiousness. see e.g., murray ( ) (chapter . ) for an introduction to such modeling approaches. another insightful extension would consist in using spatially-explicit models, e.g. reaction-diffusion models ( ) to describe the spatial spread of the epidemic, and to be able to estimate local values for the parameter r e and the number of susceptible cases. although herd immunity is far from being reached at the country scale, it is likely that the fraction of immune individuals strongly varies over the territory, with possible local immunity effects [e.g., by april the proportion of people with confirmed sars-cov- infection based on antibody detection was of % in a high-school located in northern france ( ) ]. publicly available datasets were analyzed in this study. this data can be found here: https://www.gouvernement.fr/infocoronavirus/carte-et-donnees https://geodes.santepublique france.fr and https://ourworldindata.org/coronavirus-testing. lr, ek, jp, as, and ss conceived the model and designed the statistical analysis. lr and ss wrote the paper. lr carried out the numerical computations. all authors reviewed the manuscript. world health organization. who director-general's opening remarks at the media briefing on covid- an interactive web-based dashboard to track covid- in real time the reproductive number of covid- is higher compared to sars coronavirus epidemiology 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coupled reaction-diffusion-absorption model using early data to estimate the actual infection fatality ratio from covid- in france estimates of the severity of coronavirus disease : a model-based analysis estimating the infection and case fatality ratio for coronavirus disease (covid- ) using age-adjusted data from the outbreak on the diamond princess cruise ship covid- : point épidÉmiologique du avril résidents en établissements d'hébergement pour personnes âgées en clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study temporal dynamics in viral shedding and transmissibility of covid- early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a covid- epidemic model with latency period analysis of transmission dynamics for zika virus on networks detection of sars-cov- in different types of clinical specimens preliminary estimation of the basic reproduction number of novel coronavirus to : a data-driven analysis in the early phase of the outbreak impact of non-pharmaceutical interventions (npis) to reduce covid- mortality and healthcare demand early estimations of the impact of general lockdown to control the covid- epidemic in france spatial ecology via reaction-diffusion equations cluster of covid- in northern france: a retrospective closed cohort study. medrxiv effect of a one-month lockdown on the epidemic dynamics of covid- in france this manuscript has been released as a pre-print at medrxiv ( ) . the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmed. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © roques, klein, papaïx, sar and soubeyrand. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - vh jg authors: fortané, nicolas title: antimicrobial resistance: preventive approaches to the rescue? professional expertise and business model of french “industrial” veterinarians date: - - journal: nan doi: . /s - - - sha: doc_id: cord_uid: vh jg this article focuses on the development of veterinary medicine in the industrial pig and poultry production sector. in the current context of controversies over the public problem of antimicrobial resistance (amr), the veterinary profession is tending to promote a model of preventive medicine that is supposed to reduce the use of antibiotics in livestock farming. however, veterinarians specializing in pig and poultry production (“industrial vets”) have in fact been adopting such approaches to animal health for several decades. based on interviews with pig and poultry veterinarians practicing or having practiced in western france between the s and the s, the article aims to understand how such a form of professional expertise has developed, and the business model that underpins it. contrary to public discourses which promote preventive approaches as a way to diversify professional expertise and to disconnect veterinary incomes from drug sales, it is indeed this economic model that has allowed the development of such approaches within industrial livestock farming. modern strategies for reducing antibiotic use should therefore seek less to renew the professional expertise of veterinarians than to find new ways to valorize it economically. the french veterinary profession is currently undergoing major changes; or at least it tends to see itself as being at the heart of a period of major challenges that are pushing it to reinvent itself. this is not the first time that it has had to face such a reflexivity test, even in recent history (in the british case, some historians even see a cycle-woods a), but recent literature produced by professional veterinary organizations shows the importance of what is currently perceived as a need for self-analysis and change (ondpv ; vetfuturs france ) . there are several issues that might explain why this period is favourable to such a prospective assessment of veterinary futures. one of the most important concerns the controversies and public policies that have developed over recent years with regard to the issue of antimicrobial resistance (amr), which has directly challenged the economic and professional model of farm animal veterinary practices that were setting drugs (mostly antibiotics) up as a cornerstone of veterinary activity, as a source of both income and professional expertise. cross-fertilization of research on the veterinary profession and drug regulation is not common. although veterinarians have aroused the interest of certain historians and sociologists of professions, this has essentially been in relation to the analysis of this social group's process of professionalization (berdah ; mitsuba ) , its role in animal health or food safety policies (woods b; enticott et al. ; fortané , ) , the dynamics that contribute to its specialization (gardiner ) or feminization (surdez ), or finally to knowledge and professional practices in farm (shortall et al. ; ruston et al. ) and small animal medicine (sanders ; morris ) . as for the regulation of veterinary drugs, there are also several studies by historians on the vaccination of animals against major zoonoses or epizootic diseases (woods ; berdah ) , sometimes on the veterinary pharmaceutical industry (corley and godley ) , and more recently on the amr issue (kirchhelle ). yet unlike the uses of human medicines that medical anthropology has been able to theorize and document for many years (whyte et al. ) , the uses of veterinary medicines, i.e. the conditions under which they are prescribed, dispensed and used, are rarely studied, except in interdisciplinary literature from the field of veterinary sciences (speksnijder et al. ; coyne et al. ) . this article tries to open a way to cross-fertilize these reflections. using recent debates on the amr problem, it proposes to examine the relationship between the development of professional veterinary expertise and of the drug market, based on the case of a specific segment of the profession, namely veterinarians specializing in industrial poultry and pig production in western france. it thus puts the amr issue under a broader lens as it analyses ongoing changes within the veterinary profession not as potential consequence of recent measures aiming to reduce antibiotic use, but rather as a reason for the way the problem is now framed. indeed, it is common to hear professional organizations or public authorities state that in order to reduce their economic dependence on antibiotic sales, vets must rethink their activity by favouring preventive approaches to animal health which would involve a diversified range of services and would contribute to placing vets in an advisory role with a holistic vision of livestock farming or even of the food supply chain (vetfuturs france ) . however, such a form of professional expertise, combined with a particular business model for the practices promoting it, is not fundamentally new. if it is at the heart of contemporary debates, it is because it is based and supported by far earlier dynamics that initially had nothing to do with amr, but which used the opportunity of current controversies surrounding antibiotic use, sale and prescription to reinforce and legitimize a certain vision of veterinary medicine, based on preventive approaches to animal health. poultry and pig medicine in industrial production is an especially interesting area for an analysis of these dynamics. firstly, because approaches to animal health that have developed in this field are very singular and characteristic of the intensive farming methods used in these sectors, particularly in the brittany and pays de loire regions where a large proportion of the production is located. indeed, a certain vision of preventive medicine was developed by pig and poultry vets in the s and s, even if the issue of this form of veterinary expertise has not always been raised in these terms. this article therefore aims to understand the professional knowledge, practices and economic model upon which this kind of expertise is based, and why the current amr context is an opportunity to expand it (or at least attempt to). secondly, pig and poultry vets make up an extremely small and autonomous segment of the profession (which makes it possible to draw up a fairly representative picture) although its homogeneity should not be overestimated. this article thus seeks to provide a thorough analysis of this very particular part of the veterinary profession, the specificities of which have almost never been addressed by the literature. despite their small numbers, "industrial" vets are nevertheless an essential component of the profession, because they manage the health of an economic sector which supplies a considerable share of national animal production. the article opens with a brief presentation of the political context and controversies surrounding the amr problem, and how the french veterinary profession has faced up to this by defending the preventive medicine model. it then describes this form of expertise in the professional segment studied here, showing why pig and poultry vets chose this specialization. the article then looks at the origins of these preventive approaches to animal health, both in terms of knowledge and practices, and the economic model associated with it. finally, it reviews the strategies currently developed by industrial vets to adapt to the constraints of increased control of antibiotic use in livestock. the amr problem and the preventive "solution" the problem of antibiotic use in livestock farming is not new. as soon as these molecules were introduced in agriculture in the late s, there was controversy concerning the development of resistant bacteria in animals and food, and the risks of human contamination (bud ). yet for several decades, this issue has been eclipsed by the belief in a permanent renewal of the therapeutic arsenal, consisting in thinking that the continuous discovery of new antibiotics would compensate for the development of increasingly resistant bacteria (podolsky ) . after the swann report in , a series of measures to control the use of antibiotics as growth promoters was nevertheless adopted in europe, progressively separating the molecules used in agriculture and human medicine (kirchhelle ) . but years later, during the avoparcin crisis , , , these measures were considered ineffective (in the sense that they did not prevent the transmission of resistant bacteria between humans and animals) and the use of antibiotics as growth promoters was finally banned in the european union in (kahn ) . the problem of antibiotic use in livestock farming as we know it today reemerged in the late s, this time focusing on veterinary uses, i.e. on curative or preventive uses with veterinary prescription (fortané ) . veterinarians were directly accused of being responsible for the overuse and misuse of antibiotics (and therefore for the spread of resistant bacteria) on the basis of a fairly simple argument: their supposed professional "conflict of interest". indeed, in france, since the act on veterinary pharmaceuticals, vets have had a dual monopoly on the prescription and supply of medicines (hubscher ) . even if, in theory, delivery is shared between three beneficiaries (veterinarians, pharmacists and approved co-operatives), vets capture the vast majority of the curative drug market (of which antibiotics constitute the main category) (guillemot and vandaële ) . the argument that veterinarians over-prescribe antibiotics in order to increase their incomes then became the main framing of the amr problem. this construction of the problem was in reality carried by a coalition of human health actors (doctors, pharmacists, health administration) whose political agenda was twofold. on the one hand, they defended a measure that crystallized the debates around the years - : the "decoupling" of prescription and delivery, which consists in applying the professional and economic model that prevails on the human drug market, i.e. reserving prescriptions to physicians and sales to pharmacists. decoupling basically means forbidding veterinarians from selling pharmaceuticals, as is the case in countries such as sweden or spain (fortané ) . on the other hand, this coalition supported the concept of "critically important antibiotics", the principle of which is to reserve certain molecules, in particular the latest generations of antibiotics, for human medicine. from a political and institutional point of view, this period was extremely interesting because it put the spotlight on definitional and jurisdictional conflicts between different social groups for the control of the legitimate use of antibiotics. it finally ended in a relative victory for veterinarians who succeeded, at the end of an unprecedented mobilization, in reversing the stigma that human health stakeholders assigned to them. indeed, vets have been able to impose the image of a profession that is not guilty of overusing antibiotics but which is instead accountable for their proper use. the notions of prudent, judicious, rational or responsible use, now widely used in amr debates, are thus a social construct produced by conflicts between social groups for the definition of the legitimate use of antibiotics (fortané ) . this veterinarian victory led to the withdrawal of the two emblematic measures (decoupling of prescription and delivery; ban on critically important antimicrobials) supported by the coalition of human health actors. in return, a stricter framework for the use of antibiotics in animal husbandry was implemented between and : margins on the sale of antibiotics are now limited and the retail price of antibiotics must be the same for every client, and antimicrobial susceptibility tests are mandatory for the prescription of critically important antibiotics. but once again, the most important part of this victory certainly concerns the changes regarding the image of the profession. indeed, veterinarians not only reversed the stigma and positioned themselves as guardians of antibiotics, they were also able to re-appropriate the problem by highlighting the way they could solve it. without denying that the economic model of the profession was too financially dependent on antibiotic sales, and that antibiotic use may have been too prevalent in animal care in the past, vets started to defend the development of preventive approaches which would, according to them, be the only way to ensure the transition towards an economic and professional model guaranteeing responsible use of antibiotics. this view was reinforced and supported by farmers and public authorities who also called for the development of such approaches which are usually promoted by national amr policies ( fig. ) in france and in other countries (badau ; piquerez ) . at the heart of this new prospective narrative for the profession and its role in managing the amr problem, we can observe the construction of a (supposedly) new conception of animal health, which is not based on a strictly clinical approach to diseases but on a holistic vision of animals and livestock farming (biosecurity, hygiene, nutrition, good husbandry practices, etc.). in the posters above, veterinarians are portrayed as the gatekeepers of such an approach, through their transversal role as "health advisors". this professional model, based on the knowledge and practices of preventive approaches to animal health, goes hand law for food, agriculture and forestry n° - of october th, . decree no. - of march th, . when public authorities and the veterinary profession began to conceive this campaign, a third poster was designed, saying: "my vet is much more than a mere drug supplier, he is also a teacher! he prescribes the medicines i need … but above all he talks to my farmer to make sure that i am perfectly fed, sheltered and vaccinated". however this poster was not retained for the campaign because of its relatively sensitive headline. in hand with an economic model where the incomes of veterinary businesses would be more diversified since these new "health advisors" would be able to monetize a wider range of goods and services (hygiene and nutrition products, bacteriological analyses, livestock audits, etc.) than just pharmaceuticals. could we look beyond the symbols and images of professional and political discourses and see whether these preventive approaches that might change the way veterinary medicine is practiced, and how antibiotics are used, rely on actual knowledge and practices and, if so, where and since when? the fact is that this model of preventive veterinary medicine seems in reality to be quite typical of a very particular segment of the veterinary profession, the one this article proposes to describe, namely industrial vets. the remainder of the article thus seeks to address the following two questions: is this form of professional expertise really perceptible in the field, and is its development truly linked to the global context of the amr problem and to recently implemented policy measures, or does it have other origins and raisons d'être that might actually when public authorities and the veterinary profession began to conceive this campaign, a third poster was designed, saying: "my vet is much more than a mere drug supplier, he is also a teacher! he prescribes the medicines i need … but above all he talks to my farmer to make sure that i am perfectly fed, sheltered and vaccinated". however this poster was not retained for the campaign because of its relatively sensitive headline. these two posters were used in the french amr policy ("plan ecoanƟbio"). they are a perfect illustraƟon of how the veterinarian's role was reframed towards prevenƟve approaches to animal health (in parƟcular vaccinaƟon), as a means to reduce the use of anƟbioƟcs. the first poster (on the leŌ) says: "my vet is far more than a mere emergency doctor, he is an expert contribuƟng to good husbandry pracƟces". the second poster (on the right) says: "my vet is far more than a mere hands-on man, he is an advisor, always there to prevent and vaccinate". both conclude with: "ask your vet for advice" . when poultry or pig vets are asked why they chose this specialty, they often point out a huge contrast between what they do and the way they perceive cattle (i.e. "rural") or companion animal vets. they feel that being an "industrial vet" essentially relates to four characteristics. firstly, veterinarians specialized in poultry or pig medicine attach importance to working at the heart of the agri-food system, with livestock farming professionals. many of them have agricultural family origins and believe that they chose this profession in order to maintain a strong link with the rural world. they perceive farmers as animal experts, unlike pet owners, and see their activity as teamwork alongside skilled professionals, with whom it is easier to interact and who can also provide them with knowledge about animals. their clients are therefore also their partners in animal health management: they can trust them, rely on them and delegate tasks to them. secondly, pig and poultry vets consider their work to be a permanent renewal, as opposed to the repetitive and sometimes boring work of cattle vets who constantly reproduce the same gestures and who are rarely motivated or intellectually stimulated by new situations and new challenges. most of them, whether they are young vets or already have or years of experience, tend to compare their professional activity with the image of the "emergency vet" (or "fire brigade" vet), available day and night to care for sick animals. from their point of view, this traditional activity is typical of cattle vets, corresponds to the past and relates to a type of work which is limited to clinical diagnosis, drug prescription and/or surgery (e.g. midnight calving). in their opinion, the only objective of such a way of working is to ensure that clients are satisfied with an occasional intervention and that they will once again call upon veterinary services the next time health problems occur. the point here is not to claim that this is what cattle vets are actually doing, but simply to note that this narrative is widely used to define, by contrast, the professional identity of industrial vets. i am indirectly from a rural family, that is to say that my grand-parents, my uncles, they all worked as farmers, in mixed farming and mixed animal farming, from both sides of my family. it is just my parents who had access during the postwar period to national education programmes and became teachers or researchers. so i am from the third generation but i spent a lot of time at the farm. although some of the characteristics that pig and poultry vets tend to associate with cattle vets have actually been observed in other studies, in particular the fact that cattle vets do not have a high opinion of the technical expertise of the farmers they deal with (shortall et al. ) . these opinions of their clients nevertheless depend to a certain extent on the type of farm and farmers they relate to: cattle vets have better professional relationships with "commercial farmers" (managers of large, modern and business-oriented farms) as the latter tend "to understand the need to use the vet as a disease prevention consultant rather than to treat individual sick animals: i.e. part of vets' desired move from a 'test and treat' to a 'predict and prevent' model of veterinary intervention" (shortall et al. , p. ) . in addition, this shows that a tension between "fire brigade" work and advisory veterinary work is also present within a certain section of the cattle vet profession. i became a vet to take care of farm animals because i like being outside and having an intellectual occupation. so that is the reason why. so at vet school, you didn't think about working with dogs? certainly not (laughs)! that was my nightmare! when you work in the pig sector, what is good is that you work % on farms. because when you work with cattle, most of the time you have to do some small animal work as well. and what i want is to work with professionals who are producing the animals we eat. pig farmers (well, not all of them but most of them) have very good technical skills. pig farming is a very dynamic sector, you never get bored! we often have to upgrade the farms to new standards so it's always evolving. research also moves very fast, the pharmaceutical industry innovates a lot so it's interesting. (…) and to be honest, what interests me the most is this kind of follow-up, not being a "fire brigade" vet. but i can't talk very knowledgeably about the "classic" rural vet as i never was one. but what i do think about this kind of practice is that there is a lot of emergency and "fire brigade" work and i don't think that is very challenging. in pig production, we have been evolving for quite a long time because if we only do "fire brigade" work… well, farmers expect a lot more than that! and for us, that is also what's interesting. pig farming is a batch production, so we have this tendency to always try to do something for the next batch, try to prevent future batches from getting the disease we have now. so having to try this and that is always very dynamic and challenging. so we know that there is always a new batch to come, and that is not the same in cattle farming. thirdly, pig and poultry vets describe their work as being part of an epidemiological rather than clinical approach to animal health, which they associate with the idea of a preventive rather than curative or therapeutic approach to diseases. this conception is closely linked to the two previous points. on the one hand, it refers to the professional proximity that veterinarians maintain not only with their clients but also with technical advisors, often employees of the co-operative to which the farmer belongs or sometimes of a feed mill company. indeed, in pig and poultry farming, technical advisors play an important role as they visit the farms much more frequently than vets. although their role should not involve animal health issues (but rather feeding, housing or husbandry practices), they tend to conceive these aspects as a whole, as do vets when they look at the technical factors (rather than biological and medical ones) of diseases. animal health is thus the work of a professional trio, whose theoretically distinct roles often overlap, encouraging situations of cooperation and also sometimes conflict (adam et al. ). on the other hand, and consequentially, this so-called epidemiological conception of animal health establishes a form of activity which is not limited to a clinical and individual approach to pathology but which, on the contrary, opens up professional expertise to areas often considered as not specifically veterinary, such as hygiene, nutrition, zootechnics or biosecurity. the knowledge and the toolbox of industrial vets are thus extremely varied and cannot be limited to surgery tools or prescription booklets. the veterinarians interviewed often emphasized the "evidence-based" side of their professional expertise (and legitimacy), which is based on the collection and analysis of various data and promotes intellectual stimulations regularly renewed by the diverse situations they must face. they believe all these elements to be part of preventive approaches to animal health and they tend to perceive themselves as health advisers, or "health managers" as thoms ( ) has shown in the case of german poultry vets. i graduated from veterinary school in . then i got a degree in epidemiology. what i realized and particularly interested me during my studies was the difference between individual medicine and herd medicine. so with my education and also my family farming background i realized that individual medicine was more a cost than a profit. so i tried to develop this kind of herd care and preventive approach. i started my professional life in rural medicine in a region that was quite a pioneer in this type of approach, especially regarding reproduction. we have audit grids and we have exploration tools. for example, i have a device to measure co , to dose carbon monoxide. i have a ph-meter, a conductivity meter, a laser. i have a burette to measure the water flow in the feeding system. i have a light meter in case i need it. i have a camera to explore water pipes. i always have smoke bombs in my car to check ventilation in the buildings. so really, we have exploration tools in the form of scissors and gloves to perform autopsies but also equipment for managing the buildings. but aren't you stealing work from the technicians? technicians do autopsies (laughs)! no, it's true that controlling ventilation doesn't mean that i am able to deal with the farm it system. so technicians keep their skills. but i consider that it is my job to explain to the farmer that if he has a colibacillosis it's not because a germ fell from the sky but because his ventilation system doesn't work the way it should. that's part of a vet's job, even though it is the technician who is able to fix the problem. we are doing what i might call an etiological or epidemiological diagnosis. fourthly, the characteristics of pig and poultry medicine must also be related to the type of animals with which these vets work on a daily basis. without necessarily following the epistemological principles of the "animal turn" promoted by certain branches of the social sciences (guillo ) , which consists in analysing the way in which animals themselves shape human interactions or the socio-technical devices within which they take place (notion of "animal agency"), it must be noted that taking care of chickens or pigs has different implications than is the case with cattle, dogs or cats. two elements seem central here and are directly related to the characteristics of pigs and poultry and the farming systems to which they belong. on the one hand, these animals have a relatively short lifespan: only about days for a conventionally raised chicken (between and days in organic or label farming) and about months for pigs (the sows being slaughtered after or years). it is therefore very different from a dairy cow that must remain healthy and productive for an average of years, or pets that live or years, sometimes more. in these conditions, which obviously also depend on the economic structure of the agri-food industry, animal health becomes part of biological and temporal dynamics where the slightest disorder may have pathological consequences that might be seen as "just-in-time diseases", i.e. health issues directly related to, or shaped by, the sociotechnical and economic infrastructures (including the animal bodies themselves) within which they emerged (allen and lavau ) . on the other hand, the health of chickens and pigs is not considered individually, but rather from a population perspective. these are animals that are reared in batches and it is the group of animals that constitutes the epidemiological reference unit for both the vet and the farmer. in certain cases, particularly in poultry farms where the production cycle is very short, many decisions are therefore made not for the current batch but for the following one, in order to avoid repetition of the same problem. overall, all of these aspects are typical of industrial veterinary medicine and, therefore, of this form of expertise and professional legitimacy that can be qualified as preventive veterinary medicine. to sum up, and without prejudging whether or not these elements can be found in other segments of the veterinary profession, we can say that poultry and pig medicine is characterized by (i) a vocation; (ii) a technical and preventive approach; (iii) tripartite work; (iv) animals and farming systems that "call for", or co-construct, this form of expertise. however, when listening to veterinarians talk about their profession, this way of working on industrial farms does not seem to be linked to the rebuilding claimed by the profession since its incrimination in the amr problem, even though certain policy measures recently implemented may encourage the development of this preventive and evidence-based approach, such as the obligation to perform antimicrobial susceptibility tests before prescribing critically important antimicrobials (bourély et al. ) . indeed, the roots of this particular form of veterinary expertise can be traced back to the mid- s/early s, when some pioneers began to specialize in pig and poultry farms-at that time during a massive industrialization and intensification process. the origins of preventive approaches in "industrial" veterinary medicine during the s and s in france, particularly in brittany and pays de loire, poultry and pig farms expanded rapidly (nicourt ) . the mixed crop-livestock model was gradually being replaced by specialist farms which were developing through a twofold process of intensification (increased herd size, confinement and containment of animals, rationalization and (bio)technicization of husbandry methods such as genetics, feeding and pharmaceuticals) and industrialization (concentration and vertical integration of the food chain's stakeholders, and taylorization of farm labour) (diry ) . this movement led to the emergence of new needs in terms of health management. firstly, at that time veterinarians knew little about these animals in terms of medical knowledge or techniques (poultry and pig medicine was rarely touched upon in veterinary schools back then). secondly, the confinement of animals in enclosed buildings led to outbreaks of disease, especially-but not only-infectious diseases which were unknown or at least whose management methods were no longer appropriate. a small number of vets then began to specialize in this type of production, viewing it as a promising and developing market. poultry farming was not taught at all. poultry diseases even less so and the prevention of poultry diseases even less than that. so my gateway to poultry farming was the technical-economic approach to things, and not the purely pathological approach, which i could have done at the time on sheep or cattle. except that there, i was in a completely capitalist system. as you can see, one can adapt to anything (laughs). in the pig sector, it was mainly vets employed by agricultural co-operatives who embarked on the adventure, supported in particular by the creation of the station de pathologie porcine (spp) (national veterinary laboratory specializing in pig health) in ploufragan (north brittany) in (fortané ) . in the poultry sector, which is structured more around industrial groups than around co-operatives, there were more independent vets who were orienting their practices towards this type of clientele. what these veterinarians had in common was the conviction that they could no longer do their job in the traditional way, i.e. as emergency "rural" vets, alone or in partnership in small practices serving a diversified clientele (cattle farmers and pet owners in particular, with a few occasional interventions on poultry or pig farms). moreover, they were convinced that their job was to accompany the development of intensive and industrial livestock farming by providing services adapted to the specific animal health issues of this sector, and that their own organization had to follow and mimic the development of the industry they were working for. the term "industrial vets" therefore refers not only to the kind of clientele they were (and still are) working for, but also to their own state of mind and conception of veterinary medicine, as a profession and as a business. in fact, we were the defenders, the propagators of intensive livestock production. we never denied that, that's what we were employed for. we were just saying "ok, but the conditions for success are this and this and this". at the time, there was a double-digit growth in the sector, so we said: "we must stick to the development of this sector". we were focused on poultry, we had almost given up on pigs. we said: "we have to stick to the leaders". very quickly, in - , we said: "it's x [one of the biggest poultry industrial group], we'll stick to x, we'll stick to the growth of x, like a leech, we'll never let it go". that was our aim in - and it became concrete a little later when we were working for x, even though we weren't their official vets. (…) we had developed well in - , so how could we consolidate our system, how could we really sell it, how could we duplicate it? this is where the notion of "global offer" began. here, you find three activities: the veterinary practice, including advice and training, the medicines and the analysis. when you come here, you have this "global offer": the lab part, the drug part and the advisory part. the way this pioneering generation of pig and poultry vets worked was characterized by their capacity and interest in conceptual innovation and do-it-yourself techniques for responding to the often unknown situations they had to face. on the one hand, it concerned the development of pharmaceutical products adapted to these animals and their husbandry conditions, at a time when the pharmaceutical industry was producing very few medicines for this type of livestock, not considering it to be a worthwhile market. it was therefore not uncommon for veterinarians to order pharmaceutical raw materials (rather than manufactured drugs) in order to themselves prepare a product that could be effective in terms of both pharmacological (e.g. combining an antibiotic and an anti-inflammatory) and galenic properties (e.g. designing a drug in the form of a powder to be spread in bedding rather than to be mixed with food-for dermatological infections in particular). in - , we had outbreaks of staphylococci. obviously, the animals caught it very early because they already started to have small pimples at - days. so the mother was a carrier, so we thought: "what if we use an antiseptic powder?" then we thought: "we need a powder that is very powdery, that adheres well to the skin". we worked on the galenic, excipients and everything. we developed a powder and tried it and the farmers said: "this works well". who were you doing this with? we made the powder ourselves. we had a mixer. in our pharmacy, we had a manufacturing workshop. (...) it was common at the time. we had the right to make extemporaneous preparations that were the result of our prescription and our imagination. on the other hand, this broader conception of the veterinary role could be seen in the holistic vision of health that these professionals were promoting. most of the time, they combined therapeutic intervention with bacteriological analysis and research on animal nutrition (some of these vets were also employed by feed mills). all of the larger practices of this pioneering generation developed a laboratory, sometimes a makeshift one, in order to perform the autopsies necessary for bacteriological tests. x [a pioneering poultry vet] had a very solid clientele of horses and cattle at the time, but he was interested in birds. (...) when the first industrial poultry farms, the large flocks of to chickens, settled in, he moved towards that. very quickly, he realized that the bigger the batches of poultry became, the more the diagnosis had to go through the laboratory in order to be accurate -at least regarding bacteriology and parasitology. since he's a guy who doesn't want to do things by half, he thought: "i can't make a laboratory like that, we suck as vets, i need training". he said to his partner: "i will go to pasteur". further education at that time was unimaginable. (...) then, when he came back, he started his own lab and that was really the beginning of the adventure. in the pig sector, it is interesting to note the extent to which the history of the ploufragan station is still rooted in the north brittany territory, as many generations of veterinarians, still today, have done part of their training there or continue to rely on the expertise of spp's epidemiologists or microbiologists in their daily activity (especially in the case of outbreaks of infectious diseases or to set up clinical trials). oh yes, with regard to emerging diseases, i often contacted the station when we saw pathologies that seemed new to us. this was the case for many diseases. there was streptococcal, there was respiratory coronavirus. at the spp, i called [the vet in charge] and the others, they were not convinced because at that time there was still pseudorabies in the farms. i had to talk to [a pharmaceutical company] about it and then the spp became interested. indeed, veterinary researchers from the spp had developed a preventive approach to animal health called "ecopathology" that had considerable success among pig vets and farmers in the s (fortané ) . it was mostly based on an epidemiological conception of animal health (disease outbreaks are related to multiple variables, particularly "technical", i.e. non-clinical ones), and was adapted to the specific issues and husbandry conditions of intensive livestock farming. in this regard, the type of preventive veterinary medicine which was developed at that time in france would seem to have similar characteristics to that which had flourished in the uk slightly earlier (from the s to s) (woods ) : animal health has to be conceived at the scale of the herd (and not the individual animal), be articulated to non-medical matters such as feeding, housing and genetics, and integrate the economic issues of performance and profitability within the advisory support vets provide to their clients. the most important difference during the process of institutionalizing these preventive approaches in france and uk seems to be the role played by public authorities. while in the uk, the state firmly supported the development of preventive veterinary medicine as a way to accompany the modernization of british livestock farming, in france, except for the spp which was partially founded by local authorities and state veterinary services, most of the preventive approaches that flourished in the s were supported by the private engagement of pioneering vets trying to respond to the challenges of a growing industry and, in the meantime, to capture the market of "industrial" veterinary services. all in all, the point here is not to say that contemporary pig and poultry vets are nowadays using the exact same techniques and knowledge of the preventive approaches developed in the s and s, or even to suggest that all industrial vets of that time were doing exactly the same. it is rather to demonstrate that this type of veterinary medicine has at least a to -year history in these sectors and that some aspects of this can still be seen in the way that modern industrial vets continue to work (and to perceive their job and professional identity), such as the importance of technical expertise, epidemiological knowledge and bacterial laboratory-in other words, that a diversified form of veterinary expertise is required. even though this history has more or less vanished from the profession's official memory, a few unconscious vestiges of it can still be seen in recent amr debates. for example, during the campaign promoting the amr policy in the pig sector in , an implicit link was made between this pioneering medicine and the kind of expertise which is now considered to be the future of the profession and through which veterinarians tend to legitimize their role as the guardians of antibiotics. this is demonstrated by the utilization of the ecopathology icon which expresses the holistic conception of animal health (namely the so-called "ploufragan hexagon") to promote good veterinary practices in antimicrobial use in livestock (fig. ) . all these elements show that what is now called preventive veterinary medicine and which is perceived, within the political context of the amr problem, to be the model of professional expertise towards which veterinarians must turn, is not fundamentally new, at least within the knowledge and practices of pig and poultry vets. but what about the economic model on which this kind of expertise is based and how it is actually related to the use of antimicrobials? although it is in fine difficult to associate the development of preventive approaches with the reaction of veterinarians to their incrimination on the amr issue, contrary to what the sole examination of professional discourses in the press or in various it should also be noted that in the uk the development of preventive approaches concerned farm animal medicine in general (so mostly cattle medicine), while in france this movement was located in "industrial" pig and poultry medicine -although due to local history it would seem that in some regions other forms of preventive medicine were also developed in a small number of cattle veterinary practices as from the late 's (combettes et al. ) . this is indeed the purpose of talking about "preventive approaches" in the plural, i.e. to highlight the fact that these approaches could be rooted in different local histories or individual trajectories yet still share some common principles. while pig vets were mainly referring to ecopathology by virtue of their link with the spp, poultry vets did not use a specific term (preventive, holistic or epidemiological approach, global offer, etc.). nevertheless all industrial vets were (and still are) referring to what they were (are) doing with the same kind of contrasting schemes: epidemiological vs clinical, preventive vs curative, herd vs individual, technical vs medical, etc. which i consider to be the common ground of "preventive approaches to animal health" with, in addition (cf. next section), an economic model based on free-of-charge services funded by drug sales and aimed at "capturing" clients. institutional spaces relating to amr policy-making might suggest, nowadays this model has nevertheless become more visible and contributes to the re-legitimation of the veterinary profession (badau et al. forthcoming) . however, the idea that preventive approaches will make it possible to move away from the economic model placing antibiotic sales at the heart of veterinary income seems more dubious, as the development of said preventive approaches was in fact based on this very system. by the notion of economic model, we mean the way in which veterinary services are monetized. one of the key elements of the criticism against veterinarians with regard to the amr issue has been the fact that their sources of income are largely dependent on drug sales (antibiotics in particular). however, whereas professional discourses seek to explain that the development of a preventive medicine in which antibiotics no longer hold a central place will make it possible to change the "business model" of veterinary practices, it is on the contrary clear that this economic model based on drugs sales has in fact allowed these preventive approaches to flourish. even though it is difficult to concretely assess the accountability of veterinary practices due to a lack of available data (in particular if we wish to distinguish between different segments of the ploufragan hexagon: animal health is dependent on six variables (tillon et al., ) prevenƟon campaign against anƟbioƟc misuse in pig farming, fig. the common principles of past and present preventive approaches in industrial veterinary medicine. -ploufragan hexagon: animal health is dependent on six variables (tillon ) . this famous diagram represents the way animal health (and economic performance of the farm) is conceived within the ecopathological framework: it is correlated to six variables, namely the farmer, the animal, housing, feeding, microbes and husbandry practices. at that time, it was considered quite innovative to claim that veterinary expertise should take into consideration all these aspects and not just focusing on animals and microbes (fortané ). -prevention campaign against antibiotic misuse in pig farming, . this poster used in the amr campaign perfectly reproduces the six "ecopathological" variables (here referred to as the six "pillars" of animal health). it says: "no more antibiotics than needed. with my vet, i manage the health of my animals while limiting the use of antibiotics". this poster therefore illustrates how preventive approaches within veterinary medicine are deemed to be a solution to reduce antimicrobial use profession), we can nevertheless rely on estimates from various literatures. a veterinary thesis on the structure of the drug market considered that drug sales were generating to % of the income of farm animal practices (rivière ) . thirty years later, in the midst of the amr debates, a report by the french food, agriculture and rural areas council estimated this figure to be around % for farm animal vets in general, and up to % (including a high proportion of antibiotics) for industrial vets (dahan et al. , p. ) . so one cannot help but wonder what happened during the three last decades and to what extent this evolution of the economic model of veterinary activity has in fact supported the development of preventive approaches to animal health, contrary to the prospective narrative the profession has constructed to defend its role as the guardian of responsible use of antibiotics. the answer is actually quite simple, and is a classic case in agricultural economics: the sale of inputs funds the advice. in the case of industrial vets, the context in the s and s favoured the emergence of strong competition between practices seeking to capture the growing clientele of pig and poultry farmers. this competition was also heightened by the fact that the supply of veterinary goods and services was still highly heterogeneous, due to the non-existence of standardized pharmaceutical, hygienic or nutritional products adapted to the new health problems caused by the intensification of animal husbandry. consequently, pharmaceuticals, which are the only product on which veterinarians have a monopoly for prescribing (and therefore advice) and dispensing (and therefore sales), became the main means of ensuring client loyalty. since the act, the veterinary drug market has thus been developing into a captive market, where drug sales have gradually become the only monetized exchange between vets and farmers, funding all the services actually offered by veterinarians (visits, diagnosis, analyses, prescription, audit, etc.) (bonnaud and fortané ) . indeed, preventive veterinary medicine developed on the basis of this economic model which corresponded, on the one hand, to that of the few pioneering independent practices which owned bacteriology laboratories, small drug manufacturing factories or wholesale companies and, on the other hand, to that of the agricultural co-operatives which the act established as lawful drug suppliers and which, until , employed veterinary practitioners in this regard. indeed, it was precisely by providing a whole range of "free of charge" services (visits, advice, vaccination, etc.) that these vets were able to disqualify their competitors, i.e. their "emergency" or "fire brigade" colleagues who offered little advice but were able to monetize their occasional interventions. yet were the farmers prepared to pay for their visits, as they would have done with rural vets at the time? because, even the independent vets specializing in group pathologies, so pig and poultry, like those from x [one of the pioneering veterinary practices in industrial medicine], most of the time, they did not charge for their visit, their visit was paid through drug sales. in fact, most farmers were smart enough to understand that if they were satisfied with the services of a vet or a veterinary practice, they should buy drugs to pay them indirectly, even if there were price differences. i think it was like this: "you do me a favour, i need you, you treat, you prevent, we work together, i buy drugs". it was indirect payment. (…) i've always thought that the best way to ensure farmers' loyalty is to be efficient. then they will not even talk about drug prices. there was a time when vets were very active on the farms. if farmers were happy with our services, [they didn't care about] the price of the drugs. at one point, we were a little cheaper but we weren't always cheaper. we were also known for being reasonably priced, so that there wasn't too much competition. sure, we weren't the cheapest on the market, but that's not the issue. but i'm convinced, and i know that's the way it was for a lot of people, the best way to keep them was to meet their expectations. their expectations were simple: "i have problems, i have to solve them, help me solve them, if it takes time ok it takes time". it was up to us to meet farmers' expectations in terms of health. if the farmer was satisfied, the rest would follow, he would take the feed, the genetics, the drugs. we had an indirect commercial role, but we could only do it if we were efficient. (…) but some of them stopped buying our drugs. they told me: "you're too expensive". so i answered: "if we are too expensive, you go elsewhere, but you no longer have the services". what does he want? a cheap price, but with a service that will be what it will be, it may be very good, ok, but the farmer is free to choose. however, he fully understood that he could not ask us to come ten times a year or twenty times and then buy zero medicines. moreover, i tell most people: "we provide you with important services, we know that you also like to work with other people from time to time, at x for example, so don't buy everything from us, but buy a little from us, as we come to see you regularly and we need a return". and then everything was fine, they bought some elsewhere and some from us. the construction of a captive market is therefore a central dimension of what i call here "the economic structures of professional expertise". the development of preventive approaches within industrial veterinary medicine is not only the result of the emergence of new demands or needs from farmers, but it is also and above all the consequence of the intra-and extra-professional competition that veterinarians have had to face after the act. prescribing and dispensing pharmaceuticals, combined with a holistic expertise, is a means of capturing a clientele through a simple and exclusive form of economic contracting (the sale of drugs) while providing a wide range of services (diagnosis, advice, etc.). it is indeed the articulation of these two dimensions that makes it possible to prevent, or at least reduce the risk, that clients are captured by a competitor. in this sense, preventive approaches to animal health must be read simultaneously as a professional and a business model, these two dimensions being constitutive of each other. such economic structures of veterinary expertise have been observed in another context, that of the usa in the s and s. smith-howard ( ) shows how american veterinarians, who did not have a monopoly on the sale of medicines, gradually established themselves as the main distribution channel by coupling their prescriptions with preventive services that provided real added value to farmers, compared with pharmacists or other retailers who were merely supplying the drugs. a linguistic distinction has even emerged between "dispensing" and "merchandising", contributing to (re)establishing the legitimacy of veterinarians on a new basis. in the end, it is therefore in the light of this link between professional expertise (in the sense of type of knowledge and services) and business model (in the sense of how these services are economically valorized) that we must question the current context and controversies surrounding amr. it seems finally a little too simplistic to support the thesis of the professional conflict of interest that human health actors have mobilized to point out veterinarians' responsibility in the amr problem, because of their dual monopoly on the prescription and supply of medicines. in fact, the structure of the veterinary drug market has set up the sale of pharmaceuticals as a quasi-unique way of making professional expertise profitable, even though this expertise was already based on preventive approaches (and thus diversified services) for quite a long time. the current challenge for the veterinary profession is therefore less the development of a preventive medicine than the renewal of the economic model on which it has been based until now. in this regard, it is actually interesting to note that the vets i met in this study clearly mention this issue and have already started to develop strategies to deal with it. that's exactly the issue we now have with my colleague. i'm right thinking this. historically in poultry, no charge is really made for visits. overall, payment is made through [the purchase of] medicines, which is, for me, not a bad thing if it is not excessive. at some point, when we take the example of human medicine, you pay a doctor, you pay a pharmacist, everyone is happy because there is healthcare security. the day there's no healthcare security, i don't know what we're going to do. so that's what i explain to farmers: "the day you have to pay a vet and a pharmacist it's going to cost double". so at the moment, with this system, you only have one cost, so in theory that's good. indeed, there can be misuse, someone who would systematically prescribe medicines even if there is no need. this is clearly an issue and it is difficult to explain, that's our problem. but honestly, every morning when i stand up, i don't say to myself: "i will prescribe kilos there, kilos there and kilos there, and the day is done". that's absolutely not the way i think, but i can understand… and i understand when people are telling me: "yes, but if you sell, you earn". that's all there is to say. it's been on my mind for a year. firstly, particularly in a context where profits on medicines are now more strictly controlled (and therefore more limited), it seems essential for the veterinary profession to be able to charge for services that were previously funded by drug sales. this is the case in particular for visits, technical advice and bacteriological analyses (the latter were generally charged but often below the real cost-which could be high for the veterinary practice that has to employ laboratory technicians). secondly, there are strategies consisting in investing in hygiene and/or nutrition product factories (in particular disinfectants and food additives), in order to be able to sell products other than just pharmaceuticals. it is a strategy of diversification of the professional and economic activity that pioneering independent practices or certain cooperatives employing veterinarians have been using for many years, but which is now tending to become generalized among industrial vets due to the development of franchised practices. company x is a part of group y [a pig co-operative]. [we sell] zero drugs. there is hygiene, so disinfectants and detergents, and also nutrition and probiotics, i.e. products that can be added to food or that farmers can put in drinking water. you have suppliers for these products or you manufacture them yourselves? there are products that we make ourselves, otherwise we choose products from everything we know, and from people we work with. we are doing clinical and zootechnical trials as well. we've written some articles, i did one on swine haemorrhagic dysentery. we did an international article in which we showed that we can prevent it with a certain probiotic. it allowed me to go and see farms in portugal, spain and england as well. i even went to cuba for x. trying to sell products? to preach the good word! we hold meetings on a given theme, for example it might be digestive issues, and we talk about the ecopathological approach. rather than using this or that antibiotic, there are other things that work, and we know this because we have experience with farmers from y. thanks to this experience, and because there were disastrous situations where antibiotics no longer worked, we can tell other veterinary colleagues [that these products work well]. a third strategy consists in developing various forms of contractualization with the clients. for example, the vet interviewed below tries to imagine an annual flat-rate model that would cover all veterinary services, from advice to medicines. this would be based on a form of insurance, where some farmers would ultimately pay a higher price than the actual cost of the services they have received, while others, particularly those facing more health issues, would pay a lower price (although the reverse might be true from one year to the next). in fact, this system is similar to the one set up by the so-called "veterinary groups under contract" in central and south-eastern france, but which was only developed in areas where livestock farming is not widespread i.e. in small and medium-sized cattle or sheep farms connected to local markets; this system is mainly associated with alternative agricultural projects resulting from s protest movements (combettes et al. ) . it would be interesting to know the extent to which such a model might be easily generalizable in the heart of an industrialized agri-food system where farmers are involved in more complex and constraining chains of interdependence with upstream and downstream industries. this trend resonates with certain dynamics currently observable in the uk where farm animal vets are also concerned about the sustainability of their economic model and are trying to develop similar forms of contractual veterinary services, although less than % of the clientele of the practices trying to develop these kinds of contractual schemes have adopted it so far (ruston et al. ). the economic model and the type of goods and services that veterinarians can provide are nevertheless now considered to be key criteria for the sustainability of veterinary businesses (henry et al. ) . so i've been thinking for a year. now i'm going to test a new system, a comprehensive flat-rate package. i'm testing it, it's brand new, with two or three farmers. this is a complete veterinary follow-up, which includes visits and medicines. we don't talk about fees anymore. [...] so i don't know yet. my package system might be good in theory, maybe not good if farmers... because you know, on the farms, there are major variations in antimicrobial consumption. at some point the package is going to be an average. some of the farmers will find my services a little bit too expensive whereas others will like it, but yes perhaps major antibiotic users so it might be a bad influence for them. i'm not claiming victory yet, but i'm trying to find another system. a fourth strategy consists in monetizing certain veterinary services no longer to farmers, but to co-operatives. this relates to at least two types of activity. first of all, it concerns the follow-up of "herd health plans" that co-operatives are obliged to set up if they want to be approved for selling veterinary medicines, in accordance with the act. it is of course an activity that has existed for a long time, but that many cooperatives delegated to their employed vets until . the second activity relating to this strategy is linked with the development of new roles for veterinarians, in particular those of standards controllers or certifiers. indeed, with the development of "antibioticfree" labels, more and more co-operatives are asking vets to help them implement such standards. for example, vets have to perform "pharmaceutical audits" in an attempt to recruit farmers who can comply with such specifications, or to set up protocols to monitor antibiotic use and help farmers reduce it. when the co-op announced its project [antibiotic-free pigs], some farmers called me the day after, or i called them, to be part of this project. so for those who were still using antibiotics in feed there were still some stages to go through but when a farmer calls you, you think that you're going to need him for the project so you have to find alternatives. you have to know if he really needs antibiotics and how to reduce them. so this is where you have to set up a procedure, to find ways to identify the flaws… and in the end this is the perfect way to familiarize farmers with this issue. (…) well the farmer has to be motivated but this is where the bill of specifications helps, because there is an added-value. and there is also glory and self-satisfaction because the added-value isn't much, but there is both. yet these new roles for veterinarians, or more exactly these new ways of monetizing their expertise, are associated with a redefinition of their professional identity that vets are not always comfortable with. while being a health advisor rather than a clinician is perfectly in line with their preventive conception of veterinary medicine, becoming a sort of controller and sometimes even a sales representative in charge of enrolling and accompanying farmers in quality insurance schemes is not necessarily easy. this difficulty has also been noted among uk cattle vets who felt conflicted between their role of practitioner (within the framework of their relationship with their clients) and their role of "enforcers" of biosecurity practices (while implementing policy measures to prevent bovine tuberculosis), although in this case they were acting on behalf of the government and not for market schemes of private stakeholders (enticott ) . the vet interviewed below explains the practical and ethical adjustments he has had to make to accommodate this new way of valuing his professional expertise, and the limits he personally sets to remain in what he considers to be the role of a health advisor. so luckily we have some experience [with following up on bills of specifications], especially with welfare standards. in this standard, the farmers have to use the co-op's animal feed. so our experience allows us to recognize the feed bags. and one day i was on a farm visit, and i saw a feed bag. and i thought "but this is not the feed from the co-op?". because i know that it is normally small granules, and this time it was big granules. so here, clearly, the farmer is not complying with the bill of specifications! so you see, if you look carefully, you can see things. well you can't see everything, especially when farmers don't want you to know… because in this case he even didn't think about it. leaving the bag like that… it is sure that he's going to be in trouble with the co-op. but you won't see the ones who really want to cheat! and in this example, what happened in the end? you just let him know, asked him to be careful, and let it go? or might you impose sanctions at some point? well that's not for me to decide. but the question for me is whether or not i want to report someone. one day the co-op boss asked me [about the co-op's granules] but i replied that i didn't have time to check whether every farmer in the quality insurance scheme actually uses its granules. (…) but no, what i do, mostly, is warn the farmer. i tell him that he has been found out. and i tell him that the coop is fairly meticulous and that if they find out they will take him off the scheme. so for a few months he will lose cents and he won't be happy. so that's the situation. and they are absolutely capable of doing it, without any doubt. so this is what i do, i warn him but i don't think it is my role to report him, or to impose sanctions. conclusion "if i change, i may lose my clients. but if i don't change, i might lose my job" by cross-examining the development of the drug market and the professional expertise of french "industrial" vets, this article has aimed to articulate reflections from the sociology of professions and the anthropology of medicines. the amr problem is a very interesting case-study through which to make such an articulation fruitful. indeed, numerous works of research in the anthropology of medicines invite us to analyse drug use through the lens of the global circulation of pharmaceuticals and not just by focusing on the practices and knowledge of end-users, be they farmers or patients (whyte et al. ; petersen ) . antibiotic use cannot therefore be disconnected from upstream practices (or pharmaceutical "life stages", as anthropologists of medicines would call them) such as production, distribution, prescription and sale. though little is yet known about the structure of the veterinary drug market, we have shown that as key actors at the heart of the veterinary drug chain, involved in many areas of this global circulation of pharmaceuticals, veterinarians play a major role in the regulation of antibiotic use-particularly industrial vets who are not only involved in prescription and delivery but also in some cases in production and distribution. this result is therefore interesting from the point of view of the sociology of professions as it shows that the "jurisdiction" (abbott (abbott , of veterinarians is certainly more complex than them being mere practitioners of animal healthcare. indeed, when we look at the veterinary profession as a whole it becomes quite obvious that veterinary knowledge is involved in many domains of animal health and food safety, from farm to policy and industry fortané , ) . what this article shows, in addition to describing the specificities of a relatively unknown segment of the profession, is that this highly diversified form of professional expertise, and therefore the variety of areas in which it can be exerted, can also be concentrated within a tiny group of professionals, namely pig and poultry vets. moreover, even though it still has to be more broadly demonstrated, it would seem that the trajectory of this particular segment, characterized by the development of specific approaches and economic models, is somehow quite representative and/or influential of more global changes that are now affecting the profession as a whole (at least in farm animal medicine). the expansion of the activity of industrial vets within the drug chain and the "technical" domains of livestock farming indeed corresponds to a (conflicted) evolution of veterinary jurisdictions that echoes the profession's ongoing attempts to renew itself in the context of amr controversies. all in all, articulating reflections from the sociology of professions and the anthropology of medicines has allowed us to highlight the "economic structures of professional expertise" (that is to say that professional practices and knowledge are shaped by the way they are economically valued on a given market), which is of considerable importance for the analysis of the amr problem. indeed, it underlines the fact that amr cannot be addressed by simply focusing on antibiotic use or prescription and, more importantly, it shows that the framing of the amr problem cannot be understood without reconnecting it with the global transformations of the veterinary profession (both in terms of expertise and business model). in this regard, we have shown that the veterinary profession has developed a prospective narrative to defend its role as the guardian of antibiotics (i.e. moving towards preventive approaches in order to prescribe less antibiotics and be less dependent on drug sales) that does not fully correspond to either the history or the actual practices of industrial vets. such approaches have in fact already existed for many years and it is thanks to this diversified veterinary expertise that this segment of the profession has developed. however, the economic model upon which this expertise has been based is in fact the one which is nowadays criticized within amr controversies (i.e. the sustainability of veterinary businesses being highly dependent on antibiotic sales), so that it would appear relatively doubtful, if not ironic, that the profession now brandishes preventive approaches as the undisputable solution to antibiotic overuse or misuse. indeed, drug sales were almost the only way of monetizing veterinary services in the pig and poultry farming sectors and this is how the pioneering generation of industrial vets disqualified their traditional competitors, by "enrolling" clients through the development of a captive drug market. so although promoting preventive approaches is probably the right way to encourage veterinarians to move towards a "health advisor" role where antibiotics are neither the main tool of their activity nor the main source of their income, the profession still has to develop economic models (practice accountability, contractual arrangements with the clients, etc.) that do not rely on drug sales. recent policy measures and incentives are probably pushing in this direction, as are broader trends such as the development of franchised practices and new professional roles and status, but this still has to be empirically confirmed (even though we know that over recent years antibiotic use has already started to decrease). social sciences have a major 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with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -r ygqmy authors: lapeyre-mestre, maryse; boucher, alexandra; daveluy, amelie; gibaja, valerie; jouanjus, emilie; mallaret, michel; peyrière, helene; micallef, joëlle title: addictovigilance contribution during covid- epidemic and lockdown in france date: - - journal: therapie doi: . /j.therap. . . sha: doc_id: cord_uid: r ygqmy abstract addictovigilance is a safety monitoring targeted at substances with potential for abuse and dependence. this vigilance was involved during the period of covid- epidemic due to the significant changes in access to drugs and psychological disruption caused by the pandemic and lockdown. this article aims to present the different steps implemented by the french addictovigilance network in collaboration with the french health authorities from march to may , including monitoring of potential harmful events, and scientific communication. the first events were identified through the continuity of the networking between the french addictovigilance centres and their partners: community pharmacies, general practitioners, specialized structures and emergency wards. as soon as the lockdown began, first cases of overdoses (lethal or not) were reported with opioids, mainly with methadone, and other opioids (heroin, oxycodone, tramadol or antitussive codeine). lockdown-related noteworthy events consisted in clinical cases or other relevant information for which lockdown clearly played an important role : among the many substances identified at least once, pregabalin, benzodiazepines, cannabis, cocaine and nitrous oxide were the most significant in terms of prevalence, seriousness or particularly specific to the lockdown context. despite significant decrease in the activity and travel limited to vital needs, community pharmacies continued to identify falsified prescriptions in this period, highlighting an increase in suspicious requests for pregabalin, codeine and tramadol. in parallel, the french addictovigilance network continued its communications efforts in the period, issuing a newsletter on tramadol, a press release on methadone and naloxone, and participating in the covid- frequently asked questions (faqs) of the french society of pharmacology and therapeutic website (https://sfpt-fr.org/covid ). covid- epidemic has been an important challenge for addictovigilance, and has proved that this monitoring is highly essential for alerting health professionals and health authorities to points of vigilance in the field of psychoactive substances. any safety monitoring system is part of a global approach aimed at identifying emergence or spread of a health risk. this health security approach involves the early detection of signals and their most rapid integration into an action system allowing an adapted, effective and early intervention to preserve the health of populations. in the context of pharmacovigilance and drug safety, new or unexpected adverse drug reactions should be detected as early as possible in order to further inform and secure the use of the drug, giving the general population and health professionals the opportunity of evidence-based information about these risks. in the context of covid- epidemic, the french regional pharmacovigilance centres network ensured this mission [ ] , with an assessment maintained in its continuity, based on a pharmacological and medical characterization of cases, shared with a population-based approach integrating pharmacoepidemiological methods when possible, contributing to optimizing the level of evidence. sharing and collaboration, both within and beyond the french pharmacology and therapeutics scientific community, was integral within these special weeks and beyond (see frequently asked questions [faq] at https://sfpt-fr.org/covid ) [ ] [ ] [ ] . in the addictovigilance context, the field is even wider and more heterogeneous [ ] [ ] [ ] . in the first weeks of epidemic spread, most of the interrogations were related to the disease itself and to drugs with supposed antiviral properties or interactions with the immune system. concerns about substances of abuse appeared as soon as lockdown occurred in france on march , . this article the french addictovigilance network was set up in the s, in order to benefit from a proactive vigilance system targeted at substances with potential for abuse and dependence (except tobacco and alcohol), and to participate in a proactive and coordinated manner in the activities of the world health organization (who) expert committee on drug dependence [ , ] . this vigilance is based on spontaneous notification by healthcare professionals of any serious case of misuse, abuse and drug dependence involving psychoactive substances, regardless of their nature or status [ , ] . in addition to this passive monitoring subject to under-reporting, other sources of information have been developed to improve vigilance: systematic data collection on falsified prescriptions from pharmacies ("ordonnances suspectes indicateur d'abus possible", osiap survey) [ , ] and on secure prescription forms for narcotic drug prescriptions ( "antalgiques stupéfiants et ordonnances sécurisées", asos survey) [ ] , systematic data collection from patients seen in addiction specialized structures ("observatoire des produits psychotropes illicites ou détournés de leur usage médicamenteux", oppidum survey) [ ] , analysis of toxicological data on chemical submission [ ] or on deaths in a medico-legal framework ("décès en relation avec l'abus de médicaments et de substances", drames survey) [ ] . addictovigilance can broaden the assessment of the potential for abuse and dangerousness of substances by specific analyses on large databases from the national health data warehouse [ , ] , or on ad hoc field studies [ ] [ ] [ ] [ ] [ ] [ ] . the identification of a potential signal from one or more of the sources described above makes it possible to anticipate an emerging problem and to assess its magnitude using a multi-source approach (fig. ) [ , ] . on march , in his first address on the extend of the epidemic in france, the french president announced, in a message broadcast to the nation, the implementation of travel restrictions, lockdown, and a state of emergency involving the redeployment of the entire healthcare sector to prioritize covid- care from the following day. among the different measures launched by the government, several ones were intended to ensure continuous access for care, while limiting outing to what was strictly necessary (urgent medical care). in these conditions, both public and private medical sectors (general and specialized practitioners, nurses, most of other health professionals) decreased their activities, together with addiction specialized structures, in order to insure social distancing and prophylactic barrier measures to reduce the risks of viral contamination. for example, in many areas in france, several first line harm reduction structures [ ] and addiction specialized centres modified their way of functioning, with limited access hours, redeployment of nurses and doctors for covid care, remote consultations, etc., all these changes leading to a degraded operating mode. some other structures may have also closed their doors, in particular those offering conviviality space with coffee and food for homeless and vulnerable isolated people, because of the impossibility to ensure social distancing. the rules for renewing prescriptions have been modified by several decrees (the first being published in the official journal on march , [ ] ), in order to prevent the health risks related to the abrupt interruption of chronic exposure to drugs, in a context of a reduced availability of prescribers during covid- epidemic. pharmacists were invited to issue even if the period of validity of a renewable prescription has expired, within the framework of the initially planned dosage, a number of boxes per prescription line guaranteeing the continuation of treatment, for a period not exceeding one month. these measures include specific provisions concerning medicinal products liable to be abused or misused, such as anxiolytic or hypnotic drugs, opioid maintenance drugs and other narcotic drugs or drugs falling under the regulations of narcotics. along the successive decisions of the president and government over time, these decrees were intended to be prolonged during the period of the national state of health emergency. table summarizes the different situations concerning psychotropic and narcotic drugs (at the date of may , ). from march , , some important problems rapidly appeared: because of the strict lockdown and repeated controls for any outing or trip, border shutdown for all extra european countries, but also with our immediate neighbours, drug trafficking has been drastically impacted, raising fears of an increase of episodes of withdrawal syndromes in the population of drug users. opioid maintenance treatment (omt) should be considered as an essential treatment during the covid- pandemic, as significant risks to the community exist with an interruption of the stable provision of opioid treatment. difficulties for omt drug provision have been expected with permanent changes of the prescribing and dispensing rules for narcotic drugs in the first days of lockdown, leading some patients to stock large amount of methadone at home. there may be an increased risk of opioid j o u r n a l p r e -p r o o f overdose arising from i) erratic access to omt, ii) erratic access to illicit opioid supplies and iii) increased access to takeaway doses of methadone, which would have required the systematic prescription for take-home naloxone supplies [ ] . there was also a growing concern about the risk of overdose with methadone (or of accidental exposure because of lockdown and provision of takeaway methadone at home), as methadone was already the first substance involved in drug abuse-related deaths before the disease outbreak, with an increasing trend in the last years [ ] . unfortunately, despite drug approval for forms of naloxone directly available without medical prescription in , the level of use of takeaway naloxone from specialized structures or community pharmacies remains very low [ ] . psychological disturbances may occur due to the lockdown, with an increasing risk of misuse and abuse of psychoactive drugs in the population of drug users (including patients on omt particularly vulnerable to these disruptions), but also in the general population [ ] . distress may result in some people increasing their substance use and subsequently require treatment (for example, alcohol use may increase). changes in illicit drug supply may occur due to a range of complex interacting factors, with an increased demand for services. alternately, some people who use drugs may be less likely to request services during the pandemic, with an escalation of substance use during a time of distress. some not evidence-based and potentially deleterious "guidelines" were launched in order to anticipate withdrawals, with several dangerous recipes for substitution or techniques to make provisions of narcotic drugs. such practices may bring new patterns of problematic use, including access to new psychoactive substances sold on the internet, with free home delivery services for using up stocks of illicit drugs. finally, in relation with the covid- itself, concerns arose about risk of drug-drug interactions and qt prolongation with methadone potentially combined with chloroquine and hydroxychloroquine or azithromycin. actually, when infected by sars-cov- , older people, men and those with medical comorbidities (chronic pulmonary disease, cardiovascular disease, cerebrovascular disease, diabetes and a compromised immune system) present a much higher likelihood of acute respiratory distress, renal failure and death. due to the respiratory and pulmonary tropism of sars-cov- , people who smoke or vape tobacco or cannabis products were expected to be more at risk of pulmonary complications. immune-suppressed people, for example, due to hiv infection or other chronic medication conditions, are also at increased risk for sars-cov- infection. consequently, drug users with these conditions may be a subgroup more at risk. we described the different events and facts collected and observed from mid-march to may , . in the first days of lockdown, several concerns emerged in the field. the first events were identified through the continuity of the networking between the french addictovigilance centres and their partners (i.e. community pharmacies, general practitioners, specialized structures and emergency wards). by the second week of lockdown, several cases of methadone overdoses for people at home were reported, and falsified prescription forms to obtain hydroxychloroquine and azithromycin were also identified as osiap by different pharmacies on the french territory. these early signals have been transmitted to national health authorities, leading to the implementation of a weekly specific monitoring of noteworthy cases or events related to the covid- , related to the lockdown, and of all falsified or abnormal prescription forms reported through the osiap survey during the period. this weekly monitoring was closely done between the french addictovigilance network and the ansm [ ] . the lessons of this weekly monitoring by conference calls and shared minutes of the meeting are presented in the following paragraphs. the fig. summarizes the highlights of this monitoring. detecting and identifying signals are a cornerstone for addictovigilance actors: they need to be able to label a piece of information received as a signal [ , , , , [ ] [ ] [ ] . signals suggesting a public health risk are collected and analysed in continuous manner in a surveillance process implemented by watchdog or public health structures, in a perspective of alert, anticipation and early action. in this framework, a signal is defined as a piece of information concerning a health phenomenon or exposure to a risk or hazard, which requires investigation in order to validate it and decide whether or not it should be considered as an alert. the signals observed in addictovigilance may be related to human cases (unusual deaths, symptoms or syndromes grouped in clusters); to psychoactive substances or associations thereof likely to have serious health consequences (presence of adjuvants, degree of purity, novelty of the substance or its usage) and to new ways of administration or new settings of use. monitoring such noteworthy events is an important issue in addictovigilance. simad-covid was the national periodical assessment with the aim to proactively monitor and share occurrence of fatal and non-fatal overdoses due to opioid medications (methadone, opioid analgesics) or opioid substances (heroin) or other illicit drugs (cocaine). as soon as the lockdown began, first cases of overdoses were reported with opioids, mainly with methadone, and to a less extent, with heroine and other opioid analgesics (oxycodone, tramadol) or cough syrups containing codeine. until may methadone was the most reported drug among overdoses. interestingly, several characteristics of methadone overdose have emerged: i) accessibility of methadone by storage from family/friends at home was often reported ii) occurrence of overdose among opioid naïve subjects (never previously exposed to opioids or return to use after cessation) iii) occurrence among vulnerable subjects (homelessness, migrants, patients with psychiatric comorbidities) iv) methadone used outside its labelling in france, for anxiolytic or analgesic purposes iv) take-home naloxone was exceptionally used in the period. it is important to note that during this period the price for street methadone remained relatively low, suggesting continued accessibility during the lockdown period compared to illicit drugs. heroin overdoses were also observed in several areas, often among previous heroin users (around - years old) leading to severe opioid toxidromes (acute renal failure, rhabdomyolysis, haemodialysis). the same trend was observed with cocaine leading to cardiogenic complications including a patient with covid- myocarditis. overdoses were reported among young adults after tramadol use alone or associated with other drugs (cannabis) or after concomitant codeine and promethazine use (purple drank). lockdown related noteworthy events "simad confinement"" consisted in clinical cases or other relevant information for which lockdown clearly played an important role, and concerned all other substances, whatever their nature (medications, illicit drugs, diverted drugs). during the lockdown period and until may , , reports were collected by the french addictovigilance centres all over the country, including oversea territories. among the many substances identified at least once in these reports, pregabalin, benzodiazepines (including z drugs), cannabis, cocaine and nitrous oxide (n o) were the most significant in terms of prevalence, seriousness or particularly specific to the lockdown context. -first signals of abuse of pregabalin (a gabapentinoid close to gabapentin, approved for the treatment of neuropathic pain, epilepsy and generalized anxiety disorder) were reported in france from with falsified prescriptions, medical nomadism and diverted use for psychoactive effect [ ] [ ] [ ] . the french addictovigilance monitoring of pregabalin has shown, at the end of , a dramatic increase in the number of cases of abuse, with the emergence of a population of young abusers. during the whole lockdown period and then afterwards, reports came from medical doctors who were urgently requested for prescription of pregabalin by young people, often minors, including migrants. this pregabalin addiction was not clearly identified before by these health professionals, since in the recent past reports came only from community pharmacists reporting abnormal prescription of lyrica ® . during the period, several cases of overdose were reported with pregabalin, including one requiring hospitalization with dyspnoea and hallucinations in a -year-old male. -benzodiazepines and z-drugs were expected to be highly consumed during the beginning of the lockdown in france, because of social isolation or psychological troubles due to the lockdown with the potential increase of marital conflicts and domestic violence. no withdrawal syndrome was reported (renewal of prescriptions was possible along the period), but abuse or misuse (with alcohol or other psychoactive substances) were reported. clonazepam alprazolam, oxazepam and zolpidem were the most frequently reported. -several reports concerned n o indicating persistent diverted use during the lockdown due to i) a shortage of other substances in some areas and ii) a need to consume due to inactivity. on the other hand, difficulties to easily obtain large quantities of n o cartridges led a -year-old male to abuse cocaine because of his craving. during the lockdown, it would appear that home deliveries have been made easier with internet orders. neurological complications with sensory-motor axonal polyneuropathies were also observed in the period, highlighting the spread of this new phenomenon of n o addiction that has appeared in recent months [ ] . -unexpectedly, reports concerning cocaine were numerous (more over than with heroine or cannabis), while supply constraints could be considered as the same as for other illicit substances. actually, this accessibility varied according to the regions, with cocaine easily available in some ones and with a wide disparity in cocaine concentration. the above described case of switching n o to cocaine illustrates this greater availability of cocaine, with modified supply chains (home delivery instead of buying on the street from dealers). -cannabis supply was expected to be more difficult during lockdown. some patients reported withdrawal symptoms due to supply difficulties or an increase in prices, while others abused cannabis in a context of anxiety related to the outbreak. cases of accidental poisoning in children under years of age who have accidentally ingested cannabis have been also reported. in addition to these most frequent substances, other reports confirm that after a short period of waiting, the drug trade has adapted to lockdown, and cases of abuse, misuse or deleterious consequences of use were reported with synthetic cathinone -mmc (n = ), amphetamines (n = ), lsd, ketamine and ghb (n = each). finally, even if the number of reports seems quite low, it should be borne in mind that there is often a delay in reporting (cases that have occurred since lockdown break have not been reported by may , ) and that under-reporting in this area is very significant [ ] . the two first reports collected through the osiap survey concerned out of date and falsified hydroxychloroquine prescription forms (presented during the first week of lockdown), in the context of media coverage about its hypothetic efficacy on sars-cov- [ , ] . this first signal has been forwarded to the ansm at the end of march. from this date, all suspected falsified prescription forms identified by community pharmacies and reported to the addictovigilance centres were centrally analysed weekly and compared to the information collected at the same period in . as a reminder, osiap is one of the national program implemented by the french addictovigilance network in the s to record all falsified prescriptions presented to a network of community pharmacies located all over the country [ , ] . this monitoring program has been useful to identify addictovigilance signals or characterize the abuse potential of prescription drugs [ , [ ] [ ] [ ] . usually, osiap are periodically collected each year (in may and november) on a voluntary basis by sentinel pharmacies [ ] . outside these proactive collection periods, osiap are continuously reported by community pharmacies, regional health authorities or medical/pharmacy councils. the osiap intensive data collection planned for may was cancelled due to the lockdown. between march and may , , falsified prescription forms were reported by community pharmacies to the french addictovigilance network, in a context of a significant decrease in the activity and travel limited to vital needs. this frequency must be considered with caution, as falsified prescriptions are often reported with a significant delay each year. in comparison, prescription forms were collected in the same period in , including the intensive data collection in may [ , ] . fig. presents the main frequently reported drugs during the covid- monitoring by weeks, compared to the same period in (estimated through the information available on may , ). during this period, the most frequently reported drugs were pregabalin, antitussive codeine syrup and analgesic codeine and tramadol. pregabalin and codeine syrups were mainly requested by a population of young males. this profile was similar to that observed in the covid and the lockdown noteworthy events, highlighting the emergence of a little-known population to health professionals [ ] [ ] [ ] . the french addictovigilance network has published a national newsletter on addictovigilance news for several years ("bulletin d'addictovigilance"), which was issued four times in (january, april, september and october) and once in (january). table summarizes the different topics discussed in these newsletters, which highlight the emergence or confirmation of addictovigilance signals in the recent months. in retrospect, the majority of bulletins have addressed substances that had been a problem during lockdown. throughout the lockdown and then, communication by the french addictovigilance network remained active with release of new national communications. the last issue of the national addictovigilance bulletin was entitled: "limitation of the prescription period of tramadol: how did we get there". this bulletin presented a summary of the data collected in france on tramadol between and and summarized the key elements which have led in particular to limit the duration of prescription of this drug. from april , , the maximum prescription period for analgesics containing tramadol has been reduced from to months. continuation of treatment beyond months will require a new prescription. following the results of the national addictovigilance monitoring of methadone, the french addictovigilance network has published a press release on the need to maintain access to methadone during the lockdown period, while ensuring the safety of its use. methadone is a mu opioid receptor agonist indicated for the substitution of opioid dependence. in france, for at least the past ten years, it has been the most frequently retrieved substance during the toxicological analyses of those involved in deaths linked to the excessive use of psychoactive substances (drames survey). the lockdown period may increase the risks linked to exposure to this drug in naïve-opioid subjects including children and those around them not treated with methadone. it should be remembered that the potentially lethal dose of methadone ingestion in a person who has never used opioids is estimated at mg/kg body weight. the press release focused on the risk of overdose, due to the larger dispensed quantities, methadone "storage", consumption of larger quantities of methadone or other respiratory depressants (alcohol, benzodiazepines, other opioids, etc.), resort to illegal obtaining, risk of overdose in the event of resumption of methadone after a few days off, risk of serious poisoning in children or naïve subjects. the press release also highlighted the risk of qt prolongation increased because high doses of methadone itself and because of combination with drugs or substances which also modify qt: domperidone, macrolides (erythromycin, clarithromycin, etc.), antidepressants (citalopram, escitalopram), antihistamines (hydroxyzine), antipsychotics (haloperidol, quetiapine), as well as drugs currently tested against covid- in hospitals (hydroxychloroquine, azithromycin, lopinavir/ritonavir) or other psychoactive substances such as cocaine. in order to minimize these risks, the press release insisted on warning about purchase of these drugs outside the pharmaceutical circuit, and on the need to report treatment with methadone in case of hospitalization for sars-cov- suspicion. the press release also insisted on the urgent need to increase the distribution of naloxone to methadone consumers (see brochure about where and how find naloxone; fig. ). on march , , the french society of pharmacology and therapeutics has launched a national faqs website at https://sfpt-fr.org/covid , focused on the proper use of drugs during the covid- pandemic [ ] . the french addictovigilance network has joined the scientific council and has participated to document the responses to each question related to addictovigilance. one topic of the faqs was about opioid maintenance treatment, because drugs approved in this indication (methadone and buprenorphine) should be considered as essential medications during the covid- pandemic, and significant risks to the community exist with an interruption of the stable provision of opioid treatment. another topic was related to the accessibility of naloxone take home in france. another topic gave information on the risk to switch to other substances (cannabidiol or gabapentin) to manage cannabis withdrawal or to switch to opioid analgesics outside medical management for non-cancer pain [ ] conclusion covid- epidemic has been an important challenge for addictovigilance. only part of the events that took place during this period have been reported to the french addictovigilance network, and it is likely that in the coming weeks or months the number of overdoses or deaths related to substance abuse will be higher than described in this article. this is of particular concern for methadone, heroin and pregabalin, but also for cocaine and nitrous oxide which seem to be more accessible than expected in this period. this addictovigilance monitoring has proved to be indispensable for warning health professionals at the local and regional level in order to limit the risk for users, and for alerting health authorities at the national level to points of vigilance in the field of psychoactive substances. adverse drug reactions of hydroxychloroquine: analysis of french prepandemic sars-cov pharmacovigilance data off-label" use of hydroxychloroquine, azithromycin, lopinavir-ritonavir and chloroquine in covid- : a survey of cardiac adverse drug reactions by the french network of pharmacovigilance centers french society of pharmacology t. non-steroidal anti-inflammatory drugs, pharmacology, and covid- infection genesis of an emergency public drug information website by the french society of pharmacology and therapeutics during the covid- pandemic signal identification in addictovigilance: the functioning of the french system social media mining for toxicovigilance: automatic monitoring of prescription medication abuse from twitter comment on: an insight into z-drug abuse and dependence: an examination of reports to the european medicines agency database of suspected adverse drug reactions from psychoactive medicines to addictovigilance in french public health code the french system of evaluation of dependence: establishment in a legal system safety signal detection by the french addictovigilance network: innovative methods of investigation, examples and usefulness for public health medical prescriptions falsified by the patients: a -year national monitoring to assess prescription drug diversion network of centers for e, information p. survey of forged prescriptions to investigate risk of psychoactive medications abuse in france: results of osiap survey tamperresistant prescription forms for narcotics in france: should we generalize them? surveillance system on drug abuse: interest of the french national oppidum program of french addictovigilance network french network of centers for e, information on p. chemical submission: results of -year french inquiry décès directement liés aux drogues interest of large electronic health care databases in addictovigilance: lessons from years of pharmacoepidemiological contribution ten-year trend of opioid and non-opioid analgesic use in the french adult population a capture-recapture method for estimating the incidence of off-label prescriptions: the example of baclofen for alcohol use disorder in france identification and tracking of addictovigilance signals in general practice: which interactions between the general practitioners and the french addictovigilance network? parachuting psychoactive substances: pharmacokinetic clues for harm reduction medical complications of psychoactive substances with abuse risks: detection and assessment by the network of french addictovigilance centres use of new psychoactive substances to mimic prescription drugs: the trend in france identifying life-threatening admissions for drug dependence or abuse (iliadda): derivation and validation of a model les caarud, lieux privilégiés d'émergence de signaux pour l'addictovigilance arrêté du mars complétant l'arrêté du mars portant diverses mesures relatives à la lutte contre la propagation du virus covid- intérêt de la mise à disposition de la naloxone auprès des usagers de drogues pour le traitement d'urgence de surdosage d'opioïdes améliorer la balance bénéfices/risques de la méthadone en respectant ses spécificités pharmacologiques psychopathological consequences of confinement pharmacovigilance et addictovigilance dans le contexte du covid- : une surveillance renforcée detection of signals of abuse and dependence applying disproportionality analysis early signal of diverted use of tropicamide eye drops in france pregabalin use disorder and secondary nicotine dependence in a woman with no substance abuse history patterns of gabapentin and pregabalin use and misuse: results of a population-based cohort study in france drug abuse monitoring: which pharmacoepidemiological resources at the european level? warning on increased serious health complications related to non-medical use of nitrous oxide use of multiple sources and capture-recapture method to estimate the frequency of hospitalizations related to drug abuse evidence of clonazepam abuse liability: results of the tools developed by the french centers for evaluation and information on pharmacodependence (ceip) network slow-release oral morphine sulfate abuse: results of the postmarketing surveillance systems for psychoactive prescription drug abuse in france example of an investigation of an "emergent" phenomenon in addiction vigilance: the case of methylphenidate medical prescriptions falsified by the patients: a -year national monitoring to assess prescription drug diversion pharmaciens d'officine, étudiants en pharmacie et demandes de médicaments à base de codéine : étude observationnelle disproportionality analysis for the assessment of abuse and dependence potential of pregabalin in the french pharmacovigilance database detecting the diverted use of psychoactive drugs by adolescents and young adults: a pilot study site de l'association française des centres d'addictovigilance the french addictovigilance network would like to acknowledge all persons in the addictovigilance centres who participated in the active monitoring during this period (all health professionals who reported cases during the period, and persons in charge of psychoactive drugs at the ansm (aldine fabreguettes, emilie monzon, charlotte pion, nathalie richard). authors have no competing interest to declare key: cord- -q nvn n authors: chevalier, christophe; saulnier, aure; benureau, yann; fléchet, dorian; delgrange, david; colbère-garapin, florence; wychowski, czeslaw; martin, annette title: inhibition of hepatitis c virus infection in cell culture by small interfering rnas date: - - journal: mol ther doi: . /sj.mt. sha: doc_id: cord_uid: q nvn n hepatitis c virus (hcv) infection is a major cause of chronic liver disease and hepatocellular carcinoma, yet fully efficacious treatments are missing. in this study, we investigated rna interference (rnai), a specific gene silencing process mediated by small interfering rna (sirna) duplexes, as an antiviral strategy against hcv. synthetic sirnas were designed to target conserved sequences of the hcv ′ nontranslated region (ntr) located in a functional, stem–loop structured domain of the hcv internal ribosome entry site (ires), which is crucial for initiation of polyprotein translation. several sirnas dramatically reduced or even abrogated the replication of selectable subgenomic hcv replicons upon cotransfection of human hepatoma cells with viral target and sirnas, or upon transfection of cells supporting autonomous replication of hcv replicon with sirnas. importantly, three sirnas also proved capable of strongly inhibiting virus production in cell culture. one sirna, targeting a sequence that is highly conserved across all genotypes and forms a critical pseudoknot structure involved in translation, was identified as the most promising therapeutic candidate. these results indicate that the hcv life cycle can be efficiently blocked by using properly-designed sirnas that target functionally important, highly conserved sequences of the hcv ires. this finding offers a novel approach towards developing ires-based antiviral treatment for chronic hcv infections. hepatitis c virus (hcv) infection is a major cause of chronic liver disease and hepatocellular carcinoma, yet fully efficacious treatments are missing. in this study, we investigated rna interference (rnai), a specific gene silencing process mediated by small interfering rna (sirna) duplexes, as an antiviral strategy against hcv. synthetic sirnas were designed to target conserved sequences of the hcv nontranslated region (ntr) located in a functional, stem-loop structured domain of the hcv internal ribosome entry site (ires), which is crucial for initiation of polyprotein translation. several sirnas dramatically reduced or even abrogated the replication of selectable subgenomic hcv replicons upon cotransfection of human hepatoma cells with viral target and sirnas, or upon transfection of cells supporting autonomous replication of hcv replicon with sirnas. importantly, three sirnas also proved capable of strongly inhibiting virus production in cell culture. one sirna, targeting a sequence that is highly conserved across all genotypes and forms a critical pseudoknot structure involved in translation, was identified as the most promising therapeutic candidate. these results indicate that the hcv life cycle can be efficiently blocked by using properly-designed sirnas that target functionally important, highly conserved sequences of the hcv ires. this finding offers a novel approach towards developing iresbased antiviral treatment for chronic hcv infections. hepatitis c virus (hcv) frequently establishes persistent infections in the liver, leading to the development of chronic hepatitis, and, potentially, liver cirrhosis and hepatocellular carcinoma at later stages. chronic hcv infection affects . % of the world's population and is known to be the leading factor necessitating liver transplantation in patients in developed countries. no vaccine is available for hcv and the current treatment, which consists of administering pegylated interferon α and ribavirin, has limited efficacy against certain hcv genotypes, and also produces significant adverse effects (reviewed in ref. ) . the development of alternative, specific therapies for chronic hcv infection is therefore a major public health objective. hcv, a member of the flaviviridae family, contains a singlestranded positive-sense rna genome that encodes a unique precursor polyprotein; this polyprotein is further processed into structural proteins and nonstructural proteins, thereby ensuring genome replication. the long open reading frame is flanked by and nontranslated regions (ntrs) that are highly conserved among the majority of hcv genotypes and contain elements that are essential for genome replication (reviewed in ref. ). in addition, hcv ntr contains a highly structured element, namely, an internal ribosome entry site (ires), which is essential for the initiation of hcv polyprotein translation. anti-hcv drug development has been hampered both by the lack of efficient cell culture systems that support virus replication and by the unavailability of accessible animal models. hcv subgenomic rna replicon systems have permitted the assaying of the inhibitory effect of antiviral candidates on hcv genome replication in vitro in human hepatoma cells. the recent development of stable cell culture systems permitting robust production of infectious hcv particles in vitro, based on the jfh- strain of hcv genotype a, will facilitate the investigation and testing of new antiviral strategies. in addition, alternative animal models that have recently been developed seem to show promise in evaluating candidate antiviral therapeutics. these animal models include transgenic mice engrafted with human hepatocytes, as well as nonendangered primate species (tamarins, marmosets) infected by gb virus b, a hepatotropic virus that is closely related to hcv, , or by gb virus b derivatives containing functional hcv sequences. among possible therapeutic strategies, rna interference (rnai) is an attractive path to explore. first described as a natural defense mechanism against plant viruses, rnai was subsequently shown to be a universal phenomenon of post-transcriptional gene silencing, which is initiated by double-stranded rna and leads to specific degradation of homologous rnas. this process involves the generation of -nucleotide small interfering rnas (sirnas) which, in association with a multiprotein complex named "rna induced silencing complex", are used as guides to target specific rna substrates by watson-crick base-pairing. such sirnas, when introduced directly into mammalian cells or expressed from viral vectors, can lead to the degradation of targeted sequences (reviewed in ref. ). during the past few years, rnai has been extensively investigated as an alternative specific therapy to treat cancers, genetic diseases, and infections by various human pathogens of medical importance, using both in vitro and in vivo model systems (reviewed in refs. , ) . rna viruses, in particular viruses with liver tropism such as hcv, are ideal candidates for nucleic acid-based therapies that have been shown to efficiently target the liver (reviewed in ref. ) . recently, various studies have reported variable inhibitory effects of hcv-specific synthetic sir-nas or small hairpin rnas (shrnas) expressed from viral vectors, that target sequences encoding various hcv nonstructural proteins as well as sequences within the ntr. [ ] [ ] [ ] [ ] [ ] [ ] [ ] these studies all relied on the use of hcv subgenomic or genomic replicon models in cell culture. in this study we designed sirnas targeting highly conserved sequences within the hcv ntr. the efficiency of these hcvspecific sirnas was first evaluated using previously described self-replicating hcv rnas. , this allowed us to select four sir-nas that abrogated or substantially reduced genome replication. by using these sirnas in recently described cell culture systems that support the production of genotype a jfh- in order to investigate the potency of virus-specific sirnas in inhibiting hcv infection, we selected seven sirnas that target conserved sequences of the pivotal domain (domain iii) of the hcv ires within the ntr (figure a) . this domain is particularly important for initiation of polyprotein translation, as it directly contacts s ribosomal subunits and binds translation initiation factor , eif . domain iii has also been reported to be involved to some extent in genome replication. sirnas were designed essentially according to previously described criteria; in particular, whenever possible, the asymmetric thermostability of sirna duplexes was respected. , as a messenger, hcv positive strand rna genome is an ideal target for sir-nas, but it harbors strong secondary and tertiary structures, in particular within the ires. this makes it difficult to design optimal sirnas according to the criteria referred to earlier. on the other hand, the high nucleotide conservation of this region the footprints of the seven hcv-specific sirnas selected to target the internal ribosome entry site (ires) are represented on the schematic structure of domains iii-iv of the nontranslated region (ntr) from the h strain of hcv genotype a. sirna si targets a viral sequence that contains a nucleotide change in hcv genotype a and b sequences, as indicated. (b) the two replicons used in this study, ntat aneo and nneo with ntrs derived from hcv genotype a and b strains, respectively, are schematically represented, with cell culture adaptive mutations (s i in ns a and r g in ns b, respectively) indicated by arrowheads. both replicons encode neomycine phosphotransferase (neo) c-terminally fused to either human immunodeficiency virus tat protein followed by foot-and-mouth disease virus autocatalytically cleaved a protein (ntat aneo), or to a few amino acid residues from core protein ( c in nneo). permitted the identification of sirnas that were homologous to several hcv genotypes ( table ) . selected sirnas were named according to the nucleotide position within the genome of the h strain of hcv genotype a that corresponds to the first nucleotide targeted by the sirna antisense strand: si , si , si - a, si - b, si , si , si ( table ) . sirna si targets the end of domain iii and end of domain iv involved in a pseudo-knot located upstream from the initiator codon (figure a) . the sequences of all sirnas, with the exception of si , matched both hcv genotype sequences a and b that were present in the subgenomic replicons used in the first part of the study (figure b) . two sirnas si (si - a and si - b) were synthesized with sequences homologous to genotypes a and b, respectively. when targeted to genotype b ntr, si - a formed a g:u wobble base-pairing at position of the sirna antisense strand, whereas si - b formed an a:c mismatch with the genotype a replicon (figure c ). the g:u non-canonical base-pairing is known to be non-disruptive in double-stranded rna structures and both g:u wobble basepairing and a:c mismatch, if present at certain positions of the sirna, were recently reported to be well tolerated for sirnamediated gene silencing. [ ] [ ] [ ] [ ] therefore, we proceeded to analyze the effects of si - a and si - b on both homologous and heterologous replicon targets. finally, as a non-specific, negative control in these experiments, we used an irrelevant sirna, referred to as siirr ( table ) , that had previously been used for targeting a sequence within the ntr of a poliovirus strain, and that did not share homology with the hcv sequence. first we evaluated whether selected sirnas were able to inhibit the replication of an hcv subgenomic replicon (ntat aneo) in cell culture. we did this by using a previously described system that allowed simple monitoring of rna replication by measurement of an enzymatic activity, namely, secreted alkaline phosphatase (seap) activity (see materials and methods and figure b) . all hcv sequences of ntat aneo replicon are derived from the n strain of genotype b, with the exception of the ntr, which is derived from the h strain of genotype a. following coelectroporation of en - cells with μg of in vitro-transcribed ntat aneo rna and μg of each synthetic sirna, the culture supernatant was replaced daily with fresh medium during the first days after transfection and then on day , and seap activity was measured in all culture supernatants collected. during the first days, seap signals essentially reflected translation of the input rnas, since seap levels were roughly similar in cells transfected with ntat aneo replicon and those transfected with a replication-defective rna encoding an inactive ns b rna polymerase deleted in its active site (Δgdd, see materials and methods). rna replication was then clearly detected between days and after transfection (figure a , compare seap levels in cells transfected with ntat aneo and Δgdd). cotransfection of the irrelevant sirna (siirr) did not significantly affect ntat aneo replication (figure a) . hcvspecific sirnas si and si showed only transient and nil effect on hcv replication, respectively, and seap expression levels on day after transfection were similar to those induced by transfection of ntat aneo alone (figure a) . these two sirnas were therefore not further utilized in the course of our study. in sharp contrast, sirnas si , si - a, si - b, si , and si proved capable of abolishing hcv replication in this system, yielding seap levels as low as those obtained with replication-defective rna Δgdd. in the presence of these sirnas, seap levels were even slightly lower at days , , and after transfection than those obtained with Δgdd, as a result of sirna-mediated degradation of input rnas, rendering them unavailable for translation (figure a) . the inhibitory effect of these five sirnas on hcv replication was shown to be prolonged to days after transfection (data not shown). we next monitored dose-response effects of each of the five selected sirnas using decreasing sirna doses in the range of , - ng ( - . nmol/l per μg replicon) in coelectroporation experiments with μg of ntat aneo replicon (figure b ). from these experiments, inhibition doses were calculated for each sirna (figure b) . the sirnas si - a and si proved to be the most efficient inhibitors of hcv rna replication with inhibition doses of ng ( . nmol/l) and ng ( . nmol/l), respectively, and all sirnas exhibited inhibition doses within the - ng range. the inhibition dose of si - b ( ng) was shown to be substantially higher than that of si - a ( ng), in agreement with the fact that si - b is not fully homologous to ntat aneo ntr (genotype a). this demonstrates the sequence-specificity of these sirnas. we also found that the co-administration of two sirnas targeting nonoverlapping hcv sequences, in suboptimal doses, resulted in additive inhibitory effects (data not shown). this might prove useful in future studies in which a strategy based on combined sirnas is sought to be developed in order to prevent the occurrence of escape mutations. the five sirnas (si , si - a, si - b, si , and si ) that specifically target domain iii of the hcv ires and strongly inhibit subgenomic rna replicon replication when used in the nanomolar range were retained in the remaining part of the study. next, in order to determine whether sirnas are capable of curing replicon-containing cells, we investigated whether hcv replicon rna was eliminated in sirna-treated cells in which seap activities were at basal level. total rna was extracted from cells cotransfected with replicon and sirna on day after transfection and analyzed for hcv rna. this was done by semi-quantitative reverse transcription-polymerase chain reaction (rt-pcr) using an hcv primer pair that allows amplification of a base-pair fragment spanning the ns coding region, as well as a primer pair that allows the detection of a housekeeping messenger rna (glyceraldehyde- -phosphate dehydrogenase) for the purpose of rna quantity normalization (figure c ). hcv rna was readily detected in similar abundance in cells transfected with ntat aneo or cotransfected with ntat aneo and siirr (figure c , lanes , ). very low levels of residual transfected Δgdd rna molecules could be occasionally detected under these experimental conditions (figure c, lane ) . in duplicate experiments, using cells cotransfected with ntat aneo and si - a, si , or si , viral rna was either not detected or detected at very low levels, comparable to the results obtained with Δgdd rna (figure c , lanes - ). hcv rna was consistently detected in cells cotransfected with ntat aneo and si (figure c, lanes , ) , but at levels lower than in cells cotransfected with ntat aneo and siirr. these results correlated well with inhibition levels of reporter seap activity, and confirmed that hcv rna replication is strongly inhibited, if not abolished, in cells treated with μg of the three most efficient sirnas (si - a, si , and si ). in order to work with a cell culture system mimicking an established persistent infection, we analyzed the inhibitory capacity of sirnas in en - cells that stably contain and continuously replicate ntat aneo replicon. the ongoing replication of ntat aneo replicon in these cells was reflected by seap levels that were more than tenfold higher than in transient experiments after hcv replicon electroporation. two micrograms of each sirna were electroporated into these cells, and their effect on hcv rna replication was determined at different timepoints. data obtained at day after transfection, corresponding to cumulative seap levels secreted between days and , are represented in figure . the seap activity generated by the hcv replicon in the presence of either of the homologous sirnas was reduced by % or more relative to that of ntat aneo in mock-transfected cells. when a higher dose ( μg) of sirnas was used, seap activity was reduced by - % (data not shown). the sirna-mediated inhibitory effect was not as dramatic as in cotransfection experiments, probably due, in part, to limited transfection efficiency (~ %, data not shown), thereby permitting ongoing rna replication in cells that did not receive sirna in the transfected culture. in addition, viral rnas undergoing replication within replication complexes may be less accessible than transfected viral rnas to sirnas. nonetheless, we demonstrated strong, dose-dependent inhibitory potency of hcv domain iii-specific sirnas in cells supporting continuous hcv rna replication. we next addressed the question of the efficacy of sirnas in inhibiting hcv genome replication in cells placed under selective pressure, and examined whether there was any hcv escape from specific sirna treatment. in these experiments, we used the hcv replicon-free subclone - c of huh cells, and either ntat aneo or nneo replicon. in contrast to ntat aneo, nneo replicon (see figure b and ref. ) contains a ntr derived from the n strain of genotype b and encodes neomycine phosphotransferase gene in the first cistron. - c cells were coelectroporated with either ntat aneo or nneo replicon and each sirna, then placed under g selective pressure. g resistant cell clones were counted on day after transfection. data from two or three experiments carried out with each replicon were pooled for sirnas homologous to both replicons (si , si , si ), and the mean of these data is shown in figure . a mean of , ± , resistant cell clones was obtained after transfection of hcv replicon alone. the data are represented with respect to , g -resistant clones in each experiment. treatment with each of the hcv-specific sirnas resulted in a substantial reduction in the number of g -resistant cell clones (figure ) . sirna si reduced the formation of g -resistant cell clones by ~ logs, whereas si and si reduced cell clone formation by logs or more (figure a) . this is in good correlation with results obtained in the seap reporter system. both si - a and si - b were more efficient on their homologous target than on heterologous targets (figure b) . the nature and the position of the mismatch between si and its target (g:u and a:c for si - a on hcv genotype b and si - b on genotype a, respectively, at nucleotide of the sirna antisense strand) are reasonably well tolerated by the rnai machinery, resulting in ~ log reduction in g -resistant cell clone formation, an inhibition that was, however, - . log less efficient than on homologous targets. these experiments confirmed, as already described, , , that perfect base-pairing between sirnas and targeted messenger rna is required for most effective silencing. to look for potential emergence of sirna escape mutants, several g -resistant cell clones, obtained after a single treatment with each sirna, were isolated and independently amplified. the ntrs of the hcv replicon rnas recovered from to cell clones for each sirna were reverse-transcribed and pcr-amplified. the resulting pcr products were subjected to sequencing. no nucleotide substitution was found within or in the vicinity of the sirna-targeted region of the hcv replicon rnas. these data suggest that either a single sirna treatment may not be sufficient to select for hcv replicon escape mutants in this system, or that replication competence does not tolerate nucleotide variation in this genomic region. we sought to determine whether the sirnas si - a, si , and si , shown to be the most efficient in inhibiting the replication of hcv subgenomic replicons both transiently and under selective pressure, are also capable of efficiently blocking virus infection in cell culture. we utilized the jfh- strain of hcv genotype a that was shown to infect huh and huh -derived cell lines and produce infectious particles. this in vitro infectious system therefore recapitulates the entire hcv life cycle. sequences of si - a and si were perfectly homologous to the ntr sequence of this genotype a strain. in contrast, si was shown to hybridize to the jfh- ntr with a c:a mismatch at the third position of the sirna antisense strand (see figure c) . such a mismatch at the end of the antisense strand is expected to hamper rnai. , for this reason, we also used an engineered chimeric virus that contains ntr and core-coding sequences derived from genotype a within the backbone of the jfh- genome (jfh- /c(+) (figure a) . in contrast, hcv-specific sirna si considerably reduced the number of cells infected with either jfh- or the chimeric virus (figure a) . similarly, si efficiently inhibited infection with the a/ a chimeric virus (figure a, left) , but had only a weak effect, if any, on jfh- infection (figure a , right), consistent with the existing mismatch between si and genotype a ntr (see figure c) . interestingly, si - a sirna also appeared to have a higher inhibitory effect on the infection with the chimeric virus (figure a , left) than with jfh- (figure a, right) , in spite of perfect homology with the ntr sequences of both viruses. we hypothesized that these differential effects of si - a might be linked to delayed replication kinetics of the chimeric a/ a virus, as compared to jfh- (d. delgrange, a. pillez, l. cocquerel, g. paranhos-baccala, y. rouillé, j. dubuisson et al., unpublished results), a phenotype that may impact on target/sirna ratios at early time-points following infection. the effects of the three sirnas on both viruses were also monitored by western blot analysis of e expression in infected cells (figure b) . the data obtained confirmed that si is the most efficient sirna, causing complete inhibition of e expression in cells infected with either jfh- or jfh- / c(+) - a. in order to verify whether virus production from sirnatreated cells is reduced accordingly, viral particles were titrated in supernatants from cells transfected with each sirna and infected with either jfh- or the chimeric virus, both by realtime quantitative rt-pcr (genome equivalents, figure c ) and by determination of infectious focus-forming units ( table ) . for each of the sirnas, quantification of virus production in sirnatreated cultures (figure c, table ) was in good agreement with core and e intracellular expression levels (figure a and b) . in particular, supernatants from si -treated, hcv-infected cells exhibited a > % reduction in genome equivalents/ml titer and a > % reduction in focus-forming units/ml infectious titer, as compared to mock-treated hcv-infected cells. taken together, these data demonstrate that hcv ntr-specific sirnas are capable of strongly inhibiting virus production in cell culture. in exploring rnai as a potential new therapeutic approach against hcv infection, one of the reasons for our choice to target domain iii of the hcv ntr was that domain iii contains well-conserved nucleotide sequences across all hcv genotypes ( table ) . the high degree of conservation seen in these sequences may be required for preserving virus function, since this ntr domain controls the initiation of polyprotein translation and modulates rna replication. , recent nuclear magnetic resonance and electron cryomicroscopy studies of domain iii , , have helped identify the structure-function relationships of the various subdomains of domain iii. the results of these studies suggest that subdomains iiia/c and iiid, as well as subdomain iiif that forms a pseudoknot structure (figure a) , directly contact the s subunit body and act synergistically for the proper positioning of the aug codon. in the present study, si and si (that hybridize to subdomain iiid) and si (that essentially hybridizes to the iiif pseudoknot), therefore target regions that are essential for ires structural integrity and functioning. these three sirnas proved able, at low doses in the nanomolar range, to substantially reduce or even abolish hcv subgenomic rna replication, as measured by reporter seap activities and g -resistant cell clone formation (figures and ) , and resulted in the elimination of hcv replicons from treated cells (figure c) . importantly, we also demonstrated that these sirnas, particularly si , considerably limit hcv infection in cell culture. we did this using two hcv strains that can be propagated in huh -derived cells: (i) the jfh- strain of hcv genotype a, and (ii) a chimeric derivative of jfh- carrying ntr and core sequences of hcv genotype a (d. delgrange, a. pillez, l. cocquerel, g. paranhos-baccala, y. rouillé, j. dubuisson et al., unpublished results). our data contrast with those obtained by others who concluded that domains ii and iii , of hcv ntr are relatively resistant to sirna, because there is reduced accessibility to these domains, given their association with different proteins and factors involved in translation. our data indicate that regions involved in complex secondary and tertiary structures should not be disregarded as targets for rnai. the data also underscore the importance of sirna design. , in contrast, two of the sirnas tested in the present study (si and si , targeting domains iiic and iiie, respectively) exhibited poor or only transient inhibitory effect on hcv rna replication (figure a) . interestingly, two other studies reported that sirnas having sequences identical to that of si showed relative discrepancies in their effects in cells supporting autonomous replication of hcv subgenomic replicon. , these conflicting results may be explained by the different strategies used for sirna synthesis and/or delivery. in addition, the sensitivity of the replicon systems used may have an impact on the sirna efficiencies reported. we observed some differences in sirna efficiencies between: (i) transient replication systems in which viral target rna and sirna were co-introduced into cells (figures and ) , and (ii) stable replication systems in which sirna was introduced in cells supporting ongoing replication of the viral target, whether dealing with a subgenomic replicon (figure ) or genomelength infectious rna (figure , table ). two micrograms of sirna did not inhibit viral rna replication in the stable subgenomic replicon system as efficiently as in cotransfection experiments (compare figures and ) . in addition, si - a and si caused elimination of viral rna upon cotransfection with subgenomic replicon (figure a) , whereas they substantially reduced, but did not abolish the production of virus particles ( figure , table ). this was in spite of the fact that the molar ratio of sirna to targeted hcv rna was higher in cells stably containing hcv replicons or infected with virus than in cells cotransfected with replicon and sirna. in another study, high doses ( , pmol) of sirnas proved necessary in order to strongly inhibit hcv rna replication in subgenomic replicon-containing cells. we speculate that viral rna templates engaged in the replicase complex and nascent rnas are probably not as accessible to the rnai machinery as transfected viral rnas are. our data can also be explained in part by the fact that a proportion of cells in the culture were infected or supported ongoing viral rna replication but did not receive sirna, given that the efficiency of electroporation is ~ % in en - and - c cells (data not shown). the specificity of hcv sirnas was demontrated by studying their dose-effect responses (figure b) , as well as by using si to target the replicon ntr from two hcv genotypes ( a and b; figures - ) and si to target the ntr of genotype a or a virus strains ( figure , table ). when used with heterologous targets, the sirnas si - a and si - b exhibited a noncanonical wobble base-pairing and a mismatch at the end (position ) of the sirna antisense strand, respectively (figure c) . for both a and b replicons, perfect base-pairing was preferred for maximum rnai efficacy, but a g:u wobble and an a:c mismatch were both tolerated (figure ) . this is consistent with the fact that wobble base-pairing is known to be well tolerated in double-stranded rna helices, providing stability similar to a watson-crick base-pairing. in agreement with our data, wobble base-pairs, especially when located at the end of the sirna antisense strand, were recently suggested to have no disruptive effect on rnai. , , more surprisingly, but also consistent with our data, it was reported that an a:c mismatch was not deleterious in double-stranded rna structures. contrasting with these tolerated mismatches between sirna and viral target rna, we found that a c:a mismatch at the end of the sirna antisense strand strongly reduces the inhibitory effect of si on jfh- infection in cell culture ( figure , table ) , and this is consistent with the rnai mechanism. , taken together, these data provide strong evidence against the involvement of a spurious interferonmediated mechanism of inhibition in the suppression of viral replication we observed. the sequence of rna viruses, such as hcv, is known to evolve continuously, resulting in the production of many quasispecies, because of the high error rate of rna-dependent rna polymerases with no proof-reading activity. this property allows rna viruses to escape rapidly to a selective pressure such as antiviral treatments, when sequence changes are compatible with virus functions. this holds true for anti-protease and antipolymerase compounds that are currently under development for hcv treatment. , however, we did not observe, after a single sirna treatment, the emergence of escape mutations within sirna-targeted genomic regions in rna extracted from several g -resistant cell clones. in other studies, sirna-resistant viral mutants harboring single nucleotide substitutions or deletions within or at the vicinity of the sirna-targeted sequence were reported to have emerged in cells expressing constitutively an shrna (in the case of human immunodeficiency virus infections), , and after repeated treatments with an sirna targeting the polymerase coding sequence (in the case of hcv subgenomic replicons). the lack of escape mutations observed in our study could either be due to the fact that a single sirna was used in the treatment, or to the fact that sirnas target highly conserved, functionally important genomic sequences, in which substitutions would not be compatible with translation/replication competence. additional studies will be needed in order to determine which of these hypotheses holds true. from the point of view of evaluating the potency of sirnabased antiviral strategies to eradicate persistent infections, it was recently shown that sirnas may be used to cure in vitro persistent infections caused by rna viruses such as poliovirus or lymphocytic choriomeningitis virus. , for assaying such a therapeutic strategy in animal models, synthetic sirnas need to be further chemically stabilized and formulated to be efficiently delivered. successful utilization of stabilized, lipid-encapsulated sirnas was reported in a mouse model of hepatitis b virus replication. in addition, organ or cell-type specific delivery of sirnas has been achieved through sirna binding to cholesterol or to antibodies directed against cell surface antigens. synthetic shrnas also showed a more prolonged inhibitory effect in murine liver than sirnas did. this was demonstrated upon co-delivery of shrna or sirna targeting domain iv of the hcv ires and plasmid dna encoding a reporter protein placed under the translational control of the hcv ires. alternatively, optimization of sirna delivery may rely on the use of viral vectors that allow continuous synthesis of shrnas in cells, thereby leading to sustained rnai. , it is crucial, however, to control intracellular shrna production levels, since it has recently been reported that constant, high expression of shrnas in the liver could be lethal in mouse models. nevertherless, controlled doses of sirna or expression of shrna have already been shown to be efficient and safe in several animal models of viral infection, including in a mouse model of hepatitis b virus infection, a rhesus macaque model of severe acute respiratory syndrome-associated coronavirus infection, and mouse models of west nile virus and japanese encephalitis virus infections. in the present study, we have identified promising anti-hcv sirnas (si and, to a lesser extent, si and si ) that target highly conserved sequences in domain iii of the ntr and efficiently silence hcv infection in cell culture. it has now become possible to monitor the effect of these sirnas in vivo using a tamarin/marmoset primate model of infection with a chimeric gb virus b-containing hcv domain iii of the hcv ires, that we have previously described. during revision of this manuscript, kanda et al. reported that delivery of an hcv ntr-specific shrna to hepatoma cell lines infected by hcv resulted in the reduction of viral production. hepatoma cell lines, - c, en - , and huap are derived from the hepatocarcinoma cell line huh , and were cultured in dulbecco's modified eagle medium (invitrogen, cergy-pontoise, france) supplemented with % fetal calf serum, u/ml penicillin and μg/ml streptomycin. en - cells stably express seap under the control of the human immunodeficiency virus long terminal repeat promoter, and were cultured in the presence of μg/ml blasticidin (invivogen, toulouse, france). en - cells supporting autonomous replication of ntat aneo hcv replicon were cultured in the presence of μg/ml blasticidin and . mg/ml geneticin (g , invitrogen, cergy-pontoise, france). genotype a jfh- and chimeric jfh- /c(+) - a virus stocks were generated by transfection of huap cells with corresponding in vitro transcribed genomic rnas. plasmid pjfh- containing the genome-length complementary dna (cdna) of the jfh- isolate of hcv genotype a (genbank accession no. ab , ref. ) was generously provided by t. wakita. plasmid pjfh- /c(+) - a was derived from pjfh- by substituting nucleotides - of the ntr, as well as the capsid coding sequence by corresponding sequences of the h strain of genotype a (d. delgrange, a. pillez, l. cocquerel, g. paranhos-baccala, y. rouillé, j. dubuisson et al., will be described elsewhere). plasmids pjfh- and pjfh- /c(+) - a were linearized with xbai and treated with mung bean nuclease prior to in vitro transcription using megascript t kit (ambion, courtaboeuf, france). huap cells were electroporated with in vitro transcribed rna, as previously described, and supernatants collected - days after transfection were used to re-infect naïve huap cells. supernatants collected at days after infection were used as virus stocks and stored at °c. design and synthesis of sirnas. sirnas targeting hcv ires were designed using previously described criteria. , the sequence of the sense-strand of each sirna selected is shown in table . sirnas are referred to by the nucleotide position within the genome of the h strain of hcv genotype a that corresponds to the first nucleotide targeted by the sirna antisense strand. si was synthesized with a sequence homologous to hcv subtype a or b, i.e., containing a u (si - a) or a c (si - b) residue at position of the sirna antisense strand. an irrelevant sirna, named siirr, targets the ntr of the sabin strain of poliovirus type and was used as negative control in this study. the sequences of all sirnas were compared with known genes by using basic local alignment search tool within the genbank database, and no significant homology to other genes was found. the two strands of each sirna were chemically synthesized at the institut pasteur ("plate-forme ") and annealed at a final concentration of pmol/μl, as previously described. the quality and quantity of hybridized sirnas were analyzed by electrophoresis on nondenaturing % agarose gels. hcv subgenomic replicons and in vitro transcriptions. two dicistronic, subgenomic hcv replicons that encode a selectable reporter gene, neomycine phosphotransferase (neo) were used in this study (see figure ). these replicon cdnas, kindly provided by s.m. lemon, are inserted downstream of the t rna polymerase promoter and have been previously described as pnneo/ - b and pntat aneo. we will refer to these replicons as nneo and ntat aneo, respectively, in this study. nneo and ntat aneo each carries a cell culture adaptive mutation, r g and s i, respectively. both replicon cdnas are derived from the cdna of the n strain of genotype b hcv with the exception of the ntr sequence of the ntat aneo, which is derived from the h strain of hcv genotype a. ntat aneo rna encodes human immunodeficiency virus tat protein fused to the a protease of foot-and-mouth disease virus, followed by neo. upon transfection of en - cells with ntat aneo replicon, the expression of tat- a drives the synthesis of secreted seap. cdnas bearing a -nucleotide deletion, including the three codons (gdd) of the active site of the rna polymerase ns b within the backbone of nneo and ntat aneo, , and referred to as Δgdd, were used as replicationdeficient rnas. plasmid dnas were linearized with xbai prior to in vitro transcription using megascript t kit. the dna template was removed by treatment with turbo dnase (ambion, courtaboeuf, france) and rnas were purified by phenol-chloroform extractions and precipitated with ethanol. the quality and quantity of replicon rnas were analyzed by electrophoresis on a non-denaturing . % agarose gel and by optical density measurements. cell transfections. en - or - c cells ( × ) were electroporated with μg replicon rna, or coelectroporated with μg replicon rna and various doses of each sirna, in a mm-gap width cuvette by applying one pulse at v, μf (easyject plus, equibio, kent, uk). cells were resuspended in complete medium immediately after the pulse and seeded at various concentrations. en - cells supporting autonomous replication of ntat aneo replicon were similarly transfected by electroporation with each sirna. en - transfected cells ( × ) were seeded in -well plates and supernatants were collected at , , , , and days after electroporation and replaced with fresh medium. seap activity was measured in supernatant aliquots with the phospha-light system (applied biosystems/tropix, courtaboeuf, france) using a luminescent substrate according to the manufacturer's recommendations. luminescent signals were read for second using a lumat lb luminometer (berthold technologies, thoiry, france). to select for g -resistant cell clones, variable fractions of - c transfected cells were seeded in mmdiameter dishes and supplemented with nneo-Δgdd or ntat aneo-Δgdd transfected cells to adjust the final number of cells to × cells per dish. twenty-four to forty-eight hours following transfection, cells were placed under the selective pressure of . mg/ml geneticin (g , invitrogen, cergy-pontoise, france) for weeks, with the medium being replaced twice a week. g -resistant cell clones supporting viral rna replication were fixed and stained with a . % crystal violet solution, or selected and expanded under selective pressure for viral rna sequencing. viral rna sequencing. total rna was extracted from expanded sirnaresistant clones with trizol reagent (invitrogen, cergy-pontoise, france). viral rna was reverse transcribed and amplified with hcv specific primers designed to generate pcr products spanning nucleotides - , using the superscript one-step rt-pcr kit with platinum taq (invitrogen, cergy-pontoise, france). sequencing reactions were carried out on uncloned rt-pcr products using big dye terminator version . kit (applied biosystems, courtaboeuf, france) and analyzed on a abi capillary dna sequencer (applied biosystems, courtaboeuf, france). en - transfected cells ( × ) were seeded in -well plates (duplicate wells per transfection) and cultured for days, then trypsinized and passaged at a : dilution in new -well plates. on day after transfection, total rna was extracted novel insights into hepatitis c virus replication and persistence internal ribosome entry site-mediated translation in hepatitis c virus replication replication of subgenomic hepatitis c virus rnas in a hepatoma cell line production of infectious hepatitis c 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virus required for rna replication analysis of gene function in somatic mammalian cells using small interfering rnas rational sirna design for rna interference transcripts from a single full-length cdna clone of hepatitis c virus are infectious when directly transfected into the liver of a chimpanzee sirnas can function as mirnas a systematic analysis of the silencing effects of an active sirna at all single-nucleotide mismatched target sites complete cure of persistent virus infections by antiviral sirnas a single sirna suppresses fatal encephalitis induced by two different flaviviruses virus-host cell interactions during hepatitis c virus rna replication: impact of polyprotein expression on the cellular transcriptome and cell cycle association with viral rna synthesis risc is a phosphomonoester-producing rna endonuclease genetic diversity and evolution of hepatitis c virus- years on structures of two rna domains essential for hepatitis c virus internal ribosome entry site function structure of the hepatitis c virus ires bound to the human s ribosome: remodeling of the hcv ires specificity of microrna target selection in translational repression anti-hcv therapies in chimeric scid-alb/upa mice parallel outcomes in human clinical application mutations conferring resistance to a hepatitis c virus (hcv) rna-dependent rna polymerase inhibitor alone or in combination with an hcv serine protease inhibitor in vitro human immunodeficiency virus type escapes from rna interference-mediated inhibition hiv- can escape from rna interference by evolving an alternative structure in its rna genome rna interference-mediated virus clearance from cells both acutely and chronically infected with the prototypic arenavirus lymphocytic choriomeningitis virus potent and persistent in vivo anti-hbv activity of chemically modified sirnas therapeutic silencing of an endogenous gene by systemic administration of modified sirnas antibody mediated in vivo delivery of small interfering rnas via cell-surface receptors small hairpin rnas efficiently inhibit hepatitis c ires-mediated gene expression in human tissue culture cells and a mouse model clearance of hepatitis b virus from the liver of transgenic mice by short hairpin rnas fatality in mice due to oversaturation of cellular microrna/short hairpin rna pathways using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque small interfering rna targeted to hepatitis c virus nontranslated region exerts potent antiviral effect subcellular localization of hepatitis c virus structural proteins in a cell culture system that efficiently replicates the virus functional analysis of cell surface-expressed hepatitis c virus e glycoprotein courtaboeuf, france). five micrograms of total cellular rna were heatdenatured at °c for minutes and used as template for reverse transcription with u of superscript ii reverse transcriptase (invitrogen, cergy-pontoise, france) and ng of random hexanucleotide primers (roche, meylan, france) for minutes at °c. the resulting cdnas were treated with u of rnaseh (invitrogen, cergy-pontoise, france) for minutes at °c, and purified using qiaquick pcr purification kit (qiagen, courtaboeuf, france). one fifth of the cdna was used for programming pcrs with either hcv-specific primers that allowed amplification of a fragment spanning nucleotides - of the n strain of hcv genotype b, or primers specific to cellular housekeeping gene glyceraldehyde- -phosphate dehydrogenase, so as to normalize for total rna content. pcrs were performed using platinum taq dna polymerase (invitrogen, cergy-pontoise, france) under the following cycling conditions: cycle at °c for minutes, followed by cycles of: (i) seconds at °c; (ii) seconds at °c; and (iii) . minutes at °c. pcr products were analyzed by electrophoresis on % agarose gels. huap cells ( × ) were electroporated with μg of sirna, and × electroporated cells were seeded on coverslips in -well plates and subsequently infected at hours after transfection with jfh- or jfh- /c(+) - a virus stocks at ~ focus forming unit per cell. after hours of incubation at °c, virus inoculum was washed off and cells were fed with culture medium. at hours after infection, cells were fixed and processed for core detection by indirect immunofluorescence, as previously described, using anti-core monoclonal antibody acap (kindly provided by j.f. delagneau, bio-rad, marnes-la-coquette, france). cells were counterstained with hoechst dye to enable nuclei to be detected. for titration of infectious viral particles, × huap cells were infected with : dilutions of supernatants from cells transfected with sirnas and infected with hcv. the foci of infected cells were detected by immunofluorescence with anti-core monoclonal antibody at hours after infection and counted to determine titers in focus forming units/ml. huap cells ( × ) seeded in -well plates were lyzed in mmol/l tris-hcl (ph . ), mmol/l nacl, mmol/l edta, and . % (vol/vol) igepal buffer containing a mixture of protease inhibitors (complete, roche, meylan, france) and processed for e or β-actin detection by immunoblot analysis as previously described, using anti-e monoclonal antibody / or anti-β-actin monoclonal antibody (sigma-aldrich, saint quentin fallavier, france), respectively. rnas were isolated from cell culture supernatants using qiaamp viral rna kit (qiagen, courtaboeuf, france) and quantified by real-time quantitative rt-pcr using primer pair and probe targeting a sequence spanning nucleotides - within the hcv ntr : fp -cgggagagc catagtgg- ; rp -agtaccacaaggcctttcg- ; probe -fam-ctgcggaaccggtgagtacac-tamra- . assays were performed using taqman one-step rt-pcr master mix reagents kit and an abi prism sequence detector instrument (applied biosystems, courtaboeuf, france), according to the manufacturer's instructions. key: cord- -rr h dgy authors: prague, melanie; wittkop, linda; clairon, quentin; dutartre, dan; thiebaut, rodolphe; hejblum, boris pierre title: population modeling of early covid- epidemic dynamics in french regions and estimation of the lockdown impact on infection rate date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: rr h dgy we propose a population approach to model the beginning of the french covid- epidemic at the regional level. we rely on an extended susceptible-exposed-infectious-recovered (seir) mechanistic model, a simplified representation of the average epidemic process. combining several french public datasets on the early dynamics of the epidemic, we estimate region-specific key parameters conditionally on this mechanistic model through stochastic approximation expectation maximization (saem) optimization using monolix software. we thus estimate basic reproductive numbers by region before isolation (between . and . ), the percentage of infected people over time (between . and . % as of may th, ) and the impact of nationwide household confinement on the infection rate (decreasing the transmission rate by % toward a re ranging from . to . ). we conclude that a lifting of the lockdown should be accompanied by further interventions to avoid an epidemic rebound. in december , grouped pneumonia cases have been described in the hubei province, china and sars-cov was identified on january, th as the cause of this outbreak (li et al., a; zhu et al., ) . sars-cov causes the viral disease which has been named covid- (world health organization, b) . sars-cov rapidly spread all over the world and the pandemic stage was declared on march th by the world health organization ( c). on april th , over , , cases (in accordance with the applied case definitions and testing strategies in the affected countries) including , deaths have been reported (world health organization, a) . the first case in france was declared on january, th (bernard-stoecklin et al., ) and on april th , santé publique france reported , confirmed cases and , hospital deaths due to covid- includes non-specific symptoms such as fever, cough, headache, and specific symptoms such as loss of smell and taste (gane et al., ; greenhalgh et al., ) . the virus is transmitted through droplets and close unprotected contact with infected cases. the majority (around %) of infected cases have a mild form (upper respiratory infection symptoms) without specific needs in terms of care. around % of cases need hospitalization and among those are severe forms (severe respiratory distress) which will need to be admitted to intensive care units (icu) with potential need of mechanical ventilation. the percentage of patients in need for icu care varies between % reported from china (guan et al., ) and % reported from italy (grasselli et al., ) . the number of icu beds in france was , at the end of (drees, ) (although it is currently being increased, having doubled and aiming to reach , according to the french minister of health). thus, the availability of icu beds with mechanical ventilation is one of the major issues as facilities are not prepared to deal with the potential increase of the number of patients due to this epidemic. unprecedented public-health interventions have been taken all over the world (kraemer et al., ) to tackle this epidemic. in france, interventions such as heightening surveillance with rapid identification of cases, isolation, contact tracing, and follow-up of potential contacts were initially implemented. but as the epidemic continued growing, comprehensive physical distancing measures have been applied since march th , including closing of restaurants, non-vital business, schools and universities etc, quickly followed by state-wide lockdown on march th . the president has announced on april th , a progressive lifting of the lockdown from may th onwards. in wuhan (hubei, china), the extremely comprehensive physical distancing measures in place since january rd have started to be relaxed after months of quarantine and lifted completely on april th (tian et al., ; wu and mcgoogan, ) . interestingly, these interventions have been informed by mathematical models used to estimate the epidemic key parameters as well as unmeasured compartments such as the number of infected people. another interesting outcome is the forecast of the covid- epidemic according to potential interventions. several models have already been proposed to model and forecast the covid- epidemic using compartment models (fang et al., ; tang et al., ; or agent based models (di domenico et al., a; ferguson et al., ; wilder et al., ) , its potential impact on intensive care systems (fox et al., ; massonnaud et al., ) , and to estimate the effect of containment measurements on the dynamics of the epidemic (magal and webb, ; prem et al., ) . most of those rely on simulations with fixed parameters and do not perform direct statistical estimations from incident data (massonnaud et al., ) . roques et al. ( ) used french national data but did not use a population approach to model the epidemic at a finer geographical granularity. yet, the dynamics of the epidemic can be very heterogeneous between regions inside a given country resulting in tremendous differences in terms of needs for hospital and icu beds (massonnaud et al., ) . moreover, the data collection yields noisy observations, that we deal with a statistical modeling of the observation process, rather than altering the data by e.g. smoothing such as in roques et al. ( ) . in the present study, we use public data from the covid- outbreak in france to estimate the dynamics of the covid- epidemic in france at the regional level. we model the epidemic with a seirah model, which is an extended susceptible-exposed-infectious-recovered (seir) model accounting for time-varying population movements, non-reported infectious subjects (a for unascertained) and hospitalized subjects (h) as proposed by to model the epidemic in wuhan. parameters from the model are estimated at the regional scale using a population approach which allows for borrowing information across regions, increasing the amount of data and thereby strengthening the inference while allowing for local disparities in the epidemic dynamics. furthermore, we use forward simulations to predict the effect of non-pharmaceutical interventions (npi) (such as lift of lockdown) on icu bed availability and on the evolution of the epidemic. section introduces the data, the model and the necessary statistical tools, section presents our results and section discusses our findings and their limits. because epidemics spread through direct contacts, their dynamics have a strong spatial component. while traditional compartment models do not account for spatiality, we propose to take it into account by: i) modeling the epidemic at a finer, more homogeneous geographical scale (this is particularly important once lockdown is in place); ii) by using a population approach with random effects across french regions which allows each region to have relatively different dynamics while taking all information into account for the estimation of model parameters iii) aligning the initial starting time of the epidemic for all regions. the starting date in each region was defined as the first date with incident confirmed cases of covid- directly followed by additional consecutive days with incident confirmed cases as well. this criterion of consecutive days with incident cases is needed in particular for the Île-de-france region which had consecutive days with imported confirmed case in late january which did not lead to a spreading outbreak at that time. open-data regarding the french covid- epidemic is currently scarce, as the epidemic is still unfolding. santé publique france (spf) in coordination with the french regional health agencies (agences régionales de santé -ars) has been reporting a number of aggregated statistics at various geographical resolutions since the beginning of the epidemic. during the first weeks of the epidemic in france, spf was reporting the cumulative number of confirmed covid- cases with a positive pcr test. other french surveillance resources such as the réseau sentinelles (valleron et al., ) or the sursaud r database (caserio-schönemann et al., ) quickly shifted their focus towards covid- , leveraging existing tools to monitor the ongoing epidemic in real time, making as much data available as possible (given privacy concerns). in this study, we combined data from three different opendata sources: i) the daily release from spf; ii) the sursaud r database that started recording visits to the emergency room for suspicion of covid- on february, th ; iii) the réseau sentinelles which started estimating the weekly incidence of covid- in each french region on march th . from the daily release of spf, we computed the daily incident number of confirmed covid- cases (i.e. with a positive pcr test) in each region. in . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . addition we used the incident number of visits to the emergency room for suspicion of covid- in each region from the sursaud r database using the oscour r network that encompasses more than % of all french emergency services (caserio-schönemann et al., ) . although this does not represent the full extend of hospitalized covid- cases, it is the only public data available that early in the epidemic, when the majority of covid- cases at hospitals were admitted through emergency rooms. finally, we used the réseau sentinelles network's weekly incidence estimates of symptomatic cases (including non confirmed cases) to set the ratio between ascertained and unascertained cases in each region (later denoted as r i ). table presents these observed data. of note, we studied the epidemic in the metropolitan french regions -excluding the corsican region (corse) which exhibits different epidemic dynamics, possibly due to its insular nature. wang et al. ( ) extended the classic seir model to differentiate between different statuses for infected individuals: ascertained cases, unascertained cases and cases quarantined by hospitalization. the model, assuming no population movement, is presented in figure . the population is divided into compartments: susceptible s, latent e, ascertained infectious i, unascertained infectious a, hospitalized infectious h, and removed r (recovered and deceased). this model assumes that infections are well-mixed throughout the population, ignoring any spatial structure or compartmentalization by population descriptors such as age. such assumptions make it particularly relevant to infer the dynamics of the french epidemic at the regional level (a finer geographical scale at which such hypotheses are more likely to hold). figure illustrates the dynamics between those compartments that are characterized by the following system of six ordinary differential equations (ode): cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) model parameters are described in table . of note, given a combination of parameters and initial states of the system ξ = , using a solver of differential equations, it is possible to deterministically compute at any time t the quantities s(t, ξ), e(t, ξ), i(t, ξ), r(t, ξ), a(t, ξ), and h(t, ξ). . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . observation processes in our case, none of the compartments of the system are directly observed: the only observations considered are i) the number of daily incident infectious ascertained cases denoted y , and ii) the number of daily incident hospitalized infectious cases denoted y . these observations are the only one available both before and after the initiation of lockdown. those two quantities are modeled in equation ( ) respectively as observations from the i (in) (t, ξ) = re(t,ξ) de and h (in) (t, ξ) = i(t,ξ) dq random variables, which are the numbers of new incident cases at time t given the parameters ξ in compartment i and h respectively. because these are count processes, we propose to model their observations y and y with poisson likelihoods: where k and k are the respective numbers of cases. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . the initial states of all compartments at the date of epidemic start (t = ) for region i are also important drivers of the dynamics. some of them can be approximated by quantities directly depending on the observations: i) , and iii) r i (t = ) = . others, namely compartments a and e are not directly observed, and we evaluate these initial quantities. due to variation in data collection protocols and the initial size of regional outbreaks, this estimation is particularly important. indeed, the number of daily incident cases at t = ranges from to cases depending on the region. a i (t = ) is set as the goal of this study is to model the epidemic of covid- in france, but at the regional level using a population approach. this is done using a mixed effect model. in this inference framework, baseline parameters governing the dynamics of the epidemic in each region are assumed to be drawn from a shared distribution which allows for heterogeneity between regions, known as the random effects. we use the log-normal distribution for all parameters to ensure their positivity during estimation. because public health policies changed over the time period of observation of the epidemic, we incorporate explanatory covariates such as physical distancing by lockdown (c and c ) as a time-dependent effect on the transmission of the disease b. covariate c is until - - date of the start of the policy in france and then set to . covariate c is until - - assuming that social distancing behaviours build up in a week. in other words, we have ∀i = , . . . , (where i is the region identifier): the parameters (b , d q , e(t = )) are mean shared values in the population, and can be seen as the country values for these parameters. the inter-region random-effect (u b i , u dq i , u e i ) are normally distributed and assumed independent. so the vector of parameters in the model for each region is cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . which is the factor by which transmission is modified after the start of lockdown during the first week. the factor by which transmission is modified after that first week of confinement is given by k = exp(−β − β ). the coefficients β = (β , β ) are expected to be negative as lockdown aims at reducing transmission. interestingly, with our approach, we can evaluate whether or not there is a statistically significant effect of lockdown on the transmission by testing β = using a wald test. region-specific model parameters based on the results from the theoretical identifiability analysis of the structural model of the epidemic from equation ( ) (see appendix a ), we estimate the parameters (b i , d q i , r i ) as well as the initial state (e i ) when the epidemic begins being reported in each region i. we used the monolix software version r (lixoft sas, ) to estimate those five parameters by maximizing the likelihood of the data given the model and the other fixed parameters. this software relies on a frequentist version of the stochastic approximation expectation maximization (saem) algorithm (delyon et al., ) and standard errors are calculated via estimation of the fisher information matrix (kuhn and lavielle, ) , which is derived from the second derivative of the log-likelihood evaluated by importance sampling. in addition, we use profile-likelihood to confirm that no further information can be gained from the data at hand on parameters α, d e and d i by running the saem algorithm multiple times while setting these parameters to different values and obtaining similar maximum likelihood values (meaning more data would be needed to be able to estimate those parameters). during inference, practical identifiability of the model is evaluated by the ratio of the minimum and maximum eigenvalues of the fisher information matrix, that will be referred as "convergence ratio" in the reminder of the manuscript. convergence of the saem algorithm was assessed by running multiple saem chains and checking that they all mix around similar probability distributions. we are particularly interested in the trajectories of the model compartments. we use monte carlo methods (parametric bootstrapping) to compute the confidence intervals accounting for the uncertainty in estimating the structural and statistical model parameters. for . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . all compartments c(t, ξ i ) (c being s, e, i, r, a, or h) the % confidence interval is estimated by sampling from the posterior distributions of the model parameters to simulate , trajectories, and taking the . % and . % percentiles of these simulated trajectories. we also added to it the error measurement given by the poisson distribution of the outcomes. other outcomes of interest are the number of icu beds needed and the number of death (d) in a given region at a given time. these quantities are not specifically modeled by our mathematical structural model. however, it is possible to roughly approximate them by assuming that they represent a percentage of the hospitalized cases h(t, ξ i ) and removed cases r(t, ξ i ). we assume that icu (t, ξ i ) = . × h(t, ξ i ) which is consistent with the prevalence of icu cases among hospitalized cases at the french national level. based on the estimation of the infection fatality ratio (ifr) from roques et al. ( ), we get a rough estimation of d(t, ξ i ) as . % of r(t, ξ i ). roques et al. ( ) conclude that covid- fatalities are under-reported, and using their ifr estimate we adequately fit the trend of the observed covid- deaths but with an offset due to this assumed higher ifr, see appendix a . model update furthermore β estimations can be easily updated as new data become available using parametric empirical bayes (thus avoiding the need to re-estimate the whole system). it consists in maximizing the likelihood again with respect to β while holding the other parameter distribution fixed to their previously inferred a posteriori distribution. this is how our results are updated with data after march th in this work. effective reproductive number for each region, we compute the effective reproductive number r e (t, ξ i ) as a function of model parameters: when individuals are homogeneous and mix uniformly, r e (t, ξ i ) is defined as the mean number of infections generated during the infectious period of a single infectious case in the region i at time t. this is the key parameter targeted by npis. we compute analytically its % confidence interval by accounting for all % confidence interval [x min ; x max ] of parameters and . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . trajectories x used in its definition such that: asymptomatic proportion at a given time t the number of incident unascertained cases is equal to the sum of two populations, the number of incident non-tested symptomatic individuals (nt) and the number of incident non-tested asymptomatic individuals (as): where r is the proportion of cases tested positive. collection of data from general practitioners through the re-purposing of the réseau sentinelles network to monitor covid- provides a weekly estimation of the number of incident symptomatic cases (tested or not tested) that we previously called r s . this quantity is given over a week but can be evaluated daily by averaging: where r s represents the proportion of infected cases seeing a general practitioner. combining equations ( ) and ( ) allows to compute the incident number of asymptomatic cases as a function of the compartment e: short-term predictions of attack rates we predict the proportion of infected individuals among the population in each region at a given date by computing: . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . given the values of parameters ξ i , we predict the trajectories of the dynamical system compartments using the dlsode differential equation solver in r (soetaert et al., ) and we can investigate the impact of npis such as lockdown in various scenarios. this impact is driven by two parameters: • k (k is considered fixed), the decrease ratio of transmission rate of the disease following the first week of npi, defined as this directly translates into a decrease of the effective reproductive number according to equation ( ). it reflects the fact that individual by getting confined decrease their number of contacts. of note, the current k is estimated by exp(− β − β ) (see section . . ). • τ , the duration (in days) of the lockdown during which the mixing and transmission are fixed to we evaluate the magnitude of the possible epidemic rebound after confinement according to several values for k . in particular, we predict the rates e(t, ξ i )/n i , a(t, ξ i )/n i and i(t, ξ i )/n i on may th (currently considered by french authorities as the possible start date for lifting lockdown in france). we also compute the optimal lockdown duration τ opt i needed to achieve the epidemic extinction in region i defined as e i (t, ξ i ) < and a i (t, ξ i ) < and i i (t, ξ i ) < simultaneously. in each scenario we predict the date at which the icu capacities in each region would be overloaded, this date is given by: η i denoting the icu capacities limits in region i. we additionally predict how many more icu beds would be needed at the peak of hospitalization in each scenario, as a proportion of current icu capacity (drees, ). finally we provide a rough prediction of the number of deaths for each envisioned scenario assuming a confinement duration of τ opt i days. data fitting because of the lag inherent to diagnostic testing, we also estimated the number of people already infected at this epidemic start by e (notably, the largest numbers of e in table are estimated for Île-de-france and grand est the two most affected french regions in this early epidemic). . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . on march th , the cumulative number of ascertained cases was , and the cumulative number of hospitalized cases was , , see table for a regional breakdown. our seirah model fits the data well as can be seen in figure . moreover, the stability of the estimates is good with a convergence ratio of . (see section . . ), corroborating the good identifiability of the estimated parameters (see appendix a ). table provides the regional estimates of the transmission rates (b i ). of note, regions with higher transmission rate are not necessarily those known to have the highest number of incident ascertained cases. d q i , the number of days from illness onset to hospitalization, can be quite variable between regions and likely accounts for heterogeneity in the observed data. we estimates its population mean at d q = . days with a standard deviation σ dq = . . to evaluate the validity of our structural ode model ( ) and of our inference results, we compare the aggregated predictions of the number of both incident ascertained cases and incident hospitalized cases at the national french level to the daily observed incidences (that are still publicly available from spf at the country level, even after march th -while incident ascertained cases are not openly availilable at the regional level after march th ). figure displays both predictions and observations, illustrating the added value of incorporating data after march th as those encompass new information about the epidemic dynamics and characteristics as we approach the peak in most regions (notably grand est and Île de france). of note, worse fit of the observed hospitalizations by our model can be explained by the data discrepancy: while we use the sursaud r data for our inference (which only account for patients arriving through the emergency room), figure displays the data from santé publique france which should contain all covid- hospitalizations in france (including hospitalizations not coming from the emergency room, as well as corsica and the french departements d'outre mer that are not taken into account in our model). . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . evolution of the epidemic without intervention it is also interesting to predict the percentage of infected individuals in each region at future dates, corresponding to attack rates. change of transmission rate during lockdown the parameter β and β measure the effect of the lockdown before and after a week of adjustment. both are significantly different from (p< . ) such that the lockdown reduced the transmission rate of covid- by a divisive factor k estimated at . [ . ; . ] during the first week and k estimated at . [ . ; . ] after this first week. of note, thanks to our update algorithm (see section . . ), it is possible to update those results rapidly as soon as more data are available to inform which scenario of prediction described in section . is the most likely. effective reproductive number the above quantities directly impact the effective reproductive number as described in figure displays the effective reproductive number trajectories in each region. in tables and , we vary k = , , (the magnitude of the reduction of transmission during lockdown after the first week). this gradient of simulation is important because the actual french value of k remains currently unknown. in section . we showed that we can estimate a lower bound for k ≥ k = . . from table , we show that the higher k is, the lowest . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . table : estimation of the effective reproductive ratios r e during each of the considered periods (before lockdown, during the first week of lockdown, and beyond week of lockdown) for each region with % confidence intervals. the numbers of ascertained (i), unascertained (a) and latent (e) infected individuals are on may th . however, it is not equal to , which means the epidemic is not extinct (and ready to bounce back as soon as lockdown is lifted). in table , we predict the optimal (i.e. shortest) duration of the lockdown to achieve extinction of the epidemic in each region, which is, table presents the proportion of infected individuals at various dates, i.e. instantaneous attack rates. we also predict them for three horizon dates assuming confinement would be maintained until these dates: - - , - - and - - . we predict the national french attack rate on may th to be . % [ . %; . %]. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . table : model predictions for the proportion of infected and immunized in the population (deaths not taken into account), assuming continued lockdown until then. . it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . lockdown lift on may th we simulated the effect of lifting the lockdown on may th assuming that after this date the transmission goes back to its value before lockdown. figure shows the predicted dynamics for each region. in every region, we observed a large rebound occurring either in june or july. the timing and magnitude of this rebound is largely influenced by the importance of the first wave, that is successfully contained thanks to the lockdown. these results strongly argue for enforcing other npis when lockdown is lifted in order to contain r e below and prevent this predictable rebound of the epidemic. in this work, we provide estimations of the key parameters of the dynamics of the covid- epidemic in french regions as well as forecasts according to npis especially regarding the proportion of infected when lifting the lockdown policy. the point estimates of the basic reproductive ratios for french regions fluctuated between . and . before lockdown took effect, but according to the uncertainty around these estimates they are not substantially different from one region to another. therefore, observed differences in the number of cases were due to the epicemic starting first in grand est and Île-de-france regions. these estimates were close to those reported before isolation using other models (alizon et al., ; flaxman et al., ) . the model provided estimates of the impact of the lockdown on the effective reproductive ratio and although recent data led to a substantial reduction of r e after the lockdown, it remains close to thus without a clear extinction of the epidemic. these estimates should be updated with more recent data that may lead to an estimated r below . on the other hand, it is an argument to add other measures such as intensive testing and strict isolation of cases. in addition, the model provides estimates of the size of the population of people who have been or are currently infected. as already reported (di domenico et al., a) , this proportion of subjects is around to percent, so excluding any herd immunity and control of the epidemic by having a large proportion of people already infected and therefore not susceptible. with our estimates of basic reproduction ratio, the epidemic would become extinct by herd immunity with a proportion of . % ( % ci [ . %; . %]) of infected people. interpretation of our results is conditional on the mechanistic model illustrated in figure , and careful attention must be given to the parameters set . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . table , as updated estimates are published every day. first and foremost, our model takes only two kinds of infectious cases into account: confirmed cases i, and unascertained cases a. our observation model takes i as the number of infectious cases confirmed by a positive pcr sars-cov- test. thus, a can be interpreted as . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . unconfirmed symptomatic cases that can be diagnosed by a gp visit (possibly through remote teleconsultation). this is a very simple representation of the covid- infection, which can have various degrees of severity (e.g. asymptomatic, mild, severe) that could be themselves modeled into different compartments. however, very little data is currently available to gather sufficient information to be able to distinguish between those infectious states. second, our model does not have a compartment for covid- patients in icu, and the number of occupied icu beds is simply taken as a fixed percentage ( % based on an estimate from the bordeaux chu university hospital). meanwhile icu bed capacity does not account for the recent surge of available icu beds in response to the covid- epidemic. compared to , our model does not feature an inflow of susceptibles n (and matching outflow) but population movement across regions are limited during the isolation period (see appendix a for a thorough discussion). deaths were also not distinguished from recoveries in the r compartment, but over the observation period this did not impact the main estimates. third, our model does not take into account the age-structure of the population on the contrary to the recently posted report salje et al. ( ) using french data. interestingly, although the models were different and the data not fully identical, our results were comparable. actually, our approach captures a part of the unexplained variability between regions through the random effects. this variability might be explained at least partly through the difference in age-structure and probability of hospitalization according to the age. we would like to underline the interest of making the data publicly accessible as done by santé publique france on the data.gouv.fr web portal, hence allowing our group to work immediately on the topics. furthermore, we have made our code fully available on github www.github.com/sistm/ seircovid and we are currently working on packaging our software for facilitating its dissemination and re-use. in conclusion, the lockdown has clearly helped controlling the epidemics in france in every region. the number of infected people varies from one region to the other because of the variations in the epidemic start in these regions (both in terms of timing and size). hence, the predicted proportion of infected people as of may varies, but stays below % everywhere. it is clear from this model, as in other published models (di domenico et al., b; flaxman et al., ) , that a full and instantaneous lockdown lift would lead to a rebound. additional measures may help in controlling the number of new infections such as strict case isolation, contact tracing (di domenico et al., b) and certainly a protective vaccine for which the . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . strategy of administration to the population remains to be defined (amanat and krammer, ; lurie et al., ; thanh et al., ) . the data from the sursaud r database regarding covid- is available from the data.gouv french government platform at https://www.data. gouv.fr/fr/datasets/donnees-des-urgences-hospitalieres-et-de-sosmedecins-relatives-a-lepidemie-de-covid- . the source code used for this work is available on github at www.github.com/sistm/seircovid . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . strategies to reduce social mixing on outcomes of the covid- epidemic in wuhan, china: a modelling study. lancet public health. raue, a., kreutz, c., maiwald, t., bachmann, j., schilling, m., klingmüller, u., and timmer, j. ( ) . structural and practical identifiability analysis of partially observed dynamical models by exploiting the profile likelihood. bioinformatics, ( ): - . roques, l., klein, e., papaix, j., sar, a., and soubeyrand, s. ( . using early data to estimate the actual infection fatality ratio from covid- in france. medrxiv. tian, h., liu, y., li, y., wu, c.-h., chen, b., kraemer, m. u. g., li, b., cai, j., xu, b., yang, q., wang, b., yang, p., cui, y., song, y., zheng, p., wang, q., bjornstad, o. n., yang, r., grenfell, b. t., pybus, o. g., and dye, c. ( ) . an investigation of transmission control measures during the first days of the covid- epidemic in china. science. valleron, a.-j., bouvet, e., garnerin, p., ménares, j., heard, i., letrait, s., and lefaucheux, j. ( ) . a computer network for the surveillance of communicable diseases: the french experiment. american journal of public health, ( ): - . wang, c., liu, l., hao, x., guo, h., wang, q., huang, j., he, n., yu, h., lin, x., pan, a., wei, s., and wu, t. ( ) . evolving epidemiology and . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . figure s : cumulative incidence hospitalization for covid- at france national level according to either santé publique france or the sursaud r database . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . of note, the earlier slowing down of emergency admissions for covid- suspicions observed in the sursaud r data compared to the si-vic data could lead to an optimistic biais regarding the epidemic stage and evolution in our estimations and predictions . however, since the si-vic data are not publicly accessible before march th , we are currently unable to use this data source to estimate the impact of the lockdown. under-reporting or over-estimation of covid- deaths according to roques et al. ( ) , the infection fatality ratio (ifr) for covid- is . % ( %-ci: . ; . ). figure s shows that using this ifr over-estimate compared to the death currently reported by santé publique france. figure s : observed incident number of ascertained cases and hospitalization at france national level compared to predicted ones, based on estimations with data collected up to either march (before lockdown), march th or april th (delimited by the vertical line). . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . based on ode model ( ), the statistical analysis of the population evolution can be turned into a parameter estimation problem. theoretical identifiability (i.e. the possibility of learning the true values of the model parameters) of epidemiological compartment models is rarely checked. although it relies on unrealistic hypothesis (namely that incident number of ascertained cases and hospitalized cases are observed continuously, without noise and for an infinite length of time in our case), this framework provides important guarantees for the results. to determine which parameters from table can be accurately identified from the available observations we first evaluate the identifiability of this seirah structure with the daisy software from bellu et al. ( ) (based on differential algebra results). we conclude that there is global identifiability of ξ = (b, d q , r, d e , d i ) with known α and d h , even if initial conditions are unknown -which is our case for (e , a ). practical identifiability (for which most existing evaluation methods rely on estimations and are based on fisher information matrix or likelihood profiling (raue et al., )) of these parameters will be discussed in section . . figure s present the occupancy numbers of the six compartments of the model when one parameter is varied while the others are fixed. because the epidemics start date and state is different in each region, it is necessary to estimate in priority e and a . b, d e and d i have a similar impact on the simulations, and we chose to prioritize the estimation of the transmission b as it is a important actionable driver of the epidemics, that can potentially be reduced by interventions. r and d q have little impact on i (in) but are informative for h (in) . since r can be estimated from other data sources, we prioritized the estimation of d q . in addition, there is a strong rational to estimate b and d q as they are directly involved in the computation of the reproductive number. so in the end, we assume d e , d i , and r fixed and we will estimate e , a , b, and d q . both d e and d i are set to a common value across regions, estimated from previous studies referenced in table . to set r, we used data collected from general practitioners through the re-purposing of the réseau sentinelles network to monitor covid- and provide a weekly estimation of the number of incident symptomatic cases (regardless of their confirmation status through a pcr test) at the region level. thus, for each region, r i is set to r s the ratio of observed incident number of ascertained cases over the incident number of symptomatic cases (as defined by the incident symptomatic cases reported by the réseau sentinelles network). values are provided in table . . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . the model ( ( )) is a simplified version of the ode system presented in wang et al. ( ): in the ode system ( ), consider the possibility of an exogenous flow of susceptible modeled by a sustained susceptible inflow n. while this make sense for modeling an outbreak in a region surrounded by other regions free of the epidemic (such as the first outbreak of covid- in wuhan (hubei, china) in late -early ), it is not suitable for a pandemic where the pathogen is circulating in all regions, which is the current situation in all of france covid- . nonetheless we study the possible asymptotic steady states of this model, i.e. the constant values s ( ) , e ( ) , i ( ) , r ( ) , a ( ) , h ( ) possibly reached by the system when t → ∞. without loss of generality, we assume the initial number of removed subjects is set to r(t = ) = . by definition, the steady . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . states verify: with β = d e /r ( /d q + /d i ) and β = d h /d q . in the particular case of n = , the fifth equation in ( ) can be simplified and imposes a ( ) = d i ( − r)β i ( ) /d e which in combination with the third equation leads to i ( ) = and thus a ( ) = h ( ) = e ( ) = . assuming that a(t) = i(t) = for all times t and applying the population conservation principle we identify (n, , , , , ) as a steady state. since the ode imposes that s is strictly decreasing as long as s(t) > and a(t) > (or i(t) > ) and that s and a (or i) cannot reach in finite time if a = (or i = ), this steady state (n, , , , , ) is unstable. having a = (or i = ) leaves us with s ( ) = as the only possible asymptotic steady state value and since conservation principle imposes s + e + i + r + a + h = n , we end up with r ( ) = n and we identify s ( ) , e ( ) , i ( ) , r ( ) , a ( ) , h ( ) = ( , , , n, , ), as the only possible asymptotic steady when a = (or i = ). but in presence of an inflow of susceptible n = , can we expect an asymptotic behavior with extinction of the epidemic. if we set a ( ) = i ( ) = in ( ), the first and fourth equations give us: hence the potential steady state corresponding to the extinction imposes s ( ) = n , r ( ) = and ( , , , n, , ) is no longer a possible steady state point. the only steady state (n, , , , , ) corresponds to the case where no one gets infected nor removed i.e when the epidemic does not start at all. . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . in this new setting, a ( ) , s ( ) and r ( ) are now functions of i ( ) and given by: with β = ( − r)β d i /d e . from algebraic manipulation of ( ), we derive that a necessary and sufficient condition for i ( ) to constitute a steady set is to be a positive real solution of the fourth-order polynomial equation: bd e s ( ) (nd i + (αβ + ) (n − ( + β )i ( ) ))(n − ( + β )i ( ) ) −β n (nd e + n − ( + β )i ( ) ) nd i + n − ( + β )i ( ) = . cc-by . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . sars-cov- vaccines: status report. immunity daisy: a new software tool to test global identifiability of biological and physiological systems the french syndromic surveillance system sursaud r convergence of a stochastic approximation version of the em algorithm report # : expected impact of school closure and telework to mitigate covid- epidemic in france report # : expected impact of lockdown in île-de-france and possible exit strategies statistique annuelle des établissements de santé transmission dynamics of the covid- outbreak and effectiveness of government interventions: a data-driven analysis modelling the impact of covid- upon intensive care services in new south wales isolated sudden onset anosmia in covid- infection critical care utilization for the covid- outbreak in lombardy, italy: early experience and forecast during an emergency response covid- : a remote assessment in primary care the effect of human mobility maximum likelihood estimation in nonlinear mixed effects models the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov ) monolix v r developing covid- vaccines at pandemic speed predicting the number of reported and unreported cases for the covid- epidemic in south korea, italy, france and germany. medrxiv. impact of non-pharmaceutical interventions on the outbreak of coronavirus disease the role of age distribution and family structure on covid- dynamics: a preliminary modeling assessment for hubei and lombardy coronavirus disease (covid- ) situation report - naming the coronavirus disease (covid- ) and the virus that causes it who director-general's opening remarks at the media briefing on covid- - characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of , cases from the chinese center for disease control and prevention a novel coronavirus from patients with pneumonia in china the authors thank romain greffier for his time in discussing the aggregated features of covid- patients care at the bordeaux university hospital. the authors thank the opencovid- initiative for their contribution in opening the data used in this article. bph thanks vincent pey for discussions about the clinical characteristics of the covid- infection. this work is supported in part by the gestepid inria mission covid . the authors have no competing interests to declare. mp, lw, rt and bph designed the study. mp and bph analyzed the data. mp, dd and bph implemented the software code. qc performed identifiability and asymptotic analysis of the model. mp, lw, rt and bph interpreted the results and wrote the manuscript. one of the difficulties in modeling the covid- epidemic in real-time is the reliability and comparability of data sources an reporting artifacts.differences bewteen the sursaud r database and santé publique france reports the santé publique france epidemiological reports cite si-vic as their source. si-vic is a special system that can be activated for the identification and monitoring of victims from terror attacks and exceptional health situations, and that was activated on march th for the covid- epidemic and is maintained by the french agence du numérique en santé. because si-vic is a declarative web-based plateform (with data populated by regional health agencies, emergency services and hospitals) it was likely missing some case declaration when first launched. on the contrary, the sursaud r database. the figure s illustrate the discrepencies and differences between the two database. key: cord- -pvf uon authors: zeitoun, jean-david; faron, matthieu; lefèvre, jérémie h. title: impact of local care environment and social characteristics on aggregated hospital-fatality rate from covid- in france: nationwide observational study date: - - journal: public health doi: . /j.puhe. . . sha: doc_id: cord_uid: pvf uon objectives we aimed to investigate possible differences in aggregated hospital-fatality rate from covid- in france at the early phase of the outbreak, and to determine whether factors related to population or healthcare supply before the pandemic could be associated with outcome differences. study design nationwide observational study including all french hospitals from january , to april , . methods we analysed aggregated hospital-fatality rate. a poisson regression was performed to investigate associations between characteristics pertaining to populational health, socioeconomic context and local healthcare supply at baseline, and the chosen outcome. results on april , , a total number of patients were hospitalized among the french healthcare facilities, including patients in intensive care unit (icu). a total of deaths due to covid- had been recorded, with a median mortality rate per people per department of . (iqr: . - . ). there were significant variations between the french departments even after adjusting on outbreak inception (p< . ). after multivariable analysis, four factors were independently associated with a significantly higher aggregated hospital-fatality rate: a higher icu capacity at baseline (estimate= . ; p= . ), a lower density of general practitioners (estimate= . ; p= . ), a higher fraction of activity from the for-profit private sector (estimate= . ; p< . ), and the ratio of people over (estimate= . ; p= . ). conclusions aggregated hospital-fatality rate from covid- in france seems to vary among geographic areas, with some factors pertaining to local healthcare supply being associated with outcome. first cases of coronavirus disease , the viral pneumonia related to severe acute respiratory syndrome coronavirus (sars-cov- ), were officially identified in december in china and were notified to the world health organization (who) on december , . since then, the epidemic has expanded well beyond china and the pandemic has officially been declared by the who on march , . while italy has been the earliest disease cluster in europe , france has rapidly followed. on february , , the french ministry of health issued the phase i of the national epidemic. phases ii and iii were respectively announced on february , and march , . fatality rate, defined as the number of deaths of patients in whom covid- was confirmed, divided by the total number of covid- cases, seems to vary among countries. italian reports have shown a casefatality rate ranging from approximately % to % , while other countries such as south korea have observed much lower figures. even if there is uncertainty due to variations in case recording, we lack definitive explanations for possible differences in case-fatality rates between countries. the number of tests that could be made to screen and insulate patients has been raised as a possible factor contributing to differences. also, it is not known whether this outcome varies within a country. several factors can likely explain differences such as affected population profile, healthcare environment and quality of care. there has been concern in france regarding critical care capacity with respect to the probable high number of simultaneous severe cases during the outbreak peak. it has been estimated by the french ministry of health that there were approximately , intensive care unit (icu) beds in france yet with differences between regions. estimates forecasted that this capacity would be exceeded. j o u r n a l p r e -p r o o f therefore, we sought to measure aggregated hospital-fatality rate from covid- in france, and to examine the association between populational and local healthcare supply characteristics, and this outcome. we used official and publicly available sources to retrieve and gather the needed data: we also retrieved the number of hospital beds per people, including surgery beds, medicine beds, obstetrical beds, physical medicine beds, psychiatry beds and those in long-term care facilities ( ) according to a report from the french ministry of health, and the total number of adult intensive care beds in each department at baseline, i.e. before the outbreak ( ). last, the fraction of hospital care activity as measured by hospital-days, performed by the for-profit private sector was collected ( ). for each department, the following health indicators were retrieved: overall mortality aggregated hospital-fatality rate was chosen as study outcome (i.e. for each day of the study period, the number of hospital deaths divided by the number of admitted patients). we chose not to analyze case-fatality rate since it would be unreliable in the french case. indeed, france has not performed systematic or large sars-cov- testing, and the number of recorded cases has repeatedly been recognized as being orders of magnitude below actual frequency. conversely, all serious cases of suspected covid- were required to be tested for confirmation. hospitalized cases, whether in regular wards or intensive care units (icus), therefore represent a reliable denominator for calculation. for each day of study period and in each of the french departments, the number of hospitalized covid- patients and the number of covid- patients in icus were collected. also, for each day of study sample, the j o u r n a l p r e -p r o o f cumulative number of covid- -related in-hospital deaths over study period was collected. to account for gaps in outbreak start between areas, the time origin for each department was set to the first day where at least deaths due to covid- had been recorded in total. to investigate the relationship between our covariates and the selected outcome, a mixed-effects poisson generalized linear regression was used. models were adjusted for the number of people living in the department and the corrected day since the beginning coded as a third order polynomial as fixed effects. to account for the hierarchical structure of our data, the department (grouping variable) was used as a random effect. both a random intercept and random slope (for the corrected days since the beginning) were used. any variable achieving a pvalue < . in the univariable analysis was proposed in the multivariable model. in there were a total number of healthcare facilities (including public hospitals, table . the median area of the departments was km (iqr: - km ). the study included data from january , (first french case) to april , . the details of univariate and multivariable analyses are given in table . following univariate analysis, eleven factors were included in the multivariable analysis. apart from the population, four factors were independently associated with a significantly higher aggregated hospital-fatality rate from covid- : a higher icu capacity at baseline (estimate= . ; p= . ), a lower density of general practitioners (estimate= . ; p= . ), a higher fraction of activity from the for-profit private sector (estimate= . ; p< . ) and the ratio of people over (estimate= . ; p= . ). no health indicator was associated with our outcome in the multivariable analysis. in this nationwide observational study regarding covid- in france, we found significant differences between areas in terms of aggregated hospital-fatality rate. four factors were associated with our study outcome: a higher density of icu beds at baseline, a lower fraction of hospital care activity from the for-profit private sector, a j o u r n a l p r e -p r o o f lower density of general practitioners, and a greater proportion of people over were all predictors of higher aggregated hospital-fatality rate in the current model. our study has several strengths. first, it is a nationwide analysis gathering exhaustive data from reliable sources. for most of covariates, year of availability was very recent, thereby limiting timeliness issues. in addition, the variables of interest are unlikely to significantly change across a relatively short period of time. second, we collected a very diverse set of data regarding demographics, populational health, wealth, and also characteristics of care supply and local healthcare ecosystems. populational health data were in particular critical to incorporate in the model since they are factors likely to influence disease outcome. we had very fine health data beyond age, namely prevalence of chronic conditions that have already been recognized as risk factors for covid- outcome. , , third, we used a robust statistical model to analyse the data, namely a poisson linear model as the variables were daily counts and a mixed model as the observed data were not independent (repeated measures within a department), which allows separate intercept and slopes for each department. also, time-adjustment was made so as to align all departments on a similar basis and take into account timeliness issues. our findings have implications. critical care capacity has been a matter of concern regarding covid- outbreak. it has been predicted that france did not have enough icu beds to absorb all of the patients in need along several days or weeks. yet we found no evidence that less icu beds at baseline in a given area were associated with a worst outcome. conversely, we found that areas with an initial higher density of icu beds were associated with a higher aggregated hospital-fatality rate. we do not have any certain explanation for those unexpected findings. it may be that critically ill patients were more often transferred from rural areas or smaller facilities to more j o u r n a l p r e -p r o o f comprehensive facilities. it also should be underlined that hospitals have anticipated the outbreak progression by resetting their organization and creating new icu capacity in other wards. we could not measure actual icu beds at a given time since those data were not consistently reported. this will need further investigation. we also found that areas in which the density of general practitioners was higher were associated with a better outcome. even though this should be interpreted with caution, one may hypothesize that general practitioners played a critical role in the epidemic, through adequate orientation of covid- patients to hospitals while maintaining others at home. last, it is remarkable that social and wealth factors were not associated with the chosen outcome. the relationship between wealth and health has been consistently documented by a huge body of literature. again, we cannot certainly explain why herein departments with more deprivation were not associated with a higher aggregated hospital-fatality rate yet it should be recalled that france has a very protective social system with a great safety net. perhaps it helped to attenuate the social risk in the case of the epidemic. this study has limitations. firstly, as an observational study, it cannot establish definitive causality. we cannot exclude the possibility that our results might be confounded by factors that were not measured. in particular, we cannot rule out that criteria for admitting patients were different among areas and that some hospitals had more serious cases than others, whether in regular wards or icus. also, we did not have access to age-and gender-structure of hospitalized patients. last, we did not take into account control measures implemented in the different departments even though those measures were thought to be very similar. secondly, the follow-up was intentionally limited. however, given the high urgency that many healthcare systems are currently facing worldwide, we aimed at rapidly providing a first evaluation of j o u r n a l p r e -p r o o f hospital-fatality rates from covid- in a markedly affected country. subsequent work over the outbreak course will say whether local differences and their associated factors persist. thirdly, we did not have access to hospital data or patient data. thus, we could not calculate individual hospital-fatality rate and had to deal with aggregate measures which have been updated on a daily basis at the department level over the study period. fourth, we intentionally excluded nursing home since the related data were not available across the whole study period. this represents a selection bias. last, as of march , , the french government decided to implement targeted transfers of seriously ill patients by medicalized trains or helicopters in order to improve resource allocation within the whole territory. those transfers may have interfered with our results even though we believe it is unlikely. indeed, reported counts of those transfers showed it involved very few patients as compared to the magnitude of the epidemic. it seems implausible that it significantly influenced the findings from the regression analysis, which were otherwise consistent over time. in conclusion, we found significant differences in aggregated hospital-fatality rate across french areas over the early period of the covid- outbreak. several factors pertaining to local healthcare supply were associated with a worst outcome, such as a higher icu capacity at baseline and a lower involvement from the private sector as well as a lower density of general practitioners. those findings clearly deserve further investigation with hospital-or patient-level data and over a longer follow-up. those departments have been chosen to illustrate the heterogeneity of situations across the whole french territory (see figure ). world health organization. who director-general's opening remarks at the media briefing on covid- - critical care utilization for the covid- early experience and forecast during an emergency response arrêté du mars portant diverses mesures relatives à la lutte contre la propagation du virus covid- case-fatality rate and characteristics of patients dying in relation to covid- in italy transmission potential and severity of covid- in south korea coronavirus : les simulations alarmantes des épidémiologistes pour la france health as an independent predictor of the french presidential voting behaviour: a crosssectional analysis les établissements de santé -édition the association between income and life expectancy in the united states clinical characteristics of coronavirus disease in china characteristics of and important lessons from the covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention key: cord- -mvtux x authors: pullano, g.; di domenico, l.; sabbatini, c. e.; valdano, e.; turbelin, c.; debin, m.; guerrisi, c.; kengne-kuetche, c.; souty, c.; hanslik, t.; blanchon, t.; boëlle, p.-y.; figoni, j.; vaux, s.; campese, c.; bernard-stoecklin, s.; colizza, v. title: underdetection of covid- cases in france in the exit phase following lockdown date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: mvtux x a novel testing policy was implemented in may in france to systematically screen potential covid- infections and suppress local outbreaks while lifting lockdown restrictions. , virologically-confirmed cases were reported in mainland france from may , (week , end of lockdown) to june (week ). accounting for missing data and the delay from symptom onset to confirmation test, this corresponds to , [ % ci , - , ] cases with symptom onset during this period, a likely underestimation of the real number. using age-stratified transmission models parameterized to behavioral data and calibrated to regional hospital admissions, we estimated that , [ , - , ] covid- symptomatic cases occurred, suggesting that out of cases with symptoms were not ascertained. median detection rate increased from % [ - ]% to % [ - ]% over time, with regional estimates varying from % (grand est) to % (normandy) by the end of june. healthcare-seeking behavior in covid- suspect cases remained low ( %) throughout the period. model projections for the incidence of symptomatic cases ( . [ . - . ] per , ) were compatible with estimates integrating participatory and virological surveillance data, assuming all suspect cases consulted. encouraging healthcare-seeking behavior and awareness in suspect cases is critical to improve detection. substantially more aggressive and efficient testing with easier access is required to act as a pandemic-fighting tool. these elements should be considered in light of the currently observed resurgence of cases in france and other european countries. as countries in western europe gradually relaxed lockdown restrictions, robust surveillance and detection systems became critical to monitor the epidemic situation and maintain activity at low levels . the need is to rapidly identify and isolate cases to prevent onward transmission in the community and avoid substantial resurgence of cases. in france, the surveillance strategy implemented by authorities to exit lockdown on may , was multifold , and based on an expanded case definition for covid- suspect cases to guide clinical diagnosis ; recommendations to the general population to seek healthcare even in presence of mild symptoms; prescription of diagnostic tests to suspect cases by general practitioners for systematic and comprehensive testing; isolation of confirmed cases and tracing of their contacts. the specific characteristics of covid- epidemic, however, hinder the identification of cases . large proportions of asymptomatic infectious individuals , and presence of mild or paucisymptomatic infections that easily go unobserved , present serious challenges to detection and control [ ] [ ] [ ] . this may potentially result in substantial underestimates of the real number of covid- cases in the country. here we estimated the rate of detection of covid- symptomatic cases in france in may-june after lockdown, through the use of virological and participatory syndromic surveillance data coupled with mathematical transmission models calibrated to regional hospitalizations. the study focused on mainland france where the epidemic situation was comparable across regions, and excluded corsica reporting a very limited epidemic activity and overseas territories characterized by increasing transmission . covid- epidemic management in france in the post-lockdown phase involved the creation of a centralized database collecting data on virological testing (si-dep, information system for testing) to provide indicators to monitor the epidemic over time , . , virologically-confirmed cases were reported from may (week ) to june (week ) in mainland france. after imputation of missing data (see methods), an estimated , [ %ci , - , ] cases with symptoms resulted in the study period (figure ) . the average delay from symptom onset to testing decreased from . days in week (w ) to . days in w . accounting for this delay (see methods), we estimated that , [ % ci , - , ] confirmed symptomatic cases had onset in the study period, showing a decreasing trend over time ( , in w , in w ). the test positivity rate decreased in the first weeks and stabilized around . % (average over w -w ). a digital participatory system was additionally considered for covid- syndromic surveillance in the general population , including those who do not consult a doctor. called covidnet.fr, it was adapted from the platform grippenet.fr (dedicated to influenza-like-illness surveillance since , ) to respond to the covid- health crisis in early . it is based on a set of volunteers who weekly self-declare their symptoms, along with socio-demographic information. based on symptoms declared by approximately , participants each week, the estimated incidence of covid- suspect cases decreased from about % to . % over time (figure ) , according to the expanded suspect case definition recommended by the high council of public health for testing (methods). out of suspect cases ( %) consulted a doctor in the study period. among them, ( %) received a prescription for a test, resulting in screening for individuals ( % of those given the prescription). week of symptom onset for symptomatic confirmed cases was estimated based on patients' declarations (see panel b) through a gamma distribution fitted to the data with a maximum likelihood approach. missing data about presence/absence of symptoms and declaration of onset were imputed by region and by week, by sampling from a multinomial distribution according to the observed breakdown among cases with complete information (see methods). test positivity rate was computed on cases with complete information. data for weeks - were consolidated in w . (b) breakdown of virologically-confirmed symptomatic cases by week of testing according to declared onset of symptoms, along with estimated mean time from onset to testing. (c) incidence of covid- suspect cases (estimates by week and -week moving average (thick line)), along with percentage of those seeking healthcare, estimated from participatory surveillance system covidnet.fr. (d) number of covid- suspect cases of the participatory cohort seeking healthcare, and among them those receiving a prescription, and performing a virological test given the prescription. covidnet.fr estimates were adjusted on age and sex of participants. (e) estimated change in individuals attending their workplace locations over time and by region based on google location history data we used stochastic discrete age-stratified epidemic models , based on demography, age profile , and social contact data of the regions of mainland france, to account for age-specific contact activity and role in covid- transmission. disease progression is specific to covid- , and parameterized with current knowledge to include presymptomatic transmission , asymptomatic and symptomatic infections with different degrees of severity (paucisymptomatic, with mild symptoms, with severe symptoms requiring hospitalization) , . the model was shown to capture the transmission dynamics of the epidemic in Île-de-france and was used to assess the impact of lockdown and exit strategies , . full details are reported in the methods section. intervention measures were modeled as modifications of the contact matrices, accounting for a reduction of the number of contacts engaged in specific settings, and were informed from empirical data. lockdown data came from refs. , . the exit phase was modeled considering region-specific attendance at school based on . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint ministry of education's data , partial maintenance of telework according to estimated presence in workplaces from mobile phones location history data (figure ) , reduction in adoption of physical distancing over time based on survey data (figure ) , partial reopening of activities, senior protection . a sensitivity analysis was performed on the reopening of activities and senior protection, as data were missing for an accurate parameterization. testing and isolation of detected cases were implemented by considering a % reduction of contacts for the number of virologically-confirmed covid- cases , . region-specific models were calibrated to regional hospital admission data (figure ) through a maximum likelihood approach in the phase before lockdown, during lockdown, and in the exit phase. further details are reported in the methods section. (a-c) hospital admissions over time, data (points) and simulations (median and % probability range), for Île-de-france (a), pays de la loire (b), normandie (c). hospital admission data up to w (consolidated in w ) were used to calibrate the models. (d-f) projected number of new symptomatic cases over time (median and % probability range) and estimated number of virologically-confirmed symptomatic cases by week of onset (points), for the same regions above. the estimated detection probability of symptomatic cases (%) is also shown (red points, median and % probability ranges, right y axis). projected number of cases decreased over time in all regions, in agreement with the decreasing tendency reported in hospital admissions in the study period (figure ) . overall, , [ , - , , % probability range] new infections were predicted in mainland france in weeks - (from , [ , ] in w to , [ , - , ] in w ). Île-de-france was the region with the largest predicted number of cases ( , [ , - , ] to [ - , ] from w to w ), followed by grand est and hauts-de-france (table ) . projections were substantially higher than virologically-confirmed cases (figures and ) . the estimated detection rate for symptomatic infections in mainland france in the period w -w was % [ - %], suggesting that out of new cases with symptoms were not identified by the surveillance system. estimated detection rate increased over time ( % [ ] [ ] [ ] [ ] % in w , % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] % in w ). by the end of june, . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint regions had a median detection above or equal % (figure ) , and regions detected a number of cases within the probability ranges of model projections ( table ) . all regions except pays de la loire displayed increasing trends in the estimated detection rate. we compared the projected incidence of covid- symptomatic cases in w ( . [ . - . ] per , ) with the value obtained from confirmed cases ( . per , ) and two estimates based on covidnet.fr data (figure ). the first estimate applies the measured test positivity rate to the number of self-reported covid- suspect cases (estimate # , yielding . [ . - . ] per , ); the second additionally assumes that only % would be confirmed as suspect case by a physician and prescribed a test (according to covidnet.fr data on consulting participants, estimate # , yielding . [ . - . ] per , ). our projections are in line with plausible estimates from covidnet.fr. sensitivity analysis showed that findings were robust against elements of the contact matrices that could not be informed by empirical data, and against current epidemiological uncertainties. including in the analysis also asymptomatic cases led to higher detection rates, % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] % in w compared to % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] % for symptomatic cases only. this however assumes that asymptomatic cases were detected by the virological surveillance system in the week of infection, as no additional information was available to adjust for the possible delay. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint table . number of virologically-confirmed symptomatic cases, number of projected symptomatic cases, estimated detection rate, estimated trend in detection rate, population per region. regions are ranked by decreasing number of confirmed cases in w . the trend is estimated comparing the average of the estimated detection rate in the weeks of june (w - ) with the average in the weeks of may (w -w ). despite a test positivity rate in mainland france well below who recommendations ( %) , a substantial proportion of symptomatic cases ( out of ) remained undetected in the first weeks following the end of lockdown. more than , symptomatic infections were not ascertained by the surveillance system from may to june , , according to our estimates. surveillance improved substantially over time. detection rate was estimated to be % [ ] [ ] [ ] [ ] % at the national level in mid-may, in line with estimates for the same period from a seroprevalence study in geneva, switzerland . by the end of june, it increased to % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] %, leaving about / of cases with symptoms undetected. two regions (occitanie, normandy) reported cases compatible with model projections. these figures suggest that the new surveillance framework was progressively strengthened with increasing resources and capacity over time. detection became also faster, with a % reduction of the delay from symptom onset to testing. at the same time, increasing performance benefited from a concurrent decrease of the epidemic activity in all regions. despite this positive trend, our findings highlight a critical need for improvement. some regions remained with limited diagnostic exhaustiveness by the end of june. this is particularly concerning in those regions predicted to have a large number of weekly infections, such as Île-de-france where approximately only out of cases with symptoms were detected by the end of june, and grand est ( out of ). novel recommendations since the end of lockdown require that all patients with symptoms suggestive of covid- (as well as contacts of a confirmed case) be screened for sars-cov- . almost all cases ( % since may ) clinically diagnosed by sentinel general practitioners as possible covid- cases were prescribed a test . however, only % of individuals with covid- -like symptoms consulted a doctor in the study period according to participatory surveillance data. overall, these figures suggest that a large number of symptomatic covid- cases were not screened because they did not seek medical care despite recommendations. a similar evidence emerged from a large-scale serological study in spain where only . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint between % and % of symptomatic participants with antibodies against sars-cov- reported a previous virological screening . by combining estimates from virological and participatory surveillance, together with measured rates for test recommendations by general practitioners (e.g., due to misclassification of selfreported symptoms), we extrapolated an incidence of symptomatic cases from crowdsourced data that is compatible with model projections. this finding further supports consultation for all covid- suspect cases. large-scale communication campaigns should reinforce recommendations to raise awareness in the population and strongly encourage healthcare-seeking behavior especially in patients with mild symptoms. at the same time, investigations to identify reasons for not consulting could be quickly performed through the participatory surveillance system. red tape might have contributed to low testing rates. prescription for a test was deemed compulsory in the new testing policy to prevent misuse of diagnostic resources , however the path involving consultation, prescription, and lab appointment may have discouraged mildly affected individuals not requiring medical assistance. to facilitate access, some local initiatives emerged recently that increase the number of drivethrough testing facilities, mail test vouchers to promote massive screening in certain regions (e.g. in Île-de-france ), offer temporary mobile testing facilities (buses, pavilions) to increase proximity with the population . these initiatives are particularly relevant for counteracting socio-economic inequalities in access to information and care in populations vulnerable to covid- and may be established in the longterm. given the non-uniform detection rate estimated within the country, learning from specific regional realities may aid to improve detection. the recent change in screening policy no longer requiring a prescription for testing could further improve access. screening rates remained overall well below the objective fixed by authorities for the post-lockdown phase (average weekly number of tests in may-june was , vs. target of , ), and the delay from onset to screening was still very long ( days) by the end of june, despite substantial reduction over time. the large demand for testing currently observed in certain regions, mainly as a result of imminent travels and protocols imposed by certain countries and air companies, is reportedly causing long waiting lists at overwhelmed testing sites . given pre-symptomatic transmission, notification to contacts should be almost immediate to allow the effective interruption of transmission chains . for testing to be an actionable tool for surveillance and, most importantly, for control of covid- transmission, screening rates should be radically increased and delays suppressed. the risk would otherwise be a rapid and uncontrolled resurgence of cases with potential transmission in the community , as currently reported in some french areas (e.g. mayenne district in pays de la loire region) and countries in europe . such risk is expected to increase if the reported relaxation in preventive behaviors persists, due to adhesion fatigue . aggressive and efficient testing will become increasingly more important in the fall months, as other respiratory viruses, such as influenza, rsv, rhinoviruses, will start to circulate and influenza-like-illness incidence levels will become comparable with those of covid- . reviewing the testing strategy over summer, while at low covid- epidemic activity, is an important opportunity to strengthen french response system for next season. models were region-based and did not consider a possible coupling between regional epidemics caused by mobility. this choice was supported by stringent movement restrictions during lockdown , and by the limited mobility increase in may-june , before important inter-regional displacements took place at the start of summer holidays in july. foreign importations were neglected , as france reopened its borders with eu member states on june , and the schengen area remained closed till july. covidnet.fr cohort is not representative of the general population , however the agreement found with sentinel incidence trends for influenza-like-illness suggests that these limitations have little effect once results are adjusted for lack of representativeness . underdetection may also proceed from the imperfect characteristics of rt-pcr (reverse transcription-polymerase chain reaction) tests used to identify infected cases . some cases tested for sars-. cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint cov- could have been falsely negative. this would affect the analysis presented in the manuscript and would be in line with our conclusion that a large part of cases may have been undetected. asymptomatic infections were not considered in the main analysis, as we lacked information on the likely time of infection. the median duration from first to last positive nasopharyngeal swab was estimated to be days in asymptomatic patients in china, with the longest duration at days . no such analysis has been performed in france yet. assuming that asymptomatic infections were rapidly identified through contact tracing yielded higher detection rates than estimated for symptomatic cases only. this is due to a proportionally higher fraction of asymptomatic cases among the confirmed ones. though limited by the underlying assumption, this result further strengthens the main conclusion that detection of symptomatic index cases is the key aspect that requires fundamental improvement. our findings identify critical needs of improvement to increase case ascertainment in france and the performance of the response system to monitor and control covid- epidemic. substantially more aggressive and efficient testing needs to be achieved to act as a pandemic-fighting tool. these elements should be considered in light of the resurgence of cases currently observed in some regions in france and in other countries with similar response systems. virological surveillance data and analysis. the centralized database si-dep for virological surveillance collects detailed information on patients tested in france, including (i) date of test, (ii) result of test (positive or negative), (iii) location (region), (iv) absence or presence of symptoms, (v) self-declared delay between onset to test in presence of symptoms. the delay is provided with the following breakdown: onset date occurring - day before date of test, - days before, - days before, - days before, or > days before. the si-dep database provided complete information for , ( %) out of , laboratory-confirmed covid- cases tested between week (may -may ) and week (june -july ), with an increasing trend of complete information over time. the study referred to the period from w to w ; data of w were used to account for the delays. data were consolidated in w . for cases with complete information, we estimated the number of symptomatic laboratory-confirmed covid- cases by date of onset, using the information on the date of test and the time-interval of onset-to-test delay declared by the patient. we fitted a gamma distribution to these data with a maximum likelihood approach, obtaining a shape parameter equal to . , and expected value of delay equal to days. given a symptomatic confirmed case tested on a specific date, we assigned the onset date by sampling the onset-to-testing delay from the fitted distribution, conditional to the fact that the delay lies in the corresponding time-interval declared by the patient. to account for the changes in the distribution of self-declared delays over time, we also fitted the distribution to three periods of time, obtaining no significant difference. cases with missing data were imputed by sampling from a multinomial distribution with probabilities equal to the rate of occurrence of the labels reported for cases with complete information. imputation was performed by region and by week. onset date was then estimated for the imputed symptomatic cases. participatory surveillance data and analysis. covidnet.fr is a participatory online system for the surveillance of covid- , available at www.covidnet.fr. it was adapted from grippenet.fr to respond to the covid- health crisis in march . grippenet.fr is a participatory system for the surveillance of influenzalike-illness available in france since through a collaboration between inserm, sorbonne universite and sante publique france , , , supplementing sentinel surveillance. the system is based on a dedicated website to conduct syndromic surveillance through self-reported symptoms volunteered by participants resident in france. data are collected on a weekly basis; participants also provide detailed profile information at enrollment. in addition to tracking influenza-like-illness incidence , , grippenet.fr was used to estimate . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint vaccine coverage in specific subgroups individual perceptions toward vaccination and health-seeking behavior . it was also used to assess behaviors and perceptions related to other diseases beyond influenza . participants are on average older and include a larger proportion of women compared to the general population , . participating population is however representative in terms of health indicators such as diabetes and asthma conditions. despite these discrepancies, trends of estimated influenza-like-illness incidence from grippenet.fr reports compared well with those of the national sentinel system , . all analyses were adjusted by age and sex of participants. to monitor covid- suspect cases in the general population, we used the expanded case definition recommended by the high council of public health for systematic testing and described in their april notice : • (sudden onset of symptoms or sudden onset of fever) and (fever or chills) and (cough or shortness of breath or (chest pain and age > years old)) • or (sudden onset of symptoms or (sudden onset of fever and fever)) and o (age > years old and (feeling tired or exhausted or muscle/joint pain or headache or (loss of smell without runny/blocked nose) or loss of taste) o or ((age ≥ years old or age < years old) and diarrhea) o or (age < months old and (fever without other symptoms))). figure reports two estimates obtained from covidnet.fr cohort data for the incidence of symptomatic cases in w . they are computed as follows: • estimate # = (covidnet.fr estimated incidence in w ) * (test positivity rate from si-dep in w ) • estimate # = (covidnet.fr estimated incidence in w ) * (estimated proportion screened and confirmed as covid- suspect case by a physician, and prescribed a test; estimates from covidnet.fr) * (test positivity rate from si-dep in w ) ethics statement. grippenet.fr/covidnet.fr was reviewed and approved by the french advisory committee for research on information treatment in the health sector (i.e. cctirs, authorization . ), and by the french national commission on informatics and liberty (i.e. cnil, authorization dr- - ) -the authorities ruling on all matters related to ethics, data, and privacy in the country. transmission models summary. we used a stochastic discrete age-structured epidemic model for each region based on demographic, contact , and age profile data of french regions . four age classes were considered: [ - ), [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , and + years old. transmission dynamics follows a compartmental scheme specific for covid- , where individuals were divided into susceptible, exposed, infectious, and hospitalized. we did not consider further progression from hospitalization (e.g. admission to icu, recovery, death ) as it was not needed for the objective of the study. the infectious phase is divided into two steps: a prodromic phase ( ) and a phase where individuals may remain either asymptomatic ( , with probability = % ) or develop symptoms. in the latter case, we distinguished between different degrees of severity of symptoms, ranging from paucisymptomatic ( ), to infectious individuals with mild ( ) or severe ( ) symptoms. prodromic, asymptomatic and paucisymptomatic individuals have a reduced transmissibility = . , as estimated in ref. . a reduced susceptibility was considered for younger children and adolescents, along with a reduced relative transmissibility of younger children, following available evidence from household studies, contact tracing investigations, and modeling works [ ] [ ] [ ] [ ] [ ] [ ] . a sensitivity analysis was performed on relative susceptibility and transmissibility of younger children, and on the proportion of asymptomatic infections. models calibration. models were calibrated regionally to daily hospital admission data through a maximum likelihood approach. the likelihood function is of the form . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august , . is the time window considered for the fit. calibration involved three steps, each one corresponding to a different epidemic situation: pre-lockdown , during lockdown , post-lockdown. covid- ) in the eu/eea and the uk -tenth update prise en charge par les médicins de ville des patients atteints de covid- en phase de déconfinement testing for covid- : a way to lift confinement restrictions signes cliniques d'orientation diagnostique du covid- factors that make an infectious disease outbreak controllable suppression of a sars-cov- outbreak in the italian municipality of vo' substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov ) estimates of the severity of coronavirus disease : a model-based analysis tracing and analysis of early sars-cov- infections outside china: a modeling study reconstruction of the full transmission dynamics 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children including covid- : an overview of the epidemiology, clinical features, diagnosis, treatment and prevention options in children a case series of children with novel coronavirus infection: clinical and epidemiological features sars-cov- infection in primary schools in northern france: a retrospective cohort study in an area of high transmission cluster of covid- in northern france: a retrospective closed cohort study. medrxiv this study was partially supported by anr project dataredux (anr- -ce - - ) and evalcovid- (anr- - covi- ); eu h grants mood (h - ) and recover (h - ); reacting covid- modeling and surveillance grants. we thank pascal crepey, camille pelat, edouard chatignoux, juliette paireau, daniel levy-bruhl for useful discussions. we also thank all participants of covidnet.fr system. key: cord- -ihh q f authors: nan title: posters p - p date: - - journal: eur biophys j doi: . /s - - -x sha: doc_id: cord_uid: ihh q f nan in order to understand mrna stability, we proceed to the studie of life time model system composed of the regb ribonuclease and the ribosomal protein s . the t bacteriophage life cycle is modulated by regb which mediate speci c mrna degradations. regb cleaved the mrna at the middle of the translation initiation region (shine-dalgarno sd). studies had shown that regb activity is enhanced with addition of s which is normaly required for the translation of mrna in case of unusual sd regions. s is considered as a key factor of translation's inititiation. composed of six similar domains (f -f ), it interacts with the ribosome by his f -f domains and with the mrna by f -f . we had shown that the f fragments got the same properties than the whole protein. many global architectures had been published (linear or globular conformation). then we try to understand what is the catalysis way of s in the regb activation and in rna recognition. we study the global conformation of f fragment. for that, we use nmr to determinate the domains interfaces. at rst we made the nmr backbone assignement of n/ c/ h labeled fragments (f -f ). then we analysed hsqc overlapping of each fragments in order to identify interfaces residues. the residues identi cation on s homologous model allowed us to determinate interaction region between domains. currently we study of s -rna interactions to determinate if the ligand conformation could change the interaction region and involve conformationals changes. translocation of amino acids from the membrane interface to the interior: theory and experimiment c. aisenbrey , e. goormaghtigh , j.-m. ruysshaert , b. bechinger ulp/cnrs, chemistry, rue blaise pascal, strasbourg, université libre, campus plaine cp / , brussels the interactions of a series of histidine-containing peptides with biological model membranes have been investigated by attenuated total re ection fourier transform infra red (atr-ftir) and oriented solid-state nmr spectroscopies. related peptides have previously been shown to exhibit antibiotic and dna transfection activities. the -residue lah x and lah x peptides were designed in such a manner to form amphipathic helical structures in membrane environments. four histidines and four/six variable amino acids x constitute one face of the helix whereas leucines and alanines characterize the opposite hydrophobic surface. the dichroic ratio of atr-ftir spectra, or the orientation-dependent n solid-state nmr chemical shift have been used to follow the ph-dependent transition from in-plane to transmembrane alignments upon increase in ph. a theoretical model of the topological modulations is presented and the experimental transition curves analysed in order to reveal the gibbs free energy of transition. the novel concept provides access to the free energy changes associated with the amino acids x incorporated into an extended -helix and in the context of phospholipid bilayers. for the peptides of the lah x series the gibbs free energies associated with the transition from the membrane interface to the bilayer interior follow the sequence of amino acids: l < a i < s f < t g < v w << y. here we present a novel solid-state nmr approach which allows for the accurate determination of the tilt and rotational pitch angles of peptides reconstituted into uniaxially oriented membranes. the method works with transmembrane or in-plane oriented peptides that have been labelled with , , - h -alanine and n-leucine at two selected sites. proton-decoupled n and h solid-state nmr spectroscopy at sample orientations of the membrane normal parallel to the magnetic eld direction have been used to characterize the tilt and rotational pitch angle of these peptides in considerable detail. the same samples when inserted into the magnetic eld at degrees tilted alignments provide valuable information on the rotational diffusion constants in membranes and thereby of the association and size of peptide complexes within the membrane environments. whereas monomeric transmembrane peptides exhibit spectral averaging and well-de ned resonances, larger complexes are characterized by broad spectral line shapes. in particular the deuterium line shape is sensitive to association of a few transmembrane helices. in contrast, the formation of much larger complexes affects the n chemical shift spectrum. aisenbrey ch. & bechinger, b. biochemistry , - ( ) a meccano set approach of joining trpzip a water soluble -hairpin peptide with a didehydrophenylalanine containing hydrophobic helical peptide p. chetal, v. chauhan, d. sahal international centre for genetic engineering & biotechnology,new delhi ,india a residues long, water soluble, monomeric -hairpin peptide "trpzip" [cochran et al ( ) pnas , - ], stabilized by tryptophan zipper has been linked via a tetraglycyl linker to a hydrophobic didehydrophenylalnine ( f) containing helical octapeptide. circular dichroism studies of this residues long peptide, "trpzipalpha" (ac-gewtwddatktwtwte-gggg-fal fal fa-nh ) in water have revealed the presence of both the -hairpin and the helical conformations. this is the rst instance where a f containing peptide has been found to display a helical fold in water. the uorescence emission wavelengths of tryptophan in ac-g-w-g-nh , trpzip and trpzipalpha were . , . and . nm respectively. the uorescence quantum yield of trpzip was . fold higher than trpzipalpha suggesting that proximal interactions between the -hairpin and the helix caused the quenching of tryptophan uorescence in the former by the fs in the latter. the molar ellipticity of the far uv couplet characteristic of trpzip was reduced in trpzipalpha and the cd based thermal melting temperatures at nm were c (trpzip) and c (trpzipalpha). a concentration dependent variable temperature cd study in water showed that in trpzipalpha, increasing temperature is detrimental to the -hairpin, but it augments the helical motif by intermolecular oligomerization. our results show that in water, trpzipalpha exhibits long-range interactions between two different secondary structures. structural-based differential stability in the yoeb-yefm toxin-antitoxin module i. cherny, e. gazit department of molecular microbiology and biotechnology, faculty of life sciences, tel aviv university, tel aviv , israel the speci c physiological role of natively unfolded proteins is only recently beginning to be explored. a notable case in which natively unfolded state appears to have physiological signi cance is the e. coli yoeb-yefm toxin-antitoxin (ta) module. a crucial element in proper functioning of ta systems requires physiological instability of the antitoxin in contrast to the stable pro le of its toxin partner. we have shown that yefm antitoxin is a natively unfolded protein, lacking secondary structure even at low temperatures. in contrast, its toxin partner has a well-folded conformation at physiological temperatures. we suggest that the structural-based differential thermodynamic stability between the two components is the cause for their differential physiological stability, since structural instability of the antitoxin exposes it to cellular quality-control machinery. we further revealed that yefm and yoeb interact and form tight complex and determined it stoichiometry. a potential use of ta systems is as novel antibacterial targets. indeed, we identi ed homologous yefm-yoeb systems in a large number of bacteria including major pathogens. we aim to design peptides capable of interfering with the yefm-yoeb interaction, thus releasing the toxin to execute its detrimental function. for this purpose, we identi ed a short linear determinant within yefm that is involved in yoeb interaction. this peptide motif will be optimized for development of antibacterial lead molecules. a. chatterjee, a. prabhu, a. ghosh-roy, r. v. hosur tata institute of fundamental research, mumbai, india- dynein light chain (ddlc ), a member of the cytoplasmic motor assembly exists as a monomer or a dimer (functional form) under different experimental conditions. here we report the unfolding characteristics of the monomeric ddlc at ph , due to urea and guanidine hydrochloride, by various biophysical techniques. it is observed that the unfolding pathways due to the two denaturants have many differences. urea unfolding seems to be two state, while guanidine unfolding is more complex. the nmr experiments carried out at low denaturant concentrations have enabled detailed characterization of the structure and dynamics of the near native excited states of the protein. these are similar to the native state in structure, except for the small extensions of the helices in the nterminal half of the protein. however, the local stabilities of the and -strands are perturbed and this occurs differently in the two denaturants. in the guanidine case the entire multi-stranded -sheet in the c-terminal half is destabilized. in either case the motional characteristics, seem to suggest the presence of a nite population of the dimer in the excited state ensemble. these states are suggested to be likely intermediates in the momoner-dimer transition, and their characterization here thus provides clues to the molecular mechanism of the transition. it is also envisaged that the near native excited states could play regulatory roles in the functioning of the protein. kinetic bottlenecks identi cation in different folding models f. cecconi , c. guardiani , r. livi infm -istituto di sistemi complessi -cnr, italy, centro interdipartimentale per lo studio delle dinamich complesse, università di firenze, italy, dipartimento di sica, università di firenze, italy the ww domains are a family of fast folding, compact, modular domains featuring a triple-stranded, antiparallel beta-sheet. the ww domain of the pin protein, due to the availability of a complete picture of the residues involved in thermodynamic stability and in the formation of the transition state, in particular, represents an excellent benchmark to test computational methods. the objective of the present work is to identify the kinetic bottlenecks in the folding process through md unfolding simulations at increasing temperatures. the kinetic bottlenecks are related to the establishment of contacts requiring the overcoming of a large entropy barrier and acting as a nucleus for the creation of further contacts. the key sites are therefore those involved in contacts showing a dramatic decrease in fractional occupation near the speci c heat peak. the technique was applied to the go model and to a model based on the knowledge of secondary structure, providing in both cases a picture of the folding process consistent with the experimental data. evidence is also shown that while the go model allows a more accurate prediction of the native structure, the folding pathway is better described by the other model. the protein talin plays a key role in coupling the integrin cell adhesion molecules to the actin cytoskeleton and in integrin activation. the globular head of talin, which binds -integrins, is linked to a rod containing an actin-binding site and binding sites for the protein vinculin, which regulates the dynamic properties of cell-matrix junctions. we have determined the structure of three domains which contain vinculin binding sites (vbss) and shown that each of these are made up of helical bundles. the structures of complexes between vinculin and peptides corresponding to the vbss show that the residues which interact with vinculin are buried in the hydrophobic core of the helical bundles of the talin domains. nmr studies of the interaction of one of these domains with vinculin shows that it involves a major structural change in the talin fragment, including unfolding of one of its four helices, to make the vbs accessible. while the observation of folding of unstructured regions of a protein on interaction with a 'partner' is quite common, this kind of major unfolding to permit a protein-protein interaction is much less common. ways in which it may be regulated will be discussed. conformational changes of eye lens proteins studied by combined saxs and high pressure s. finet , f. skouri-panet , a. tardieu european synchrotron radiation facility, rue horowitz, bp , grenoble france, institut de minéralogie et de physique des milieux condensés, rue de lourmel, paris france, protéines: biochimie structurale et fonctionnelle, case , quai st-bernard, paris france -, -and -crystallins are the main components of vertebrate eye lenses, with exceptional structural and associative properties. the crystallins are known to be exceptionally stable in vivo since they have to last the lifetime of the organism. they therefore represent an extreme case of stability versus unfolding and aggregation. these proteins are mainly beta strands. -crystallins are kda monomers (from to % sequence identity), and -crystallins are large hetero-oligomers of about kda. -crystallins are molecular chaperone; they belong to the ubiquitous superfamily of small heat shock proteins, shsps. here, the conformation and the stability of -and -crystallins were investigated by small angle x-ray scattering (saxs) and high pressure, depending upon temperature and ph. at room temperature, -crystallins have shown a partially reversible change in size from to kb, and this effect was enhanced by the combination of temperature and pressure. in the case of -crystallins and in the pressure range up to kb, the pressure was combined with temperature and ph. the results depend upon the different itself. structural studies on pore-forming peptides s. m. ennaceur , d. bown , j. m. sanderson chemistry department, university of durham, biology department, university of durham the resistance of pathological microorganisms to conventional antibiotic drugs has created a need for new antibacterial agents. biologically active antimicrobial peptides that act as primary defense agents in a large variety of species are thought to have the potential as precursors for a new range of drugs from antibiotics to cancer treatments. this study has attempted to analyse the structural properties of membrane peptides and proteins through the use of model systems that have been designed to mimic their natural counterparts: we have successfully synthesised model membrane peptides with a beta-sheet structural motif and have used a wide range of techniques to analyse their interactions with phospholipid (pc) membranes. the synthetic peptides were very hydrophobic and only soluble in uorinated alcohols such as hfip and to a lesser extent tfe. we found hfip to have a very strong af nity for pc membranes and carried out a series of experiments to investigate this af nity. binding of hfip to pc membranes was found to be reversible and we exploited this property in d crystal trials of our synthetic peptides. we over expressed the c-terminal domain of brka, a gram negative autotransporter protein, which forms a beta-barrel channel in the outer membrane (omp), for comparison with our model peptides. we performed d crystal trials on the omp and imaged the resulting protein arrays by stem and afm. a protein spontaneously folds into a unique native structure in physiological conditions. this process accompanies a huge loss of the conformational entropy (ce). our major concern is to specify the factor that can compete with the ce loss. the previous discussions concerning protein folding have been focused on contributions to the free energy of folding from the interaction potentials in a system. a view lacking in earlier studies is that the folding is critically in uenced by the translational movement of water molecules. when solute particles contact each other in a solvent, the excluded volumes for the solvent molecules overlap, and the total volume available to their translational movement increases. this leads to a gain in the translational entropy (te) of the solvent. this type of te effect should be much stronger in protein folding where the tight packing of the side chains occurs. an elaborate statistical-mechanical theory is employed to analyze the te of water in which a peptide or a protein molecule is immersed. it is shown that the te gain upon folding is large enough to compete with the ce loss. when water is replaced by another solvent whose molecular size is larger, the te gain decreases to a remarkable extent. we suggest that the entropic loss accompanying the self-assembly and the formation of ordered structures in a living system is compensated mostly by the te gain of water, highlighting an aspect of the crucial importance of water in sustaining life. domain ii of ribosome recycling factor is required for disassembly of the post-termination complex p. guo, l. zhang, y. feng, g. jing institute of biophysics, chinese academy of sciences, national laboratory of biomacromolecules, beijing, china ribosome recycling factor (rrf) consists of two domains and, in concert with elongation factor ef-g, triggers dissociation of the post-termination ribosomal complex. however, the exact function of the individual domains of rrf remains unclear. to clarify this, two rrf chimeras, ecodi/ttedii and ttedi/ecodii, were created by exchanging the rrf domains between the proteins from escherichia coli and thermoanaerobacter tengcongensis. the ribosome recycling activity of the rrf chimeras was compared with their wild-type rrfs by using in vivo and in vitro activity assays. the experiments show that like wild-type tterrf, the ecodi/ttedii chimera fails to complement the rrf ts phenotype of e.coli lj (frr ts ) strain and has no polysome breakdown activity. however, under the same conditions, the ttedi/ecodii chimera complements the rrf ts phenotype and has polysome breakdown activity equivalent to that of wild-type ecorrf. the results indicate that domain ii of rrf is the functional domain that is mainly responsible for the disassembly of the post-termination ribosomal complex, and the speci c interaction between rrf and ef-g on the ribosomes mainly depends on the interaction between domain ii of rrf and ef-g; while domain i of rrf is the main contributor for binding ribosomes and maintaining the stability of the rrf molecule. this study provides direct genetic and biochemical evidence for the assignment of individual functions of rrf domains. self-assembly of natural somatostatin into liquid crystalline nano brils w the natural neuropeptide somatostatin- is a cyclic tetradecapeptide hormone, with broad inhibitory effects on both endocrine and exocrine secretions. we report the self-assembly of somatostatin in solution, into stable liquid crystalline nano brils, based on the neuropeptide bioactive backbone conformation. the system was studied as a function of peptide concentration, milieu composition and time, using optical and electron microscopy, x-ray scattering, vibrational spectroscopy and sec/rp-hplc. in pure water, the formation of twisted nano brils (around nm wide and a few microns long) was characterized. their structure relies on the native somatostatin -hairpin and on intermolecular antiparallel -sheets networks. the nano brils were observed to laterally associate further with increased concentration and time, as well as to generate hexagonal phases. increase in ionic strength (sodium chloride, phosphate) was found to signi cantly favor the self-association process. the soft conditions of formation of the somatostatin nano brils support biological relevance, for instance to the biological mechanism of storage of the neuropeptide hormone. unraveling the physical origin of the structure of fully denatured ubiquitin s. golic grdadolnik, f. avbelj national institute of chemistry, hajdrihova , ljubljana, slovenia the structure and dynamics characterization of non-native states of proteins is crucial for understanding the mechanism of protein folding. recently many experimental studies have shown variations of conformational propensity and exibility along the backbone chain of fully denatured proteins. it has been supposed that areas of residual structure may serve as initiation sites of protein folding. however, the physical origin of these variations is still unclear. we analyze the structure of fully urea-denatured ubiquitin. the experimental veri cation of conformational propensities of protein backbone is obtained through structurally dependent nmr parameters. although the secondary structure of ubiquitin under strong denatured conditions is not detectable and no correlation with the native overall topology is found, the variations of nmr parameters along the backbone follow the secondary structure elements of its native state. we show that these variations are in accord with the recently developed electrostatic screening model of denatured proteins ( ). in this model, the backbone conformations of residues in unfolded protein are determined by local backbone electrostatic interactions and their screening by backbone solvation. many virulence factors from gram-negative bacteria are autotransporter proteins. the nal step of autotransporter secretion is c nterminal threading through the outer membrane (om), followed by folding. this process requires neither atp hydrolysis nor a proton gradient. pertactin, an autotransporter from bordetella pertussis and the largest -helix structure solved to date, folds much more slowly than expected based on size and native state topology, yet folding intermediates are not aggregation-prone. equilibrium denaturation results in the formation of a partially folded structure, a stable core comprising the c-terminal half of the protein. examination of the pertactin crystal structure does not reveal the origin of the enhanced c-terminal stability. yet sequence analysis reveals that, despite size and sequence diversity, all autotransporters are predicted to fold into parallel -helices, suggesting this structure may be important for secretion. for example, slow folding in vivo could prevent premature folding of in the periplasm prior to the assembly of the om porin. moreover, extra stability in the c-terminal rungs of the -helix may serve as a template for the formation of the native protein during secretion, and formation of the growing template may contribute to the energy-independent translocation mechanism. coupled with the sequence analysis, these results suggest a general mechanism for autotransporter secretion. thermal and functional properties of e.coli outer membrane protein-receptor fhua fhua is e.coli outer membrane protein, which transports iron into the bacterial cell and also serves as receptor for several phages. in order to get more deep information about structural properties of fhua, we've studied its thermal properties by means of calorimetry. we've also investigated the interaction between t , ira phages and directly fhua by means of viscometry. the calorimetric result of heat denaturation of membrane protein fhua and next deconvolution of the recorded calorimetric curve with two transitions has shown that in a chosen conditions the structure of fhua consists of domains. though both t and ira phages grow on the common bacterial strain (e.coli k ho ), expressing fhua the results of viscometric investigation show that under direct interaction of phages with fhua the receptor activity of protein revealed only for t phage. therefore, we conclude that other than fhua protein serves as receptor for ira phage. it should be mentioned that the phage dna ejection process induced by receptor was observed for the rst time by us in an incessant regime. electron spray ionization mass spectroscopy (esi-ms) is a powerful tool for the investigation of the protein folding or proteins non-covalent interactions in solution since charge state distributions (csds) in esi-ms are affected by the conformational state and mass relates on the association state. we used this tecnique to inquire at different ph and different conditions the dimerization process of the porcine and bovine -lactoglobulins that share a high sequences similarity and close d structures. dimerization oflactoglobulins is reversible and involves both electrostatic and hydrophobic interactions. it was possible to detect simultaneously both the monomeric and dimeric form of the proteins in solution, pointing out the different dimerization behaviour of the two isoforms. we assessed the maximal stability of the dimeric structure at ph for the porcine protein and ph for the bovine one. moreover we showed that bovine lactoglobulin has a stronger dissociation costant than the porcine protein. further we showed that it is possible to modulate the dimerization equilibrium of the bovine isoform at ph both increasing temperature and adding methanol without inducing denaturation of the protein. a possible novel method of protein structure prediction; a. ikehata division of structural bioinformatics science of biological spramolecular systems graduate school of integrated sciences mechanism of protein folding has been mysterious since an nsen's dogma was sugested.here i would like to propose a possible novel method of protein structure prediction; origami method. the method comes from a protein backbone property of uctuation and the residue hydrophobicity. l. marsagishvili , m. shpagina , z. podlubnaya institute of theoretical and experimental biophysics ras, pushchino state university amyloid brils are formed by proteins or their peptides in the result of a conformational transition from alpha helix into beta-sheet structure. despite the different nature of proteins-precursors their amyloids have common properties: beta-pleated sheet structure with individual beta-sheets oriented parallel to the main axis; insolubility in vivo; speci c binding to congo red and thyo avin-t. amyloid deposits are observed in different diseases such as myositis, myocarditis, cardiomyopathies and others. we showed that sarcomeric cytoskeletal proteins of titin family (x-, c-, h-proteins) of rabbit skeletal muscles are capable to form amyloid brils in vitro. these proteins already contain % of beta-sheet structure necessary for formation of amyloids. the amyloid nature of their brils was conrmed by electron, polarization and uorescence microscopy. as x-, c-, h-proteins form amyloid brils easily in vitro, there is a danger of fast growth their amyloid deposits in vivo. taking into account common properties of amyloids formed by different proteins, our results clear the ways for conducting by amyloidogenesis in human organs and tissues. work is supported by rfbr grants - - , "universities of russia" . . and program of the presidium ras "fundamental sciences for medicine". probing the folding capacity and residual structures in -and -residue fragments of staphylococcal nuclease d. liu, x. wang, y. feng, l. shan, j. wang national laboratory of biomacromolecules, institute of biophysics, chinese academy of sciences n-terminal fragments of staphylococcal nuclease (snase) with different chain lengths were used as a model system in the folding study. the detailed characterization of conformational states of - and - residues snase fragments (snase and snase ) and their v w and g w mutants can provide valuable information on the development of conformations in the folding of snase fragments of increasing chain lengths in vitro. in this study, the presence of retained capacity for folding and residual structures in snase and snase is detected by cd, uorescence, ftir, and nmr spectroscopy. snase is represented as an ensemble of interconverting conformations. the uctuating nascent helix-and âsheet-like structures, localized in regions of a -a and t -v , respectively are transiently populated in snase . the native-like tertiary conformations are obtained for g w and v w and for snase in the presence of . m tmao. analysis of the results of such studies indicate that folding of snase fragments is dominated by developing the local and non-local nucleation sites from native-like secondary structures and by intensifying the longrange interactions of residues at nucleation sites with residues further removed in sequence. thermal disruption of a spanning network of hydration water and conformational changes of elastin a. krukau , i. brovchenko , a. oleinikova , a. geiger international max-planck research school in chemical biology, otto-hahn str. , d- , germany, physical chemistry, university of dortmund, otto-hahn str. , d- , germany hydration water strongly in uences the structural and dynamical properties of biomolecules. the existence of a spanning hydrogenbonded network formed by the hydration water enables the function of biomolecules at low hydration levels. we can expect that the formation/disruption of the spanning network formed by the hydration water in solution also affects crucially the protein properties. we present the rst computer simulation study of the thermal disruption of a spanning network formed by the hydration water of a biomolecule (elastin-like peptide). this process obeys the laws of d percolation transition, similarly to the formation of a spanning water network with increasing hydration level [ ]. the spanning water network transforms into an ensemble of small water clusters with increasing temperature: it is still permanent at k and exists with probability % at k. in the same temperature interval, the conformation of the peptide changes noticeably: its radius of gyration increases sharply (by %) at about k. these two phenomena may be related to the "inverse temperature transition" at about k, where an elastin solution separates into two phases. in our simulations, the displacement of hydration water by the addition of a denaturant (urea) or by other peptide molecules causes an even stronger increase of the radius of gyration (up to % lipidic cubic phases formed by distinct water and lipid volumes provide bicontinuous d bilayer matrices that have speci c and controllable water channel sizes and large surface areas. these systems have proven to be also valuable as membrane mimetic structures, as promising matrices for controlled-release and delivery of proteins, vitamins and small drugs in pharmacological applications, and they offer a d lipid matrix for successful crystallization of membrane proteins which do not easily crystallize in bulk solution. the present study is directed towards a better understanding of the interplay between curved cubic lipid phases and the protein entrapped within their aqueous channel structures. as model systems, we have chosen a cubic ia d phase formed by an uncharged lipid, monoolein, and incorporated different proteins, such as cytochrome c, -chymotrypsin and insulin, in its narrow water channels. we show that the protein secondary structure and unfolding behaviour may be in uenced by the con nement and, vice versa, the topology of the lipid matrix may change as a function of protein size and concentration. in fact, even new cubic lipid structures may be formed that are not known in pure lipid systems. furthermore, we compare the aggregation scenario of insulin in bulk solution and in the narrow water channels of the cubic lipid matrix and discuss the differences found in terms of the geometrical limitations imposed by the con nement. after endocytosis, i.e. at acidic ph, the t domain inserts in the membrane of the target cell and helps the translocation of the catalytic domain into the cytoplasm. therefore, the t domain has a key role in the strategy of internalization of the toxin. the study of the interaction of the t domain with membranes and its ph dependence is important for a better understanding of the diphtheria toxin translocation mechanism. at least, two steps can be distinguished during the membrane insertion of the t domain. the rst step involves hydrophobic interactions with the membrane and is related to the ph-induced stabilization in a molten-globule state. in the second step, electrostatic interactions are preponderant and the ph-sensitivity comes from changes of the balance between repulsive and attractive electrostatic interactions. the role of the n-terminal part of the t domain in the second step has been investigated by studying peptides corresponding to the amphiphilic helices found in this part of the domain. the results are correlated with those obtained with a single trp mutant probing the n-terminal region of the whole domain. the translocation mechanism will be discussed in view of the physico-chemical properties of the peptides. in complex systems with many degrees of freedom such as peptides and proteins there exist a huge number of local-minimumenergy state. one way to overcome this multiple-minima problem is to perform a simulation in a generalized ensemble where each state is weighted by a non-boltzmann probability weight factor so that a random walk in potential energy space may be realized (for reviews, see refs. [ ] [ ] [ ] xas spectroscopy results show that there are two different structures of the metal binding site in the a peptide according to whether they are complexed with cu or zn ions. while the geometry around copper is suggestive of an intra-peptide binding with three histidine residues bound to the metal, the zinc site geometry is compatible with an inter-peptide aggregation mode. this result reinforces the hypothesis that assigns opposite physiological roles to the two metals, with zinc favouring and copper blocking peptides aggregation and consequent plaque formation. effect of pressure on the conformation of proteins. a molecular dynamics simulation of lysozyme a. n. mccarthy, r. grigera instituto de física de líquidos y sistemas biológicos (iflysib), conicet-unlp-cic, university of la plata, argentina the effect of pressure on the structure and dynamics of lisozyme was studied by md computer simulation at bar ( pa) and kbar using gromacs package. all-atoms (ff gmx) force eld were used for the minimization process and for all the md simulation and kept all protein bond lengths constrained (lincs algorithm). water molecules (spc/e model) were constrained using the settle algorithm. for the electrostatic forces we applied the reaction field method. lennard-jones interactions were calculated within a cut-off radius of . nm. the results have good agreement with the available experimental data, allowing the analysis of other features of the effect of pressure on the protein solution. the studies of mobility show that although the general mobility is restricted under pressure this is not true for some particular residues. from the analysis of secondary structures along the trajectories it is observed that the conformation under pressure is more stable, suggesting that pressure acts as a 'conformer selector' on the protein. the difference in solvent accessed surface (sas) with pressure shows a clear inversion of the hydrophilic/hydrophobic sas ratio, which consequently shows that the hydrophobic interaction is considerably weaker under high hydrostatic pressure conditions. direct observation of mini-protein folding using uorescence correlation spectroscopy h. neuweiler, s. doose, m. sauer applied laser physics & laser spectroscopy, university of bielefeld, bielefeld, germany the "trp-cage" motif represents the smallest and one of the fastest folding mini-proteins known to date. the globular fold is characterized by a hydrophobic core burying a single tryptophan (trp) residue. here, we report on the direct observation of trp-cage folding kinetics using uorescence correlation spectroscopy (fcs). our method is based on the selective uorescence quenching of oxazine dyes by trp which becomes ef cient only upon contact formation between the dye and the indole moiety of trp. by sitespeci cally labeling the dye to trp-cage, temporal uorescence uctuations of the dye-peptide conjugate, caused by intramolecular contact formation between dye and trp, directly report on conformational dynamics and folding transitions of the peptide chain. in order to measure uorescence uctuations directly in solution we used fcs on a confocal uorescence microscope setup. fcs allows us to reveal conformational dynamics with nanosecond timeresolution, under thermodynamic equilibrium conditions, and in highly dilute solutions (i.e. at nano-molar sample concentrations). our method con rms microsecond folding kinetics of the trp-cage motif, previously estimated with non-equilibrium temperature-jump techniques. we further investigated stability and folding rates under denaturing conditions and at various temperatures, giving further insight into structural transitions during the folding process. identi cation and mutagenesis of a region of tnt required for the stability of tnt-tni coiled-coil evolutionarily conserved heptad hydrophobic repeat (hr) domains present in troponin subunits tnt and tni are involved in alpha helical coiled-coil formation. using recombinant peptides from fast-skeletal tnt and tni, we examined the contributions of amino acid residues within these hr domains as well as anking these domains, to the stability of the coiled-coil interaction. a series of tnt fragments were tested for their ability to form coiledcoil with tni hr domain. we show that the tnt region - , although remains outside of the coiled-coil domain, is absolutely required for the stability of the coiled-coil. interestingly, the region tnt - contains few absolutely conserved residues that are potential candidate for ionic interaction, as predicted by molecular modeling. using single point mutants we show that among all the conserved residues, residue lysine is most important in stabilizing the coiled-coil interaction, whereas others play accessory role. we propose that the lysine initiates the stabilization of the coiled-coil interaction and then the other residues acts in a zipper like fashion. magnesium promotes conformational switching of ca sensor s. mukherjee, k. v. r. chary tata institute of fundamental research the importance of mg , one of the most abundant metal ion in the cell cytoplasm, relating to the calcium sensor mechanism is demonstrated. in this study it has been shown that the amino acid long calcium binding protein from entamoeba histolytica (ehcabp) can exist in three different forms namely, the calcium-free (apo) form, the magnesium bound (apo-mg) form and the calcium bound (holo) form. these three forms have been characterized using chromatographic, calorimetric as well as various spectroscopic techniques. there is a radical difference in the stability between the calcium free and ca bound forms. the calcium free form has molten globule like characteristics. mg stabilizes the closed conformation of the apo form, where the hydrophobic core remains buried. the presence of mg signi cantly alters the calcium binding cooperativity thereby increasing the cooperativity of the conformational switching between the open and closed conformation which is an important aspect of such regulatory proteins. a structural model for the molten-globular form of apo-ehcabp and its equilibrium folding towards completely folded holo state in presence and absence of mg will be presented. intermediate states of formin binding protein ww domain: explored by replica-exchange simulation y. mu school of biological science, nanyang technological university, singapore ww domain of formin-binding protein (fbp) is a model system for beta strand folding study. although it is small, only amino residues in total, the folding kinetics of fbp ww domain proved to be biphasic. an extensive molecular dynamics-monte carlo hybrid method, called replica-exchange simulation strategy is employed to study the folding/unfolding of the fbp ww domain in explicit water model. begin with randomly chosen conformations from high temperature unfolding trajectory distributed in replicas ( different temperatures covering from k to k), the simulation lasts nanoseconds in each replica. in the end an interesting distribution of conformations shapes up. we nd that there are three distinctive subgroups, one being the unfolded conformations with rmsd averaging around Å , one being native conformations with rmsd . Å, more interestingly, the third group having rmsd Å, an intermediate folding ensemble. by checking the intermediate ensemble in detail, we nd that it is quite heterogeneous and the heterogeneity mainly comes from the exibility of the c-terminal loop region. our ndings provide a microscopic picture of folding kinetics of the ww domain: the stable intermediate states with mis-registered hydrogen bonds on the c-terminal beta strand make this peptide folding as a three-state folding model rather than a usual two-state model. h. rezaei, f. eghiaian, j. perez, y. quenet, y. choiset, t. haertle, j. grosclaude institut national de la recherche agronomique, france in pathologies due to protein misassembly, low oligomeric states of the misfolded proteins rather than large aggregates play an important biological role. to get better insight into the molecular mechanisms of prpc/prpsc conversion, we studied the kinetic pathway of heat induced amyloidogenesis of the full length recombinant ovine prp (arq) at ph . . according to the size exclusion chromatography experiments, three sets of oligomers were generated from the partial unfolding of the monomer. the effect of concentration on the oligomerization kinetics was different for the three species obsreved suggesting that they are generated from distinct kinetic pathways. limited proteolysis and peptide analysis of the two best separated peaks showed a difference in the accessibility of the c-terminal domain of these two oligomers, and allowed the identi cation of regions undergoing a structural change during the conversion process. the analysis of correlations between oligomer populations as well as numerical kinetic modeling led us to propose a multi-step kinetic pathway describing the evolution of each species as a function of time. the existence of at least three distinct oligomerization pathways on one hand and the differences in the accessibility of the two puri ed oligomers on the other hand re ect the structural plasticity of the prp protein. studying the mechanism of retention of ovine prion protein in soils will tackle the environmental aspect of potential dissemination of scrapie infectious agent. the conformational transition from the monomeric cellular prion protein prp c in -helical structure into the aggregated -sheet-rich multimer prp sc is supposed to be responsible for the so-called prion diseases. it is commonly admitted that the recombinant prp could serve as model of conversion of the normal prion protein prp c . fundamentally, the interaction of proteins with surfaces either uid or solid involves both protein binding and unfolding. our goal in studying protein adsorption is to determine the nature and the amplitude of the structural changes occurring during non-speci c adsorption. the protein-clay interaction depends on several parameters such as protein hydration, net charge, charge distribution on the protein surface. ftir spectroscopy is well-suited to probe structural changes of proteins at a molecular level at aqueous/solid interfaces. the conformational states of the full-length ovine prp adsorbed on the electronegative clay surface are compared to its solvated state in deuterated buffer in the pd range . - , using ftir spectroscopy. during the intoxication of a cell, the diphtheria toxin binds to a cell surface receptor, is internalized and reaches the endosome. the translocation (t) domain from the toxin interacts with the membrane of the endosome in response to the acidic ph found in this compartment. this process drives then the passage of the catalytic domain of the toxin through the membrane into the cytoplasm. the interaction of the t domain with the membrane involves at least two steps. in the rst step occurring at ph , t adopts a molten globule conformation, which is able to bind super cially to the membrane through hydrophobic interactions by the c-terminal region (helices th and th ). in a second step occurring between ph and , penetration into the membrane involves electrostatic interactions. this step leads to a functional inserted state. trp was mutated to phe in order to use trp located in helix th as a probe of its behavior during the interaction with the membrane as a function of ph. we found that the second step is correlated with the reorganization of the n terminal region in the membrane and is controlled by electrostatic interactions. peptide conformational search using generalized simulated annealing method p. g. pascutti , f. p. agostini , c. osthoff , k. c. mundim , m. a. moret ibccf -ufrj -brazil, lncc -brazil, iq-unb -brazil, ceppev -fundação visconde de cairú / df-uefs -brazil the three-dimensional structure of proteins is mainly determined by the sequence of amino acids, making possible the development of ab initio methods for peptides and protein structure prediction. we proposed a stochastic method based on a classical force field and the generalized simulated annealing (gsa), which utilizes tsallis generalization of boltzmann-gibbs statistics. we have applied this method for peptide conformational search and as a complement for comparative modeling of proteins, searching the conformation for loops and mismatched sequence alignments. the gsa ef ciency depends on the right choice of the parameters involved in the conformational calculation: the qv parameter which de nes a function for visiting the molecular energy surface; and the qa parameter de ning an acceptance probability, both according with tsallis statistics. to avoid the conformational trapping in local energy minima we introduced a new parameter in this work, qt, to control the temperature decreasing. to investigate the qv, qa and qt best parameters set, we used the -alanine and -alanine peptides, which have a known alpha-helical structure in low dielectric environment. the global minimum energy occurs for the alpha-helix folded structure, and was found for qv ranging from . to . , qa from . to . , and qt from . to . . we observed also that convergence values for qv decrease while for qa and qt increase for folded structures. p. s. santiago, É. v. de almeida, m. tabak instituto de quimica de são carlos-usp cp são carlos sp brasil extracellular hbgp is a giant hemoglobin, similar to other annelid hemoglobins, having a molecular weigth of . mda. the effect of ctac in the oligomeric protein structure was assessed by optical absorption and emission spectroscopies. optical absorption spectra of hbgp . mg/ml as a function of ctac concentration from up to . mm evidentiate changes both in soret band at nm, and at nm associated to light scattering which appears at low ctac concentration. below mm of surfactant extensive light scattering occurs together with signi cant shifts of max of soret band from nm to around nm, which is probably due to oxidation of the original oxyhbgp. light scattering reaches a maximum value disappearing for higher ctac concentrations. fluorescence spectra show a signi cant increase in intensity ( fold) upon titration with ctac. this is consistent with the dissociation of the oligomer with signi cant reduction of intrinsic quenching of tryptophan uorescence due to the heme groups. similar data were obtained at protein concentration of mg/ml. in this case a signi cant increase of light scattering is observed with protein precipitation at a narrow ctac range followed by re-disolution at higher ctac concentration. differently from anionic sds surfactant, cationic one induces protein aggregation. support: cnpq and fapesp. in situ formation of silk protein nano-particles studied by small angle x-ray scattering m. w. roessle , c. riekel , d. i. svergun european molecular biology laboratory; outstation hamburg/germany, european synchrotron radiation facility; grenoble/france silk threads from the mulberry silkworm bombyx mori are used for the production of textiles since centuries. in modern applications silk proteins are chosen because of their good tensile strength, their high biocompatibility as well as of the resorbability for the human body. however, the processing of silk by the silkworm is barely understood. the silk proteins are stored as a solution in the glands of the silkworm and processed during the spinning into a co-block polymer like ber. in a rst step the random coil silk proteins are transformed into molecules with beta-sheet subdomains, which provide a protein-protein interface for the ber assembly. this transformation can be mimicked by a rheometer applying a shear force to the silk protein solution. applying combined small/wide angle x-ray scattering (saxs/waxs) transient formed silk nanoparticles upon increasing shear force were found. further details of these macromolecules were derived by xing the transient state with chemicals such as polyethylene oxide (peo). the resulting data can be analyzed in detail by saxs data evaluation software and low resolution models of the found nanoparticles were derived. moreover, the internal structure of the particles was explored as well as suggestions for the silk processing of the silkworm could be made. on the three-dimensional information of a protein sequence v. a. risso, l. g. gebhard, r. g. ferreyra, j. santos, m. noguera, m. r. ermacora universidad nacional de quilmes conicet in this work, lactamase of b. licheniformis (es l) was used as an experimental model to (a) study the conversion of sequential information into d structure and (b) to investigate the distribution of conformational information in the polypeptide chain. by a novel approach, over thirty connectivity variants of the polypeptide chain were prepared; witch were also nterminally truncated to a variable degree. the variants produced in e. clil were puri ed to homogeneity, refolded, and its structure content analyzed by circular dichroism, hydrodynamic behaviour and aggregation state. several variants were dimeric in solution, suggesting a possible general inespeci c stabilization mechanism. most variants were compact and had different degrees of secondary and tertiary structure. a strikingly large number of variants showed native like spectroscopic signatures and signi cant enzymatic activity, which means that the very elaborate active site of beta lactamase is formed, at least in fractions of the molecules, despite the absence of long stretches of sequence. these ndings are discussed in the light of the current knowledge of the protein folding process. var and lgg contributed equally to this work. on the three-dimensional information of a protein sequence v. a. risso, l. g. gebhard, r. g. ferreyra, j. santos, m. e. noguera, m. r. ermacora universidad nacional de quilmes conicet in this work, lactamase of b. licheniformis (es l) was used as an experimental model to (a) study the conversion of sequential information into d structure and (b) to investigate the distribution of conformational information in the polypeptide chain. by a novel approach, over thirty connectivity variants of the polypeptide chain were prepared; witch were also nterminally truncated to a variable degree. the variants produced in e. clil were puri ed to homogeneity, refolded, and its structure content analyzed by circular dichroism, hydrodynamic behaviour and aggregation state. several variants were dimeric in solution, suggesting a possible general inespeci c stabilization mechanism. most variants were compact and had different degrees of secondary and tertiary structure. a strikingly large number of variants showed native like spectroscopic signatures and signi cant enzymatic activity, which means that the very elaborate active site of beta lactamase is formed, at least in fractions of the molecules, despite the absence of long stretches of sequence. these ndings are discussed in the light of the current knowledge of the protein folding process. var and lgg contributed equally to this work. unfolding of the extrinsic proteins of photosystem ii ( kda, kda and kda) induced by pressure unfolding of the three extrinsic proteins of spinach photosystem ii induced by pressure has been systematically investigated. thermodynamic equilibrium studies indicated that these proteins are very sensitive to pressure. at o c all the proteins show a reversible unfolding transition by about mpa for kda, mpa for kda and mpa for kda. the ph and temperature dependence of pressure unfolding of these proteins were explored. the stabilization effect of reagents sucrose etc on the proteins was found to associate not only with the increase in the unfolding free energy, but also with the reduction of the absolute value of v u . pressure-jump studies of unfolding of kda protein revealed a negative activation volume for unfolding and a positive activation volume for refolding, indicating that in terms of system volume, the transition state lies between the folded and unfolded states. comparison of temperature dependence of v u # , v f # and v u indicated that the thermal expansivities of the transition state and the unfolded state are similar and larger than that of the folded state. aggregation-prone intermediate protein structures on the refolding pathway l. smeller , j. fidy , k. heremans semmelweis university dept. biophysics and radiation biology, budapest, hungary, department of chemistry, katholieke universiteit leuven, belgium the folding of the polypeptide chain into a native conformation can be studied by experimental systems, where the environmental parameters causing the denatured state can be easily and fast eliminated. one of such parameters is the high hydrostatic pressure. refolding of the unfolded protein can be studied after decompression. the refolding pathway can contain several intermediate states. on the other hand, the destabilization of the native proteins can populate conformations, where the polypeptide chain is not completely folded. these metastable conformations can easily aggregate. deposition of insoluble protein aggregates plays a crucial role in the conformational diseases (parkinson, alzheimer's disease, amyloidozis, etc.) stability and conformation of the above mentioned metastable conformations were investigated in case of myoglobin, apo-horseradish peroxidase, lipoxygenase. fluorescence and infrared spectroscopy and light scattering experiments were used to explore not only the structure but also the aggregation af nity of the intermediates in case of all the above mentioned proteins a well de ned temperature range could be determined, where the metastable not completely folded structures were populated considerably during the refolding process. these intermediate conformations were significantly more aggregation prone, than the native conformers existing in the same temperature range. aggregation processes in beta-amyloid peptides: effects of molecular chaperons a. sgarbossa, d. buselli, f. lenci cnr istituto bio sica, pisa, italy several neurodegenerative pathologies, like parkinson's, hungtington's and alzheimer's diseases, are related to the formation of small peptides aggregates, which amyloid brils originate from. understanding the molecular mechanisms responsible for these processes can, therefore, contribute to clarify the origin and, hopefully, to control the development of the afore mentioned diseases. here we report the results of an in vitro study aiming to affect the aggregation kinetic of - and - beta-amyloid peptides by means of an endogenous chaperone-like protein (alpha-crystallin) and an exogenous polycyclic aromatic pigment (hypericin) that can perturb the aggregation process through stacking interactions with the peptides aromatic residues. because of the well known problems in getting reproducible and reliable results, particular attention has been devoted to carefully check the preparation procedures of the samples. the effects of both alpha-crystallin and hypericin on the self-assembly process have been examined at different times of the aggregation kinetics. the results are discussed in relation with the involvement of different molecular structures in the amyloid brillation phenomenon. remodelling the folding of thioredoxin by removal of the c-terminal helix j. santos, j. m. del no iquifib and departamento de química biológica, facultad de farmacia y bioquímica, uba, junín , c aad buenos aires, argentina e. coli thioredoxin (trx) is a monomeric / protein of amino acids with a fold characterized by a central beta sheet surrounded by alpha helices. two subdomains are topologically noticeable, but it is unclear whether their folding occurs in a concerted fashion. subdomain trx - has been extensively studied as a model of a partially folded, with no tertiary or persistent secondary structure. this work describes the expression and characterization -by circular dichroism (cd), uorescence emission, size exclusion chromatography, chemical cross-linking and light scattering-of a novel engineered fragment (trx - ) lacking the last stretch of amino acids. after refolding from inclusion bodies, trx - shows a strong propensity to form soluble oligomers endowed with distinctive optical properties unlike those observed for the full protein. although trx - also shows signi cant changes in secondary structure, trp residues appear to occupy rigid and apolar environments. these ndings support the existence of an alternatively folded form for trx - . in addition, the secondary structure content of chemically synthesized peptide trx - and its ability to complement fragment trx - upon refolding were also evaluated by cd. taken together, the data herein presented shed light upon issues such as the distribution of information content relevant for folding along the polypeptide chain in regard to conformational stability. with grants from anpcyt, ubacyt and conicet. thermal aggregation of two "beta-protein" models at different ph values v. vetri, f. librizzi, v. militello, m. leone physical and astronomical sciences, univ. of palermo, italy & infm the structural stability of proteins strongly depends on the environment and the lost of this stability may trigger a partial unfolding, leading in turn to the formation of aggregates. such processes have been extensively studied also in view of their biotechnological and medical implications. in fact a large number of diseases is associated with protein misfolding and aggregation. conformational changes play a keyrole in the aggregation processes and have their onset under particular external conditions. the aggregation pathways and the topology of the obtained supramolecular structures sizeably depend on the details of the involved conformational changes, which are determined by the details of the external conditions. here we present an experimental study on thermal aggregation processes of two model proteins mainly composed from structures: -lactoglobulin and concanavalin a, at different ph values. the conformational changes of the proteins (whose association state depends by ph) and the aggregation pathways were monitored by intrinsic and suitable external dyes uorescence. at the same time, the growth of supramolecular structures was followed by measuring the rayleigh scattering of the excitation light. secondary structure changes were followed by circular dichroism measurements. the results show that at different ph values the aggregation processes of both proteins follow different pathways determined by the variations in the native structure and by the details of the involved conformational changes. a. verma , t. herges , w. wenzel iwr, forschungszentrum karlsruhe, po box , , karlsruhe, germany, int, forschungszentrum karlsruhe, po box , , karlsruhe, germany ab-initio protein structure prediction and the elucidation of the mechanism of the folding process are among the most important problems of biophysical chemistry. investigations of the protein landscape may offer insights into the folding funnel and help elucidate folding mechanism and kinetics. we investigate the landscape of the internal free-energy of the amino acid villin headpiece with a modi ed basin hopping method in the all atom force eld pff , which was previously used to fold several helical proteins with atomic resolution. we identify near native conformations of the protein as the global optimum of the force eld. more than half of the twenty best simulations started from random initial conditions converge to the folding funnel of the native conformation, but several competing low-energy metastable conformations were observed. from , independently generated conformations we derived a decoy tree which illustrates the topological structure of the entire low-energy part of the free energy landscape and characterizes the ensemble of metastable conformations. these emerge as similar in secondary structure content, but differ in the tertiary arrangement. exploring the free energy landscape of a folded protein by means of afm stretching experiments m. vassalli , b. tiribilli , l. casetti , a. torcini , a. pacini , a. toscano isc -cnr, florence -italy, csdc, univ. of florence -italy, anatomy dept, univ. florence -italy the aim of our work is to study the mechanically induced folding and refolding of single proteins by means of an atomic force microscope (afm). the resulting data will be analyzed with theoretical methods, both to determine the folding pathway and to gain information on the energy landscape of real systems. recently, experiments employing atomic force microscopy have shown that mechanical and thermal unfolding share several common features. we are using an afm to perform mechanical stretching experiments on single biomolecules. the experiments that can be performed are particularly well-suited to reconstruct the folding-unfolding pathways as well as the free energy landscape of the examined protein. in particular we are interested in the free energy pro le associated to titin and elastin, by considering a periodic loading of the afm cantilever, instead of the usual linear ramp, and measuring the force as a function of displacement. these experiments will be complemented by theoretical and numerical studies. molecular dynamics simulation of simple models but including the experimental geometry, will allow to examine in detail the effect of different experimental procedures (periodic loading versus linear ramp) proposed to reproduce equilibrium energy landscapes. moreover, we will investigate the limit of applicability of the jarzynski's equality which has been claimed to be able to be used to extract equilibrium results from non equilibrium measurements. association of subunits is a prerequisite for formation of the native structure of the dimeric ipmdh a. varga, É. gráczer, i. hajdú, p. závodszky, m. vas institute of enzymology, biological research center, hungarian academy of sciences, budapest, hungary to answer the question whether subunits are autonomous folding units, or their association at an early stage of folding is required for formation of the native protein structure, denaturation-renaturation experiments were carried out with the dimeric isopropylmalatedehydrogenase (ipmdh). denaturation was induced by guanidine hydrochloride, renaturation was initiated by dilution and followed by activity measurements and uorimetry. reactivation is a complex process with an initial lag phase, indicating the presence of an inactive intermediate. the kinetics of the process is independent of protein concentration, suggesting that association of the two polypeptide chains takes place much faster than the rate limiting rst order isomerisation step(s). restoration of protein uorescence during renaturation is also protein concentration independent, biphasic process, however the initial lag phase is replaced by an even faster burst increase of uorescence. the rst step leads to formation of an intermediate with a native-like uorescence spectrum. based on our experiments the following mechanism is proposed for refolding of ipmdh: d+d i i n , where d is denaturated monomer, i and i are inactive dimer intermediates, n is native dimer, that means initial association of the polypeptide chains during refolding is a prerequisite for formation of the native dimensional structure of ipmdh. we describe a new beamline for optical biophysic in construction on the synchrotron soleil. the high briliance of the synchrotron beam, associated with its tunability on a broad part of the electromagnetic spectrum make it an excitation source of choice for several biophysical optical techniques. the disco beamline we present will consist of three endstations : . the circular dichroism (cd) endstation will bene t from the inclusion of the energies accessible in the vuv wavelength range ( - nm) and from the natural polarization of the synchrotron beam. cd spectra of proteins covering a broad range of wavelenghts will enable better and ner structural analysis. moreover, new biological chromophores such as sugars which absorb in the deep uv will be accessible in cd. . the mass spectrometry endstation,will bene t from an ionisation beam with even greater energies (down to nm) comprising nebulisation at atmospheric pressure. photoionisation of macromolecular bio-structures without any solvent restriction will produce perfect analytes for mass spectrometry, . the multiparametric imaging endstation, build on a confocal microscope, will use the great tunability of the synchrotron radiation ( - nm) to excite samples at many wavelenghts simultaneously. the temporal component of the beam will allow natural lifetime imaging by phase modulation -demodulation. predictive all-atom protein folding with stochastic optimization methods the prediction of protein tertiary structure, in particular based on sequence information alone, remains one of the outstanding problems in biophysical chemistry. according to the thermodynamic hypothesis, the native conformation of a protein can be predicted as the global optimum of its free energy surface with stochastic optimization methods orders of magnitude faster than by direct simulation of the folding process. we have recently developed an all-atom free energy force eld (pff ) which implements a minimal thermodynamic model based on physical interactions and an implict solvent model. we demonstrated that pff stabilizes the native conformation of several helical proteins as the global optimum of its free energy surface. in addition we were able to reproducibly fold several helical proteins (the amino acid (aa) trp-cage protein, the villin headpiece ( aa), the conserved headpiece of the hiv accessory protein ( aa), the headpiece of protein a ( aa) and the -helix bacterial ribolsomal protein l ( aa), as well as several beta-sheet peptides. we used several stochastic optimization methods: the stochastic tunneling method, an adapted version of parallel tempering, basin hopping techniques and distributed evolutionary optimization strategies. we discuss advantages and limitations of this approach to de-novo allatom protein structure prediction. the independent thermal unfolding simulations of gb have been performed. physical property parameters of protein structure were chosen to construct a -dimension physical property space. then the -dimension property space was reduced to dimensions principle component property space. under the property space, the unfolding pathway ensemble of gb was obtained. the pathway ensemble likes a funnel that was gradually emanative from the native state ensemble to the unfolded state ensemble. the unfolding trajectories have the similar variable trend during the native state and the transition state ensemble. during the unfolded state, the unfolding trajectohries were divided into two types that one includes only one trajectory and the other include trajectories. the rst type of unfolded state was a discontinuity step distribution model, which is not random distribution. the second type of unfolded state was a near ellipsoid distribution model and a near random. there were substantial overlaps of unfolded state, indicating that thermal unfolded state consists of a con ned set of property values that makes the number of unfolded state of protein to be much smaller than that was believed before. the protein circular dichroism data bank (pcddb): a bioinformatics and spectroscopic resource b. a. wallace , l. whitmore , r. w. janes dept. of crystallography, birkbeck college, university of london, school of biological sciences, queen mary, university of london we describe the development and creation of the protein circular dichroism data bank (pcddb), a deposition data bank for validated circular dichroism spectra of biomacromolecules. its aim is to provide a resource for the biological and bioinformatics communities, by providing open access and archiving facilities for circular dichroism spectra. it is named by parallel with the protein data bank (pdb), a long-existing valuable reference data bank for protein crystal structures. it will permits spectral deposition via userfriendly web forms and will include automatic reading of a range of data formats and data mining from le headers to facilitate the process. it will be linked, in the case of proteins whose crystal structures and sequences are known, to the appropriate pdb and sequence data band les, respectively. a series of validation tools that will provide reports on data quality are included (and are accessible as stand-alone software). it is anticipated that this data bank will provide readily-accessible biophysical catalogue of information on folded proteins that may be of value in structural genomics programs, for quality assurance and archiving in industrial and academic labs, as a resource for programs developing spectroscopic structural analysis methods, and in bioinformatics studies. the relation of n-terminal residues and structural stability of l-chain apoferritin k. yoshizawa , k. iwahori , y. mishima , i. yamashita crest/jst, atrl-matsushita, nara institute of science and technology the denaturation of apoferritin by acidic solution was studied. ferritin, the ubiquitous iron storage protein, represents a well known polymeric assembly that is highly resistant to chemical and physical denaturants. it is a cage-shaped protein which is composed of subunits. natural vertebrate ferritins are copolymers of two different subunits, l-and h-chains. in the recombinant h-chain apoferritin (rhf), the structural stability is decreased by deletion of the n-terminal residues. we studied the effect of n-terminal residues of recombinant l-chain apoferritin (rlf) on the acidic ph denaturation and re-assembly. we constructed rlf and mutant rlfs which are lack of (fer ) or (fer ) amino acid residues from the n-terminus and investigated their stability by cd spectra. among three, fer has the least endurance against ph decrease. in the case of fer , the re-assembly of subunits into apoferritin can be performed by increasing solution ph without causing the by-product while huge aggregations are caused in fer and fer . the structural comparison of three mutants indicates that the hydrogen bonds of inter-and intra-subunits decrease by the loss of the n-terminal residues. therefore, it is elucidated that the hydrogen bonds of inter-and intra-subunits from n-terminal residues affect the molecule stability and re-assembly of l-chain apoferritin. pressure perturbation calorimetic studies of solvation, unfolding and aggregation of proteins r. winter university of dortmund, physical chemistry, otto-hahn-str. , d- dortmund pressure perturbation calorimetry (ppc) was used to study the solvation and volumetric properties of various proteins in their native and unfolded state. in ppc, the coef cient of thermal expansion of the partial speci c volume of the protein is deduced from the heat consumed or produced after small isothermal pressure jumps, which strongly depends on the interaction of the protein with the solvent or cosolvent at the protein-solvent interface. the effects of ph and various chaotropic and kosmotropic cosolvents (glycerol, sucrose, urea, guhcl, salts, etc.) on the solvation and unfolding behavior of the proteins was also investigated, and the observed volume and expansivity changes are correlated with further thermodynamic and spectroscopic properties of the systems. depending on the type of cosolvent and its concentration, speci c differences are found for the solvation properties of the proteins, and the volume change upon unfolding may even change sign. taken together, the data obtained lead to a deeper understanding of the solvation process of proteins in different cosolvents in their native and unfolded states. in addition, the effects of con nement and crowding on the solvational properties of the proteins were studied. finally, the use of ppc for studying intermolecular interactions and aggregation (amyloidogenesis) phenomena of proteins (e.g., insulin, prp) will be discussed. the in uence of semisynthetic derivatives of phenolic lipids on activity of yeast abc pumps phenolic lipids are the natural amphiphilic long-chain homologues of orcinol ( , -dihydroksy- -methylbenzene). they occur in numerous plants and microorganisms. resorcinolic lipids exhibit high af nity for lipid bilayer and biological membranes and are able to modify the activity of membrane enzymes (e.g. pla , ache). the in uence of semisynthetic derivatives of phenolic lipids on yeast pdr protein activity was studied by spectro uorimetric method using the potentiometric uorescence probe dis-c ( ). the probe is expelled from s. cerevisiae by abc pumps and can conveniently be used for studying their performance. two pump-competent s. cerevisiae strains and different pump-free mutant strains were used to experiment to check the effect of the semisynthetic derivatives of phenolic lipids on activity of abc transporters. two of these derivatives, named . and . , seem to affect the activity of pdr pumps. their in uence on activity of yeast plasma membrane multidrug resistance abc pumps is concentration-dependent. in uence of lipid membrane composition on pglycoprotein activity k. bucher, s. d. krämer, h. wunderli-allenspach department chemistry and applied biosciencies, eth zurich, switzerland p-glycoprotein (p-gp), a membrane atpase expelling many structurally unrelated compounds, is one of the major contributors to multidrug resistance. it is proposed that substrates bind to it within the membrane and are exported from there out of the cell. p-gp substrates are generally hydrophobic and their binding to the transporter is governed by their ability to partition into the membrane. the intimate association of both p-gp and its substrates with the membrane suggests that p-gp function may be regulated by the composition of the lipid bilayer. as detergents in uence the membrane properties and have been shown to affect p-gp atpase activity, we developed virtually detergent-free proteoliposomes to investigate the in uence of the membrane environment on the atpase activity of p-gp. the basal and substrate induced atpase activity was dependent on the cholesterol level of egg phosphatidylcholine (pc) membranes. the compound concentration at half maximal activation of p-gp (k m ) in proteoliposomes correlated with the af nity of the respective compound to liposomes consisting of the same lipids as the proteoliposomes tested. in conclusion, the basal and drug-induced atpase activity of p-gp is strongly dependent on the cholesterol content in detergent-free p-gp/egg pc/cholesterol proteoliposomes. m. berchel , j. jeftic , t. benvegnu , j.-y. thepot , d. plusquellec enscr umr cnrs , campus de beaulieu, rennes, université de rennes , umr cnrs , rennes bipolar lipids found in archaebacterial membranes, generally termed bolaamphiphiles, induce increased stability in membranes exposed to environments such as acidic conditions, high temperatures, high salt concentrations and/or absence of oxygen. we have synthesized a spin labeled unsymmetrical bolaamphiphile that selforganises in water solutions in multilamellar vesicles and shows slow ip-op phenomenon in comparison to conventional liposomes. generally, the ip-op from the exovesicular to the endovesicular membrane surface is a relatively slow process, which is due to the high energy barrier in transferring the polar amphiphilic heads through the lipophilic membrane. it can be involved in membrane transport mechanisms and in facilitating the transport, cells have evolved to use various supramolecular strategies. the half-life of the ip-op is estimated to more than twelve hours. we are now modulating the ip-op rate by incorporating chemical modi cations such as addition of cyclopentanes, double or triple bonds into the bridging chain of the molecule, in order to control the membrane transport via the ip-op mechanism. transport activity of the monocarboxylate transporter is increased by carbonic anhydrase h. m. becker, j. w. deitmer abt. allgem. zoologie, tu kaiserslautern, kaiserslautern the enzyme carbonic anhydrase (ca), which catalyses the conversion of co and h o to bicarbonate and protons, is present in nearly all animal cells, and is highly expressed in astrocytes. it is known that ca can bind to several membrane transporters, forming a transport metabolon and thereby enhancing the transport activity of the protein. in this project we have studied the functional interaction of the enzyme with the monocarboxylate transporter (mct ), which transports lactate and other monocarboxylates together with protons and is believed to play a pivotal role in the metabolite shuttling between astrocytes and neurons. therefore we expressed mct and then injected ca into xenopus oocytes. our results indicate a direct binding of ca to the mct , leading to a ca-induced increase in acid/base ux mediated by the transporter. interestingly, the effect was insensitive to the ca inhibitor ethoxyzolamide and to the nominal absence of co /hco , but disappeared when binding of ca to the mct was hindered. it seems, that ca, bound to the mct , mediates local buffer capacity by removing protons transported into the cell via the mct . this helps to stabilise the proton gradient close to the cell membrane, and thereby enhances the transport activity of the mct . these ndings suggest that ca can enhance metabolite-acid/base transport, by forming a transport metabolon with the mct . melibiose permease of e.coli (mel b) is a membrane bound ioncoupled sugar symporter that uses the favorable na , li , or h electrochemical potential gradient to drive cell accumulation ofor -galactosides. cysteine scanning mutagenesis, electrophysiological (ssm -solid supported membranes) and uorometric measurements, were used in order to better understand the role of speci c parts of the protein in the function of this symporter. the ssm technique combines a rapid solution exchange with the high sensitivity of planar lipid membranes. it employs a solid supported membrane as a capacitive electrode and allows the time resolved investigation of charge translocation during the catalytic cycle of such transporters as na /solute symporters. in order to obtain some more precise information about the function of mel b symporter, starting from the c-less melb, the mutant g c was constructed and from electrical, spectro uorimetric and fret measurements carried out on this mutant, in the absence and in the presence of speci c inhibitors, conclusions were drawn about the possible role of the helix iv in the function of the symporter. two-dimensional crystallization of co-reconstituted ca +-atpase, phospholamban and sarcolipin the sarcoplasmic reticulum ca -atpase and its regulators phospholamban (plb) and sarcolipin (sln) form a primary control mechanism in the recovery of resting state calcium levels in the myocardium. defects in the regulation of ca -atpase by plb and sln are central determinants in cardiac contractility and disease states such as cardiomyopathy. given the signi cance of these proteins, the structural details of their regulatory mechanisms remain an important future goal for the clinical improvement of heart disease. using co-reconstitution into proteoliposomes at low lipidto-protein ratios, we have examined the effects of mutation on the functional properties of plb and sln, revealing novel insights into calcium pump regulation. in addition, these same co-reconstituted proteoliposomes have been used for structural studies by electron cryo-microscopy. in an attempt to better de ne the structural interactions between plb, sln and ca -atpase, we have sought methods for the production of large two-dimensional crystals suitable for high resolution electron crystallography. we previously utilized the co-reconstituted proteoliposomes to produce long, tubular crystals suitable for helical reconstruction. our new procedure comprises three steps -co-reconstitution, membrane fusion, and crystallization -producing large two-dimensional crystals suitable for high resolution structural studies. herein, we will present our latest results characterizing the structural interaction between plb, sln and ca -atpase. t. v. demina , n. s. melik -nubarov , h. frey , e. e. pohl state university, chemistry department, moscow, russia, institute of organic chemistry, johannes-gutenberg-university, mainz, germany, humboldt university, charite, institute of celland neurobiology, berlin, germany multidrug resistance (mdr) of tumours is associated with overexpression of the p-glycoprotein responsible for an active drug ef ux from cells. block copolymers of ethylene oxide and propylene oxide (pluronics) are known to cause a pronounced chemosensitization of tumour cells. the effect may be due either to speci c polymer -protein interactions or to unspeci c lipid bilayer disturbance. we have shown recently that amphiphilic copolymers with various hydrophilic and hydrophobic blocks can disturb lipid bilayers. importantly, that block copolymers of propylene oxide and glycerol (ppo-pg) with hyperbranched "corona" induced larger effects then pluronics with linear polyethylene oxide chains. in the present work we have shown that ppo-pg copolymers increase dox cytotoxicity towards human erythroleukemia (k is , k /dox) and breast carcinoma (mcf /dox) resistant cell lines. using confocal and two-photon microscopy, we demonstrated that these copolymers accelerated dox penetration into resistant cells, inhibited efux and caused drug redistribution into nuclei. a clear correlation between the ability of the polymers to disturb lipid bilayers and favour drug accumulation in mdr cells was disclosed. this nding points to an unspeci c mode of the copolymers' chemosensitizing activity. voltage dependence of processes related to electrogenic membrane transporters electrogenic membrane transporters, such as the sodiumbicarbonate cotransporter (nbce ), may induce dependence on membrane potential upon processes which are innately voltageindependent. we tested this hypothesis by heterologously coexpressing electrogenic nbce from human kidney in oocytes of the frog xenopus laevis with the electroneutral rat monocarboxylate transporter mct . the apparent intracellular buffer capacity was increased by nbce expression and became voltage-dependent by mm/ mv membrane depolarisation. lactate transport via the mct not only became enhanced after co-expression with nbce , but also dependent upon membrane potential. injection of carbonic anhydrase caii from bovine erythrocytes into oocytes enhanced the ef cacy of nbce activity, identifying an additional, ca-sensitive, membrane current via nbce . our results show that nbce adds voltage-dependent buffer capacity to the cytosol; this is suggested to be the prime cause for enhancing acid/base-coupled transport and conferring membrane potential dependence on transporters which are stoichiometrically electroneutral. these interactions may have functional consequences for cells and tissues, where electrogenic and electroneutral processes interact, such as in brain, heart and muscle. . by using carboxy-snarf- as ph-sensitive uoroprobe and microspectro uorimetry, we now show that nhe activation is due to jun kinase (jnk) activation, resulting from reactive oxygen species (ros) produced during metabolism of b(a)p and might involve lipid raft. when analysing b(a)p-induced apoptosis, we have found that cariporide signi cantly reduces both nuclear fragmentation and caspase- like activity. we further show that nhe activation and/or alkalinization affects the mitochondrial ros production detected during the apoptotic cascade, likely via an effect on the complex iii of the electron transport chain. altogether, our results suggest that apoptotic xenobiotics, such as benzo[a]pyrene, induce an early activation of nhe that might play a signi cant role in the subsequent mitochondria-dependent apoptosis. conformational dynamics of a lactose proton symporter j. c. holyoake, m. s. sansom biochemistry department, university of oxford, south parks road, oxford, ox qu, u.k. transporter proteins are an essential class of proteins, catalysing the transfer of molecules across membranes. increasing numbers of transporter structures are becoming available, opening the way to study their dynamic properties using computational techniques. we present a study on the conformational dynamics of the lactose proton symporter lactose permease using molecular dynamics simulations. these simulations exhibit large scale conformational changes from the initial intracellularly open conformation to a more closed conformation that may be signi cant to the transport mechanism. the conformational change is analysed to identify the contributing motions. effects of nortriptyline and chlorpromazine on anthroylouabain-labeled na,k-atpase e. a. guevara , m. l. barriviera , a. hassón-voloch , s. r. louro departamento de física, puc-rio, rio de janeiro, brazil, instituto de biofísica carlos chagas filho, ufrj, rio de janeiro, brazil the effects of nortriptyline and chlorpromazine (cpz) on the uorescence properties of anthroylouabain (ao)-treated na ,k -atpase of electrocyte membranes from e. electricus are studied. na ,k -atpase oscillates between two major conformations e and e during ion transport cycle. the cardiotonic steroid ouabain speci cally inhibits this enzyme binding to the e conformation. the uorescent label ao presents increased uorescence when binding to the ouabain site of na ,k -atpase. tricyclic drugs such as the antipsychotic cpz and the antidepressant nortriptyline inhibit na ,k -atpase activity in the micromolar range. for the e enzyme, but not e , nortriptyline was found to increase the uorescence in a concentration dependent manner, suggesting a further stabilization of e . for both conformations, cpz induces negligible uorescence change up to µm. the uorescence of atpasebound ao, however, strongly increases upon ultraviolet exposure after cpz treatment at concentrations around µm. fluorescent products of cpz-photodegradation were studied in pure buffer and in the presence of membranes. the results suggest that cpz binds to na ,k -atpase and photolabels amino-acid residues near the ouabain binding site. how important is protein exibility for transport through ion channels? a. a. gray-weale university of sydney certain ion channels are selective for k+ over other ions, but the geometry of the pore does not explain selectivity because thermal uctuations are too large. i extend the usual treatment of ion channels with molecular dynamics simulation by calculating the static and dynamic pair correlations between monovalent ions and ion channels (gramicidin-a and kcsa), and also between certain small, complex cations and the gramicidin channel. this means not only the radial-distribution functions or the density pro les, but also correlations between the ion and the mass and charge densities of various regions of the protein. the advantage of this approach is that it systematically identi es the elements of the protein and modes of motion that contribute to selectivity, and illustrates the decay of correlations. recently, noskov et al. [ ] showed that thermal uctuations protect selectivity. my results on the interaction of ions with carbonyl groups agree with theirs, but take the analysis further to higher correlations. the key new element is the study of the time-correlation functions that describe the motion of the ions through the channel, borrowing methods originally developed for the study of dense or even supercooled liquids. the surface-enhanced infrared absorption spectroscopy (seiras) is used for the investigation of two membrane proteins, the cytochrome c oxidase (cco) and the bacteriorhodopsin (br). of central interest are the transport-mechanisms of electrons (cco) and ions (br). the main parts of the setup are an infrared light source, a hemispherical si-crystal, in which the beam is internal re ected and a plexiglas cell with buffer solution (see schematic scetch). the beam is totally re ected on the inner at surface of the crystal, but the evanescent wave excites surface plasmons in a chemically adsorbed gold-layer on the crystal (attenuated total re ection spectroscopy-atr). the protein to be analysed is attached at the gold surface and can absorb certain wavelengths. the gold is need for the surfaceenhancing effect. cco plays a major role in the respiratory chain, the retinal protein br is a photosynthetic protein. the cco is immobilized on the gold surface via the af nity of its histidine-tag to a nickel-chelating nitrilo-triacetic acid (nta) surface. for the br we incorporate the protein in a lipid membrane, which is attached on the gold surface by the sulfuric bindings of , -di-o-phytanyl-sn-glycerol- -tetraethylene glycol-d,l-lipoic acid ester lipid (dptl). the sensitivity of this method is further enhanced by modulation of an external parameter, like the electric potential. effects of copper ions on the escherichia coli growth and proton-potassium exchange copper ions are required for the function of many important enzymes in escherichia coli but can cause a number of toxic cellular effects also. it's interesting to reveal the in uence of copper ions on the growth of bacteria and proton-coupled membrane systems. upon transition of e. coli mc wild-type culture to stationary growth phase a decrease in redox potential (e h ) from the positive values ( + mv) to the negative ones (of - to - mv), resulting h production by formate hydrogenlyase (fhl) have been studied. copper increased a latent growth phase duration as well as delayed a logarithmic growth phase in concentration-dependent manner. during the anaerobic growth, the production of h was strongly inhibited in the presence of cucl ( mm). . mm cucl was inhibited h production under experimental conditions with glucose. the inhibitory effect of copper ions ( . mm) on n',n'dicyclohexylcarbodiimide (dcc)-sensitive h /k exchange was also observed: k uptake was decreased and the stoichiometry of dcc-inhibited ion uxes varied. interestingly, these effects on h and k uxes were absent for the mutant hd (hyc-operon for hydrogenase was deleted). we suggest that copper ions, inhibiting the activity of fhl, have an effect on h /k -exchanging mechanism which is the proton f f -atpase associated with k uptake trk system. this effect may be due to the relationship of fhl with the ion-exchanging mechanism above under fermentation at alkaline ph. lateral diffusion in tethered bilayer membranes m. jung, v. atanasov, w. knoll, i. koeper max planck institute for polymer research, mainz, germany tethered bilayer membranes (t'blm) provide a useful platform for the investigation of bilayer membranes as well as embedded membrane proteins. we have developed a modular system, which is suitable for surfaces providing gold and oxide surface coatings. these systems serve as a quasi natural environment for the study of membrane proteins, being functionally incorporated into a lipid bilayer, which is covalently bound (tethered) to the substrates. functionality could be shown using electrochemical methods. here we present a study of these systems, basically the membrane itself, using uorescence recovery after photobleaching (frap) studies in order to investigate lateral motion in the lipid bilayers. lateral mobility is essential for successful incorporation of large membrane protein complexes. we will present rst results of experiments that try to differentiate motions hindered due to the tethering from diffusion in free oating or suspended bilayers. the information gained in this study will serve for improvements in the chemical structure of the tethered molecules. we will develop the system as a basis for bio sensing applications, where embedded proteins will serve as actual sensing elements. a. d. ivetac, j. campbell, m. s. p. sansom biochemistry department, university of oxford, south parks road, oxford, ox qu, u.k. atp-binding cassette (abc) transporters form an important superfamily of membrane proteins which couple atp hydrolysis to the active transport of diverse compounds across the cell membrane. their biomedical relevance is highlighted in examples such as multidrug resistance to antibacterial and anticancer agents, and cystic brosis. the availability of crystal structures of three complete bacterial abc transporters provides an opportunity to study structurefunction relationships at the atomic level. in this work, we carry out multi-nanosecond molecular dynamics simulations of the vitamin b importer from e. coli (btucd), with both the complete multimeric transporter embedded in a phospholipid bilayer and the soluble subunits in a membrane-free environment, in an attempt to elucidate some of the conformational changes which arise during the transport event. atp-bound and atp-free structures are used to investigate the effect of nucleotide on the system. a range of analytical techniques have been applied to assess the dynamic behaviour of the protein during the simulations, which includes measurements of: conformational drift, residue exibility, transmembrane domain (tmd) movement, concerted protein motions, nucleotide-binding and translocation pathway changes. an in-vitro method was designed to measure transmembrane transport rates. liposomes were prepared by extrusion with dipalmitoyl phosphatidylcholine (dppc) and optionally cholesterol, and loaded with a peptide (zinc-insulin tagged with a uorescent group or bsa). after removal of the non-encapsulated peptide from the liposome solution by gel ltration, the release of the peptide from the liposomes was monitored by uorescence as a function of time at various temperatures. the transport was greatly accelerated by the presence of a speci c proprietary excipient molecule (cyclopentadecanolide -cpe ™), which effectively triggered the release of the peptide. a mathematical model was developed to quantify these results. a semi-empirical nonlinear equation involving four parameters ts the protein release pro les. then a neural network predictions model was used to correlate the different release condition parameters and the four semi-empirical tting parameters based on the experimental data sets. most release data t well with the mathematical model, further supporting our theory of a two step release mechanism. phenyltin the well known group of organotin compounds that exhibit toxic properties in relation to the biological systems are phenyltins. no studies have been performed as yet to establish directly whether organotins such as diphenyltin dichloride ( dpht) and triphenyltin chloride (tpht) cross the lipid bilayer. we have performed experiments that showed transfer of those compounds across the lipid bilayer using the stopped-ow technique and desorption of those compounds from a monolayer using the langmuire technique. obtained results demonstrate that dpht and tpht rst adsorb onto the lipid bilayer surface, in diffusion controlled manner, within a very short time ( . s), whereas the membrane passing was observed in a minute's time range. the long time kinetics show a complex dependence on the kind of compound, its concentration and the presence of cholesterol in the membrane. the desorption of both compounds from the monolayer to water subphase occurs in a minute's time range. these observations may explain the known fact, that the in uence of organic, amphiphilic tin (and also lead) compounds is more toxic than that of inorganic ones.the phenyltins much easier (compared with tin or lead ions ) penetrate e.g. blood -brain barrier. [ under the resting conditions ca concentration in agonistsensitive ca stores re ects a balance between active uptake mediated by a ca -atpase (serca) and passive ef ux of ca . this ca leak appears to be a common property of ca -storing organelles, but the nature of the leak in submandibular acinar cells remains unclear. we have studied the ca leak pathways in the endoplasmic reticulum (er) of acinar cells of rat submandibular salivary gland by directly measuring concentration ca in the er ([ca ] er ) in mag-fura /am preloaded cells while [ca ] i was clamped at a resting level with a egta/ca mixture. we have shown that thapsigargin (tg) or ca -free buffer treatment completely blocked ca uptake by serca after the rst minute of superfusion and caused a ca leak represented by continuous decline in [ca ] er . this ca leak from the er was not sensitive to tg, heparin and ruthenium red and therefore appears to be independent of the serca, the insp receptor and the ryanodine receptors. however, treatment with puromycin ( . - mm) to remove nascent polypeptides from er-ribosome translocon pores increased ca leak from the er by a mechanism independent of the serca, insp or ryanodine receptors. thus we conclude that basal ca leak from the er of submandibular acinar cells occurs through translocon pores in the er membrane. r. b. kishore, j. reiner, e. edgu-fry, a. jofre, k. helmerson physics laboratory, national institute of standards and technology we have developed a procedure to make lipid and polymer nanotubes of up to one cm long and nm in diameter, from the surface of giant liposomes and polymersomes, using micro uidics and optical tweezers. the liposomes and polymersomes were formed, using elcetroformation method, from phospholipids and amphiphilic diblock copolymers, respectively. the polymer tubes were made extremely robust by cross-linking them using chemical reactions. we are currently studying the transport of molecules in the crosslinked nanotubes for use in nano uidic networks. p-glycoprotein (p-gp) is an active membrane transporter capable of expelling out of the cell a large number of potentially cytotoxic amphiphilic molecules with unrelated chemical structures. as a consequence, p-gp may be responsible for multidrug resistance of tumors against chemotherapy (mdr); it also plays a key role in absorption, biodisposition and elimination of many pharmaceuticals. to understand the molecular mechanisms of the transmembrane drug transport mediated by pgp, it is highly desirable to design a convenient assay for measuring both p-gp atpase activity and p-gp transport function. to do so, we used inside-out native membrane vesicles, prepared from mdr cells and containing high amounts of p-gp. we took advantage of the speci c property of a uorescent dye, the carbocyanin jc- , known to be expelled out of mdr cells: above a critical concentration (the "cjc"), this dye forms j-aggregates which emit a uorescence at a wavelength very different from that emitted by the monomer. in the presence of mgatp, the p-gp-containing vesicles accumulated jc- , which exceeded locally the cjc and thus formed intraluminal j-aggregates; these aggregates allowed accumulated jc- both to be sequestered inside the vesicles, by dramatically slowing down its passive backdiffusion, and to be speci cally detected. kinetic characterization of this transport suggests that jc- is rst translocated to the exoplasmic lea et of the vesicle membrane before its internalization into the aqueous phase of the vesicle lumen. interaction between the energy metabolism and externally applied electric elds in yeast cells electric elds are often used for biophysical or biomedical treatment of biological cells, e.g. cell fusion or killing of cells. however, only a few data about the possible mechanisms of electrosensitivity of biological cells are available. since electrostimulation always induces depolarization of biomembranes, an impact of the energy metabolism is obvious due to the regeneration of electrochemical gradients by the expenditure of cellular energy. our aim is to investigate the interactions between externally applied electric elds and the aerobic/anaerobic energy metabolism of yeast cells. for this, we have constructed a new electrical interface for local stimulation of biological cells with variable duration and amplitude. when applying short lasting electrical pulses to yeast cells, we nd a direct response of the energy metabolism (measured by nadh-uorescence) to these pulses. a sudden and fast decrease in nadh is followed by a slower recovery of the uorescence signal. these nadh-signals are abolished in the presence of antimycin a or kcn, demonstrating the importance of mitochondrial energy production for this phenomenon. we attribute these changes to the immediate break down of atp as a consequence of the regeneration of the membrane potential (atpases) and the slower regeneration of atp by mitochondrial respiration. new insights of hypericin blood transport and its incorporation into the plasma membrane b. m. macri , g. stoian , m. l. flonta department of animal physiology and biophysics, faculty of biology, university of bucharest, department of biochemistry, faculty of biology, university of bucharest, bucharest, romania hypericin (hyp) is one of the active compounds from hypericum perforatum (an herb usually prescribed as antidepressant). the bioavailability of this hydrophobic molecule is a very important issue for medical applications. the goal of our work was the study of hyp blood transport mechanisms. techniques of absorbtion spectroscopy, electrophoresis and uorescence microscopy were used in order to de ne the properties of hyp-albumin and hyp-lipoproteins complexes and to explain the action of hyp at the plasma membrane level. hyp bind to several electrophoretic (sds-page) bands evidenced by mice plasma migration. both albumin and lipoproteins bind to hyp, forming complexes, during the blood transport process. different types of lipoproteins from males and females plasma mice were evidenced by gradient electrophoresis to bind hyp. hyp-albumin complex was also identi ed by absorbtion spectra, and the ratio a /a has a ph-dependence. hyp interaction with plasma membranes was also examined on cell culture by uorescence microscopy, and hyp plasma incorporation is a dose-and incubating time-dependent process. our results partially elucidate the plasma fractions that bind hyp, contributing to its blood transport. this study proposes a new mechanism of hyp cellular insertion, discussing its plasmatic membrane penetration due to its high hydrophobicity. the overexpression of p-glyoprotein (p-gp) is one of the major causes of multidrug resistance (mdr) in cancer chemotherapies. many p-gp inhibitors have been designed to reverse the mdr effect, but the structure-activity relationships of the p-gp inhibitors and substrates still remain largely unknown. until now, it is still very challenging to obtain the high-resolution p-gp structures and currently only low-resolution electron microscopy structure is available. this has caused the structure-based design of "p-gp-ignoring" therapeutic agents a remote goal. however, the recent determination of x-ray crystal structures of bacterial lipid a transporter, msba, has provided eligible structure templates for homology modeling of p-gp. we have therefore conducted explicit solvent molecular dynamics simulations of the fulllength ef ux pump, human p-gp, in an excessively hydrated popc bilayer to re ne the homology model. both free and atp-bound forms of p-gp have been simulated. the entire system consists of more than , atoms. our molecular dynamics simulations have shown that the overall architecture of p-gp remained very stable for tens of nanoseconds, while the observed membrane undulation was rather large. the simulation results have allowed us to investigate the conformational changes of p-gp upon atp binding in the ef ux process and to predict the possible binding site of various known substrates and inhibitors. the re ned structure models of p-gp by our simulations could be used as the basis for further drug design. electroporation (ep) is a phenomenon where increased permeability of cells exposed to an external electric eld is observed. the induced transmembrane voltage presumably leads to the formation of aqueous pores in the phospholipid bilayer, which increases permeability of the membrane for molecules and ions. ep is currently used in many biomedical applications including transfer of genes and electrochemotherapy of tumors. still, the molecular mechanisms of the process are not fully explained. recently it was proposed that ep could be monitored in real-time by measuring electric conductivity of tissue. so far the studies focused mostly on a single pulse, however in biomedical applications usually several pulses are used. in our study we used a train of electric pulses to analyse the relationship between electric conductivity and cell permeabilization. current-voltage measurements during and after pulse application were performed in dense suspension of cells. conductivity changes were analysed numerically using nite elements method and compared with the percentage of permeabilized cells. we obtained a transient increase in conductivity above a certain voltage with complete relaxation in < s. substantial changes in conductivity are also due to the diffusion of ions through membrane pores and osmotic swelling. we further show that relation between conductivity and permeabilization level is indirect. interaction of quinolones with bacterial porin ompf: uorescence quenching studies quinolones are widely used antibiotics witch develop their antibacterial action by inhibition of important bacterial enzymes. consequence of the internal location of their target of action, the translocation of this drugs trough the outer membrane is an essential step for their antibacterial action. in vivo studies have been showing that ompf is important for the entrance of some of these antibiotics, but the exact degree of involvement of this protein in the transport of the different members of this group of antibiotics, remains unknown. in this study, the quenching of the intrinsic tryptophan uorescence of ompf, in presence and in the absence of the drugs and by two distinct quenchers, was used as a rst approach, to elucidate ligandinduced structural changes and consequently prove the differential involvement of ompf in the entrance of these antibiotics in the bacterial cell. the results obtained reveal that the degree of interaction with the protein is related with the hydrophobicity of the different antibiotics. this kind of evidence suggests that the entry by the porin channel is not the only path used by these antibiotics and that it is more important for the latest generations of this group because of their increased hydrophilic characteristics. inhibition of multidrug resistance-associated protein mrp and kv channels by natural polyphenols k. michalak, a. teisseyre, b. ania-pietrzak department of biophysics, wroclaw medical university, poland resistance to cytotoxic agents remains a major obstacle to successful chemotherapy in cancer. best-characterized form of drug resistance is caused by the overexpression of genes encoding membrane drug pumps, like p-gp or mrp . in present study, activity of several plant polyphenols ( avonoids and stilbene) against mrp has been studied using functional assay based on ef ux of mrp uorescent substrate. very recently, a role of kv . potassium channels in proliferation of various cancer cells was suggested. in our study the effect of the plant polyphenols on voltage-gated potassium channels kv . was investigated by patch-clamp electrophysiological method. some of studied compounds were found to be active inhibitors of multidrug resistance-associated protein mrp and voltage-gated potassium channels, and their properties are promising for further research in the eld of anticancer activity of natural products. the transport mechanism of melibiose permease: a study using electrical measurements and uorescence techniques k. meyer-lipp , c. ganea , t. pourcher , g. leblanc , k. fendler max planck institut für biophysik, frankfurt, germany, c. davila medical university, bucharest, romania, cea, université de nice so a antipolis, nice, france the melibiose permease (melb) of escherichia coli is a membrane bound carrier that uses the favorable na+, li+, or h+ electrochemical potential gradient to drive cell accumulation of alfa-galactosides (melibiose, raf nose) or beta-galactosides (methyl- -thio-beta-dgalactopyranoside). electrophysiological techniques have proved to be extremely useful tools to investigate the mechanism of ion transfer across the membrane by ion-coupled transporters. using a solid supported membrane (ssm) as a capacitive electrode a rapid solution exchange can be combined with the high sensitivity of planar lipid membranes and allows time resolved investigation of the charge translocation during the catalytic cycle of na+/solute symporters. this technique has been combined with uorescence measurements, which report on structural changes during the substrate transport process of the carrier. we have used time resolved tryptophane uorescence, uorescence energy transfer with a uorescent sugar substrate and site speci c uorescence of a label attached to a cysteine residue on the protein. this allowed us to identify conformational transitions during the reaction cycle of the melibiose permease. we could assess their electrogenicity and determine rate constants. a kinetic model for na+ and melibiose binding and transport is presented. electrophysiological characterization of the vast number of annotated channel and transport proteins in the postgenomic era would be greatly facilitated by the introduction of rapid and robust methods for the functional incorporation of membrane proteins into dened lipid bilayers. we present an automated method for reconstitution of membrane proteins into lipid bilayer membranes, that substantially reduces both the reconstitution time and the amount of protein required. we have applied this well-de ned system to the characterization of a novel mitochondrial uncoupling protein, ucp and demonstrated that ucp exhibits protonophoric function exclusively in the presence of fatty acids, similar to that previously shown for its homologue ucp . the membrane conductance was proportional to the concentration of the reconstituted ucp in presence of oleic acid or eicosatrienoic acid, and was inhibited by atp. amphipols are amphipathic polymers designed to replace or supplement detergents in membrane protein solution studies. for the study of the ca -atpase from sarcoplasmic reticulum, previous experiments have revealed both advantages and disadvantages to the use of a polyacrylate-based amphipol, a - . these issues have been reinvestigated using four different amphipols. size exclusion chromatography showed that, although a - aggregates in the presence of millimolar concentrations of calcium -an effect that probably accounts for most of the aggregation of atpase/a - complexes observed in our previous work-, aggregation can be avoided by resorting to a sulfonated version of a - . we also found that all amphipols tested slowed down the rate of calcium dissociation from its binding sites and reduced atpase activity, while protecting the solubilized protein against denaturation. this suggests that association with the polymer may damp the protein's dynamics, perhaps due to the multipoint attachment of the polymer to its hydrophobic transmembrane surface. such a "gulliver" effect could contribute both to the protection of membrane proteins against denaturation and to the reversible inhibition of serca a. clc proteins are found from prokaryotes to mammals. they function as plasma membrane chloride channels or provide neutralizing anion currents for v-type h -atpases that acidify compartments of the endosomal/lysosomal pathway. vesicular clcs have been thought to be cl -channels, in particular because clc- and clc- mediate plasma membrane cl -currents upon heterologous expression. we have shown, however, that these two mainly endosomal clc proteins rather function as electrogenic cl /h exchangers, resembling the transport activity of the bacterial clc-e that has been crystallized. neutralization of a critical glutamate residue not only abolished the steep voltage-dependence of transport, but also eliminated the coupling of anion ux to proton counter-transport. clc- and clc- may still compensate the charge accumulation by endosomal proton pumps, but are expected to tightly couple vesicular ph-gradients to cl -gradients. calorimetry and mechanics of ca transporting systems in rat myocardial bigeminies a series of new n-oxides of tertiary amines (nta) was checked for its biological activity. individual compounds differed in the length of substituted alkyl chain. the primary goal was to nd if they can be used as effective antioxidants and, to what degree they modify used model (liposomes) and biological (erythrocytes, algae and cucumber) membranes. various methods were used in order to do that. a mechanism of the interaction between nta and membranes was studied by measuring their potency to hemolyse erythrocytes, to in uence a phase transition temperature in dppc liposomes, to change a membrane potential of algae cells. the measure of the interaction of nta with cucumber cells were potassium leakage, chlorophyll content and inhibition of growth of hypocotyls. antioxidative abilities of nta were determined by measuring their ef ciency to protect erythrocytes against membrane lipid oxidation induced by uv irradiation and by comparising their antioxidative ef ciencies with that of trolox (vitamin e analogue) in chromogen experiments. the mostly widely employed mechanism of drug extrusion in bacteria is via membrane transport proteins called ef ux pumps. in gram-negative bacteria, multidrug resistance is conferred by tripartite complexes, rather than by a single transport protein. through these systems, a wide range of substrates is expelled from the cytoplasm, through the periplasmic region, to the exterior of the cell. among these complexes, the acrab/tolc system in escherichia coli is formed by an inner membrane ef ux pump, acrb, an outer membrane protein, tolc, and a periplasmic protein known as an adaptor, acra. the components of this complex are studied, in order to provide insights into drug transport in bacteria. here we present a dynamics study on mexa, homologue of acra from pseudomonas aeruginosa. the protein has been studied by molecular dynamics simulations in bulk water. a structural adjustment by the periplasmic protein is required in order to engage both the bottom part of the om protein and the top region of im protein. the dynamics on mexa reveals a exible behaviour of the protein in water. the major concerted motions observed are the hinge-bending of the two domains, and the rotation of the -barrel domain. these can be related to the adaptation of mexa (and acra) to the om and im proteins during the process of assembly in forming the complex, and during the opening of the channels. electrophysiologic study of ap in chara corallina -indication of its biochemical nature the speci c conductance's of aqueous solution of electrolytes (viz.naf, nacl, nano , na so , kf, kcl, kno , k so , mgcl , cacl , fecl , mncl ,crcl ,cucl , cocl , )have been measured across peritoneum at temperatures between( - ) c.conductance attains a maximum limiting value at higher concentrations for each electrolyte due to a progressive accumulation of ionic species within the transmembrane region. the membrane becomes more and more conductive to incoming ions and attaining a limiting value due to the fact that an electrically neutral pore, which is speci c for a particular ion, is unlikely to contain more than one type of ion. consequently, at high electrolyte concentration, the pore saturates and the conductance's approaches a limiting value. the values of speci c conductance measured follow the sequence for anions; so > cl > no > f . whereas for the cations the sequence is k > na ; ca > mn > co > cu > mg ; cr > fe . the energy of activation for the cations as well as for the anions follows the sequence (for cations): the low temperature ( k) chlorophyll uorescence, photochemical activity, oxygen ash yield and oxygen burst decay of thylakoid membranes with different organization of the light-harvesting chlorophyll a/b complex of photosysytem ii (lhcii) were investigated after freeze-thaw cycle in criotoxic and cryoprotective medium. the increase of lhcii oligomerization, which is associate with signi cant reduction of the surface charge density of the thylakoid membrane, correlates with lower extent of freezing damage of the photosynthetic apparatus, when the procedure is carried out in cryotoxic medium (nacl). in the presence of the cryoprotective compound (sucrose) freezing damage is less pronounced and is not affected by the degree of the lhcii oligomerization. the mechanisms of damage and protection of photosynthetic apparatus in the process of freeze-thaw treatment are discussed. spectral and redox characterization of the novel heme ci in the cytochrome b f complex . this is an indication of different spin delocalization in the primary donor, for the mutant being typical of a monomeric oxidized bchl. considering the fact that the properties of both isolated and membrane-associated mutant rcs were similar, we conclude that missing bchl molecule from the mutant rc was the result of the introduced mutation but not of the protein puri cation procedure. authors acknowledge the support by the russian brf. we created two site-directed mutants, a s and l i in the d protein of photosystem ii in thermosynechococcus elongatus. both mutations are within the binding pocket of the primary quinone acceptor (q a ). we investigated the effects of the mutations in vivo and in isolated psii. while the l i mutant exhibits characteristics similar to the wild type, the a s mutation effects q a charge recombination measured by thermoluminescence and uorescencedecay. these results strongly indicate that the a s mutation induce a shift in the redox potential of q a . the a s accelerates the rate of photoinhibition, an effect consistent with the negative shift in the redox potential. epr was used to measure the temperature dependence of the electron transfer from q a to q b in the a s mutant. it was found to be indistinguishable from the wild type despite the difference in the midpoint potential of q a . this is taken as an indication as a gating mechanism on the acceptor side of psii similar to that in bacterial reaction centers. protochlorophyllide oxidoreductase takes an abnormal reaction pathway below the glass transition g. durin , d. j. heyes , c. n. hunter , d. bourgeois ibs and esrf, grenoble, france, krebs institute and r hill institute for photosynthesis, shef eld university, shef eld, uk motions through the energy landscape of proteins lead to biological function. at temperatures below a dynamical transition ( - k), the activity of some proteins cease. in this work, we describe an enzyme that, instead, engages into a non-productive pathway below k. protochlorophyllide oxidoreductase (por) catalyzes the reduction of protochlorophyllide (pchlide) into chlorophyllide (chlide), a key step in chlorophyll biosynthesis. por is one of the two enzymes known to require light for catalysis. when illuminated with gentle light at k, the complex of t. elongatus por with pchlide and nadph transforms into a nonuorescent intermediate. upon warming, several uorescent intermediates develop, and at k chlide is released. when illuminated at temperatures below k, por behaves differently. if gentle light is used, the reaction can not start. instead, if a blue laser source is used, the initial complex disappears, like at k. however, upon warming, a new intermediate develops that uoresces at nm and leads to a dead-end product. by using uorescence microspectrophotometry, we have measured the solvent glass transition temperature of the system to be k. the solvent glass transition, possibly controlling a por dynamical transition, may be the determinant that switches the enzyme reaction pathway from a non productive to a productive one. the nonproductive pathway results from a two-photons absorption mechanism, whereas the productive pathway is a one-photon mechanism. sensory rhodopsin ii from n. pharaonis (npsrii) forms a complex with its cognate transducer nphtrii in a : stoichiometry . light activation of npsrii leads to a movement of helix f which triggers a rotation of tm in nphtrii . the mechanism of signal transduction through the hamp region to the cytoplasmic domain of the transducer is still unknown. structural information exists for the transmembrane and cytoplasmic regions, however the hamp domain is not yet characterized. in order to obtain structural information on this domain, twenty-four residues in the membrane adjacent region ( - ), and six residues in the following region were spin labeled and investigated by cw and pulsed x-band epr. to analyze the overall architecture of the complex, doubly spin labeled variants between the transducer and the receptor were also engineered. depending on their function, the absorption spectra of rhodopsins can be tuned by the protein over a wide range. a major determinant for spectral shifts between different rhodopsins are electrostatic interactions between the chromophore retinal and the protein. we compute and compare the classical electrostatic potential at the retinal of three archaeal rhodopsins: bacteriorhodopsin (br), halorhodopsin (hr), and sensory rhodopsin ii (srii). these proteins are an excellent test case for understanding the spectral tuning of retinal. the absorption maxima of br and hr are very similar, while the spectrum of srii is considerably blue shifted. we nd that the electrostatic potential is similar in br and hr, but differs signi cantly in srii. a quantum mechanical model of a particle in a box with a step potential can qualitatively relate the differences between the electrostatic potentials of the proteins to the relative shifts of their absorption maxima. by decomposing the electrostatic potential into contributions of individual residues, we could identify six residues that are responsible for the differences in electrostatic potential between the proteins. three of these residues are close to the retinal, while the other three residues are more then angstroem away from the retinal. the counterion of the schiff base, which is frequently discussed to be involved in the spectral tuning, does not contribute to the dissimilarities between the electrostatic potentials. effect of uv-a radiation on thylakoid membranes with different organization p. ivanova, a. dobrikova, t. markova, s. taneva, e. apostolova institute of biophysics, bulgarian academy of sciences, so a, bulgaria the effect of uv-a ( - nm) radiation on the energy transfer and the photosynthetic oxygen evolution of thylakoid membranes from pea mutants was investigated. the membranes have different pigment composition, stoichiometry and organization of pigment-protein complexes. the aim of our work was to nd out whether uv-a induced damage is affected by the altered content and/or oligomerization of the main light-harvesting chlorophyllprotein complex (lhcii) in thylakoid membranes. the data for the effect of uv-a radiation on the oxygen evolution demonstrate that: (i) the inhibition of photosystem ii (psii)-mediated electron transport and ash-induced oxygen yields strongly depend on the amount of lhcii; (ii) the increase of the s o populations of psii centers in darkness is more pronounced in thylakoid membranes with smaller amount of lhcii; (iii) the inhibition of the oxygen evolution is related to the reduced number of the functionally active psii centres; (iv) the degree of impairing of active psii centres depend on the amount and oligomerization of lhcii. the results also show that the altered content and organization of lhcii in uence the uv-a light-induced changes in the energy transfer between psii and psi and within the supramolecular lhcii-psii complex. the effects of uv-a radiation on leaves and isolated thylakoid membranes are compared. sudden polarisation -a large change in the electric dipole moment between the excited and the ground state -is a well-known phenomenon for retinal chromophore. some early models of the energy transduction mechanism in bacteriorhodopsin (br) even attribute a primary functional role of that. however, it was apparently unrecognized that the maxwell theory intuitively predicts the appearance of an ultrafast transient electromagnetic radiation due to this dipole moment change. here we show that the existence of this type of radiation can be derived from semiclassical quantum electrodynamics as a second order phenomenon. in optical terms it corresponds to the previously unstudied resonant case of optical recti cation. recently we experimentally observed a major component in the fs coherent infrared emission of oriented purple membranes of br corresponding well to this effect (groma et. al, proc. natl. acad. sci. , , ). our theory predicts that such a signal holds detailed information on the dynamics of excited state polarization, opening a new branch of impulsive spectroscopy on asymmetric systems. beyond optical recti cation we found a complex phase a coherent oscillation living for a few ps, i.e. much longer than the excited state of br. fitting analysis resulted in at least seven vibrating modes in the - cm region, while windowed fourier transform indicated time-dependent frequency distribution. a. ghignoli, g. cercignani, s. lucia, g. colombetti istituto di bio sica, cnr -pisa, dipartimento di fisiologia e biochimica, università di pisa, italy the life cycle of ophryoglena ava, a histophagous ciliate dwelling in fresh waters, reportedly includes several stages that feature morphology changes and different phototactic responses. previous studies on the phototactic responses in o. ava during its phase of maximal positive phototaxis led to an action spectrum with two main peaks at and nm, and a minor peak at nm. starting from those results, we analyzed the phototactic response at various cell ages, using three broad-band interferential lters (fwhm = nm) centred respectively at , and nm, and constructed dose-effect curves for each band. a higher photosensitivity at nm, and lower photosensitivies with the other two lters ( and nm) have been observed at any cell age. however, the photosensitivities in the blue and orange regions show a different time course vs. cell age with respect to the photosensitivity in the green region. measures were also carried out on cells whose feeding cycle was altered by a -day starvation (a double time with respect to the standard protocol) before being fed at t = . the maximal photoresponse values reached by starved cells are lower than the highest values reached with standard cultures; in other words, a general reduction of the phototactic response is observed. these results suggest that, while feeding optimally induces cell division, it does not generally reset all cellular functions. a. quaranta , f. lachaud , y. pellegrin , p. dorlet , m.-f. charlot , s. un , a. aukauloo , w. leibl service de bioénergétique, cea-saclay, bât. , gif-sur-yvette cedex (france), laboratoire de chimie inorganique, bât. , université paris-sud, orsay (france) coordination complexes based on a photoactive rutheniumpolypyridyl moiety linked to simple, rigid ligands with binding sites for transition metals, are developed to mimic the light induced charge separation and water oxidation processes taking place in the photosynthetic apparatus. inspired by the structure around the donor side of photosystem ii a family of phenanthroline based ligands holding an imidazole, a phenol or an indole unit simulating the amino acids histidine, tyrosine and tryptophan in the oxygen evolving complex, were developed as models for proton-coupled electron transfer. in some of the molecules investigated the hydrogen bonding interaction present in the natural system is reproduced. combined data from photophysical, spectroelectrochemical studies and dft calculations evidenced the photogeneration of a phenoxyl or a tryptophan radical upon excitation of the chromophore in presence of an external electron acceptor, therefore mimicking the electron trade between p and tyrz-hist . finite element model to predict the electric potential distribution in ps i containing vesicles c. p. a. pennisi , e. chemineau , e. greenbaum , k. yoshida center for sensory motor interaction, aalborg university, denmark, chemical sciences division, oak ridge national laboratories, usa, facultad de ingeniería, uner, argentina photosynthetic reaction centers (rc) are integral membrane proteins and molecular photovoltaic structures. recently, it was suggested their use as triggers of voltage-gated ion channels in excitable cells, where a certain voltage threshold has to be reached to evoke a response (kuritz et al., ieee trans. nanobiosci. in press ). experimental studies with rc's reconstituted in lipid vesicles have shown different values of transmembrane voltage, depending on parameters like light intensity, rc concentration and membrane passive properties. ultimately, the purpose of this work is to have a tool to estimate the proximity, number and density of rc's required near a voltage-gated channel to activate an excitable cell. as a starting point, we aim to predict the spatial distribution of the membrane potential in vesicles. a nite element model was realized using a commercial package (femlab, comsol a/s). the three-dimensional distribution of the electrical potential near a single rc in the surface of a spherical vesicle was calculated. in terms of density, in conditions of saturating light, a minimum of , e rcs/cm is needed to develop a potential of mv, capable to activate voltage-gated sodium channels. microsecond time-resolved x-ray diffraction study of purple membrane t. oka , k. inoue , m. kataoka , n. yagi faculty of science and technology, keio university, japan, japan synchrotron radiation research institute (jasri), japan, nara institute of science and technology, japan the structural changes in the photoreaction cycle of bacteriorhodopsin, a light-driven proton pump, was investigated at a resolution of Å by time-resolved x-ray diffraction experiment utilizing synchrotron x-rays from an undulator of spring- . the xray diffraction measurement system, used in coupling with a pulsed yag laser, enabled to record diffraction pattern from purple membrane lm at a time-resolution of µsec over the time domain of µsec to msec. the low temperature ( k) chlorophyll uorescence, photochemical activity, oxygen ash yield and oxygen burst decay of thylakoid membranes with different organization of the light-harvesting chlorophyll a/b complex of photosystem ii (lhcii) were investigated after freeze-thaw cycle in cryotoxic and cryoprotective medium. the increase of lhcii oligomerization, which is associate with signi cant reduction of the surface charge density of the thylakoid membrane, correlates with lower extent of freezing damage of the photosynthetic apparatus, when the procedure is carried out in a cryotoxic medium (nacl). in the presence of a cryoprotective compound (sucrose) freezing damage is less pronounced and independent of the degree of the lhcii oligomerization. the mechanisms of damage and protection of photosynthetic apparatus during the freeze-thaw process are discussed. we have studied the effect of a cytokinin meta-topolin (mt, m) on senescence-induced changes in the photosynthetic apparatus of detached primary leaves of wheat (triticum aestivum l. cv. hereward). the senescing leaves were kept under continuous light conditions. mt signi cantly slowed down the senescenceinduced decrease in chlorophyll content and markedly stimulated violaxanthin zeaxanthin (z) conversion. the high z content was maintained even after an hour in darkness. mt treatment caused also the appearance of an emission band f peaking at - nm. this emission band is attributed to aggregates of lightharvesting chlorophyll a/b-binding proteins (lhc), the production of which is associated with a higher z content. the presence of lhc aggregates in mt treated leaves was documented also by electron microscopy imagines. besides the lhc aggregation, mt induced also a decrease in photosystem i content which was documented by electrophoresis and k-uorescent spectra. supported by grants frvs / and msm . recently high resolution images of bacterial photosynthetic membranes have revealed the organization of membrane proteins in these native membranes. the organization revealed is remarkable, and all the more so when we realize that these specialized, protein rich, membranes differentiate from the cytoplasmic membrane which has a more complex composition and is richer in lipids. analysis of the protein organization in these specialized membranes from several different bacteria suggest that the organization results from a phase separation of several different contiguous phases. in order to better understand our observations we have undertaken an examination of the different phase behaviors that are possible for membrane proteins considered as a two dimensional colloid. monte-carlo modeling of the phase diagram of this system shows the importance of interaction distance in the determination of system behavior. transcription of our observations on the model systems to the photosynthetic membranes suggests that electrostatic and elastic forces in the membrane are of particular importance in determining the high level order of membrane proteins. the recent crystal structure of photosystem i (psi) from synechococcus elongatus shows two quasi-symmetric branches of potential electron transfer cofactors including primary donor (dimer of chlorophylls p ), monomeric chlorophylls a and a and quinone a , bound to the psaa/psab heterodimer. so far, it is not clear if both potential electron transfer pathways are active in this process or only one of them. to solve this issue, we studied a set of mutants with methionine coordinating the primary electron acceptor, a , replaced by histidine, leucine, or serine in either of two branches. our results obtained with a technique of femtosecond transient absorption spectroscopy show that both branches are equally active in electron transfer. mutation in either branch slows the forward electron transfer between a and a from ps in wilde type psi to - ns in all these mutants. this strong effect is explained by signi cant change in the redox midpoint potential and change in the position of a by the mutations. i. s. zaharieva department of biophysics and radiobiology, faculty of biology, university of so a an approach to the investigation of structural and functional properties of the photosystem ii supramolecular complex in native photosynthesizing objects based on the registration of delayed chlorophyll a uorescence is developed. using a disc phosphoroscope, we register simultaneously: i) changes of the intensity of millisecond delayed uorescence (decayed in . - . ms time range) during the transition of the photosynthetic apparatus from dark to lightadapted state; ii) changes of the intensity of delayed uorescence decaying in different subintervals of the investigated time range; iii) dark relaxation curves at different moments of the transition; iv) changes of the intensity of prompt chlorophyll a uorescence. the analysis of these data allows the correlation of the delayed uorescence characteristics to particular processes occurring in the photosystem ii complex -proton or electrical gradient accumulation, changes in the redox state of quinone acceptors, changes in the pigment-protein complexes caused by different stress factors, for example temperature. three-dimensional structure of major light-harvesting antenna of photosystem ii from cucumber h. yan , z. liu , k. wang , t. kuang , j. zhang , l. gui , x. an , w. chang national lab of biomacromolecules, institute of biophysics, chinese academy of sciences(cas), datun road, chaoyang distr., beijing , china, lab of photosynthesis and environmental molecular physiology, institute of botany, cas, nanxincun, xiangshan, beijing , china the major light-harvesting antenna complex of photosystem ii (lhc-ii), the most abundant integral membrane protein, functions in light capture, energy transfer/distribution and photoprotection. lhc-ii from different species or conditions shows different spectral properties and variation in polypeptide and pigment components. this indicates some speci c function-related alterations in the organization of lhc-ii. here we report a . -Å crystal structure of cucumber homo-trimeric lhc-ii, organized in a perfect virus-like icosahedral particle. the electron-density map shows the reasonable existence of a chlorophyll (chl) a/b mixed binding site in the complex. the occurrence and locus of lactucaxanthin (lac) was seen directly for the rst time. based on the credible structure information, a mechanism of the energy transfer, regulation and excess excited energy dissipation under high light condition was proposed. coherent anti-stokes raman scattering microscopy (cars) is a new approach for chemical imaging of molecular systems within cells and tissues, with high sensitivity, high spatial resolution, and three dimensional sectioning capabilities, without using uorophores that are prone to photobleaching. this technique permits to map selectively molecular species, by using vibrational properties of their chemical bounds. the epi detected (e-cars) and forward detected (f-cars) intensities depends on the shape, the size of the sample, as well as the index of the solvent. in this presentation, after introducing the cars microscopy technique, we show the rst cars studies of the refractive effect of the sample, comparing the e-cars and f-cars signals for different diameters of polystyrene beads, in different refractive index solvents. we present several simulations, comparing forward-detected and backward-detected signals in different sized polystyrene beads, embedded in different index solvents, and we show that, the backwardre ected f-cars dominates the experimentally epi-detected signals. furthermore, we demonstrate experimentally and theoretically that the maxima of forward and epi-detected signals are generated at different positions along the z axis in the sample. we nally discuss how index mismatch in cells can alter cars images. the effects of static magnetic elds on humans have been the subject of continuous investigations. since one of the major static magnetic eld sources is nuclear magnetic resonance imaging (mri), the present study aimed to investigate the effects of . t magnetic eld that is produced by mri on humans. the study is carried out with voluntary and healthy young men from to years of age. the subjects informed about the purpose of the study at the beginning. the subjects exposed to minutes of . t static magnetic eld by means of putting the subjects into the magnetic resonance unit. ml blood was taken from each subject one minute before and one minute after exposure. t and t relaxation times and trace elements were measured in of pre and post exposure plasma of the subjects. the obtained post exposure values were compared with pre-exposure values of the subjects. pre and post exposure results were analyzed by means of student t-test. evaluation of tumor response of breast cancer patients by diffusion weighted mri k. a. danishad , v. seenu , u. sharma , p. k. julka , g. k. rath , n. r. jagannathan department of nmr, department of surgery, department of radiotherapy, all india institute of medical sciences, new delhi, india diffusion weighted mr imaging (dwi) measures the diffusion of water molecules in tissues and is quanti ed by apparent diffusion coef cient (adc). dwi can be used to differentiate tumors from normal tissue and also can be used to monitor the response of tumor to chemotherapy. thirteen healthy volunteers and twelve patients were recruited for the study. dw images were obtained prior to therapy (n= ) and after three cycles of therapy (n= ). the mean adc value of tumors ( . x . mm /s) was signi cantly less (p < . ) compared to the normal tissue ( . x . mm /s). decrease in adc in tumor is due to an increase in the cellularity which restricts the diffusion of water molecules. in patients receiving neo-adjuvant chemotherapy, the adc values were higher ( . x . mm /s) and were closer to that of the normal tissue (p < . ), indicating response of the tumor to chemotherapy. the post-therapy increase in adc is due to the cell damage caused by the therapeutic agents which increases the fractional volume of the interstitial space, causing an increase in the mobility of water. the study showed that dwi can be used non-invasively to assess the response of breast cancer patients to neo-adjuvant chemotherapy. quanti cation by optical imaging of gene electrotransfer in mouse muscle and knee optical imaging was evaluated for monitoring and quanti cation of the mouse knee joint and tibial cranial muscle electrotransfer (et) of a luciferase encoding plasmid. the substrate of luciferase (luciferin) was injected i.p or locally in the muscle or the knee joint. luminescence resulting from the luciferase-luciferin reaction was measured with a cooled ccd camera. luminescence of the knee joint and muscle were higher after local than after i.p injection of luciferin, but both measurements were highly correlated. local injection procedure was adopted. a signi cant correlation was observed between measurements in vivo and in vitro on the same muscle. reproducibility of individual luminescence measurements was also veri ed, and the luminescence levels were clearly dependant of the amount of plasmid injected. in vivo luciferase in the electrotransfered knee joint was detected for two weeks. intramuscular electrotransfer of . or µg of plasmid led to stable luciferase expression for days, whereas injecting µg plasmid resulted in a luminescence fall two weeks after electrotransfer. these decreases were, at least partly, related to the production of antibodies against luciferase. thus, optical imaging was shown to be a relevant technique to quantify variations of luciferase activity in vivo in one given tissue. furthermore, evaluating the effective amount of luciferase in tissues from in vivo luminescence levels requires calibration since it relies on conditions of the enzymatic reaction and light absorption. acute effect of corticosterone on nmda receptormediated ca + elevation in mouse hippocampal slices m. saito , s. sato , h. osanai , a. hirano , y. komatsuzaki , s. kawato department of physics and applied physics, college of humanities and sciences, nihon university, department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo corticosterone (cort) is a principal glucocorticoid synthesized in the rodent adrenal cortex and secreted in response to stress. we examined the rapid effects of cort on n-methyl-d-aspartate (nmda) receptor-mediated ca signals in adult mouse hippocampal slices by using ca imaging technique. application of nmda caused a transient elevation of intracellular ca concentration followed by a decay to a plateau within sec. the min preincubation of cort induced a signi cant decrease of the peak amplitude of nmda-induced ca elevation in the ca region. the rapid effect of cort was induced at a stress-induced level ( . - µm). because the membrane non-permeable bovine serum albuminconjugated cort also induced a similar rapid effect, the rapid effect of cort might be induced via putative surface cort receptors. in contrast, cort induced no signi cant effects on nmdainduced ca elevation in the dentate gyrus. in the ca region, cort effects were not evaluated, because the marked elevation of nmda-induced ca signals was not observed there. in vivo subcellular structures recognized with phase k. nagayama , r. danev , n. usuda , y. kaneko , k. nitta , a. nakazawa , k. atsuzawa , m. tanaka , m. setou okazaki institute for integrative bioscience, okazaki, japan, fujita health university, school of medicine, toyoake, aichi, japan, saitama university, saitama, japan, tokyo metropolitan institute of gerontology, itabashi-ku, tokyo, japan phase contrast transmission electron microscopy has been developed to enable a high contrast and a high resolution observation for unstained ice-embedded samples. to enhance the image contrast, two methodologies have already been developed; i) scattering contrast for stained samples with small aperture diaphrams and ii) defocus contrast for unstained or stained samples with deep defocusing. the former prevails in histochemical sciences and the latter is popular in electron crystallography. both methods, however, have a common drawback that the contrast is only improved by impairing the image quality. this drawback can be removed with use of the phase contrast method using phase plates, which has traditionally been used in visible light microscopy. due to the severe obstacle of the charging of phase plates, however, the idea has not yet been materialized. we have solved the phase-plate charging problem. an experiment kv with tem for a whole cell from cyanobacterium unstained and ice-embed ful lled the expectation. only weak and vague contrast was obtained for the conventional image of the cell even with a very deep defocus. contralily a high-contrasted image has appeared for phase contrast images, where various ne structures are clearly recognized. this may be a rst example to observe nanometer scale structures in details in the intact cell. other examples including intact state intravesicular structures will be shown. j. lichtenberger, p. fromherz max-planck-institute for biochemistry we cultured bovine chromaf n cells on an array of electrolyteoxide-silicon eld-effect transistors (eos fet) and monitored granule secretion. by stimulation with barium chloride, vesicles are released into the narrow sheet of electrolyte between the chip surface and the plasma membrane. the interaction of released protons with the silicon dioxide surface of the chip alters the threshold voltage of the transistor and gives rise to a measurable signal. simultaneously performed measurements with a carbon bre showed a correlation of the transistor signals and amperometric current traces. we conclude that the transistors are able to monitor exocytosis on a single vesicle level. to elucidate the role of protons, we destroyed the proton gradient across the vesicle membrane by nigericin and valinomycin. as a result a massive reduction of the transistor signals was induced, whereas there was only little change of the amperometric records. we conclude that released protons are responsible for the detection of vesicles with transistors. the individual transistor records of vesicle exocytosis can be explained by combining the dynamics of the exocytotic event with the diffusion in the cell-chip junction. transistor recording of exocytosis does not depend on the electrochemistry of transmitters. as many kinds of exocytotic vesicles contain a large amount of buffered protons it can be applied to numerous kinds of exocytotic events, independent on the nature of the transmitter. we tried various solvents for the solubility of the uorescent product, and found that the product was insoluble in water and most organic solvents. a quite bright uorescence emitted by the particles was observed by uorescent microscope when emitted by uv nm. sem indicated that the size of the particles was µm µm, depending on the reaction time and phospholipid concentration in hexane solution. endothelial cells from human vein grew better on the surface prepared from the particles than the culture plate, implies a possible application as a new type of biomaterial as a coating material for medical devices, and as a uorescent tracer for human bodies. confocal microscopy of the phototactic ciliate f. salina. fabrea salina is a marine heterotrich ciliate, which dwells in salt ponds. in previous works we have described the phototaxis and the uorescence properties of a hypericin like endogenous pigment in an albino strain. we have recently obtained a heavily pigmented strain from the saline of torre colimena (taranto, italy). we have used confocal microscopy to characterize the uorescent properties of this strain and to compare them with those of the albino strain. the results obtained by one and two photon confocal microscopy show that, as in the albino strain, the uorescence intensity of the pigment is higher in dead cells than in the living cells. the excitation and emission spectra are quite similar in the two strains and this is also true for the uorescence lifetime, which is about ns. all together, these measurements indicate that the pigment of the new strain belongs to the family of hypericin-like chromophores. the analysis of different confocal planes shows that the pigment is localized not only in granules under the somatic membrane in the cellular body, as currently thought, but also in the cilia. some experiments of fotobleaching "in situ" con rm this result, that might have important implications in the understanding of the mechanisms of the photomotile responses of f. salina and probably of other heterotrich ciliates. there is no doubt that modern physician should have the knowledge of basic sciences as physics, chemistry and biology. furthermore, the biophysics is incorporated into the curriculum of most european medical schools. at medical school in zagreb, the course of physics and biophysics is positioned in rst and forth year of study. the students learn basic physics phenomena of structure of matter, mechanics, thermodynamics, electromagnetism, optics and acoustics applied on the human body. additionally, the interactions of the body with the surrounding are thoroughly discussed as the basis for different diagnostic methods. the arguing at our school is still going on where to include the content of this course. should it be the autonomous course or the part of physiology and radiology courses in the problem based learning approach?! so far at zagreb medical school the biophysical courses are autonomous structured according to the biophysics programs at other european universities. the highlighting is on seminar work and lab, encouraging the students for individual learning. the seminars are made more vivid and instructive for students by inclusion of different model devices constructed in our department learning on science is also learning how scienti c knowledge is produced. in this sense, issues related to the dynamics of science should be brought into focus in science education. the idea has support in the "declaration on science and the use of scienti c knowledge", particularly in the statement that "science curricula should include science ethics, as well as training in the history and philosophy of science and its cultural impact". thinking on rst year undergraduate students, we are interested in an integrated approach of that kind of themes. this presentation describes a practical teaching module included in the basic biophysics course for biochemistry majors. organized in case studies, it deals with stories of biophysics (and biochemistry), and addresses the role of the biophysical approach in the progress of the life sciences. the module follows the whole course, and consists in small exercises on the way a given understanding has been constructed explored within the practice trend of contemporary science studies. examples of the chosen stories -anchored in the subjects covered in the lectures of the course -relate to the search for the mechanism of energy production through atp-synthase, the development of radioactive labelling techniques, and the discovery of protein water channels. beyond their value as cultural legacy and as motivating tools, the insight they might provide is vast. axiomatic theory of biophysics q. zhao china rehabilitation research centre, beijing, china. until now, all approaches to interpret biology by applying principles of physics have been announced failure. the achievement of biophysics is limited in area to provide essential tools for biological research and biophysics is far from to be the basic theory of biology. to resolve it requires the fundamental research about the logic features of biophysical processes of biology. we promoted system logic and then protein thermodynamics structure theory. based upon it, the axiomatic theory of biophysics and biology could be developed. our result shows that the real understanding of biology or biophysics must be constructed based upon new thinking methods. a successful ligand-receptor docking methodology depends strongly in the ef ciency of the global optimization algorithm used to explore the ligand conformational space. in this work we have implemented and analyzed the performance of a new exible ligand-receptor docking methodology. this methodology uses as optimization method a multisolution version of the generalized simulated annealing algorithm adapted to problems with box constraints. a grid-based methodology, considering the receptor rigid, and the gromos classical force eld are used to evaluate the ligand-receptor scoring function. the methodology was tested in redocking (ligand within it own protein conformation) and cross-docking (ligand within another protein conformation) experiments for ve hiv protease-ligand complexes with known threedimensional structures. all ligands tested are highly exible, having to conformational degrees of freedom. the implemented docking methodology was able to redock successfully all exible ligands with a success ratio % and a mean rmsd lower than . Å with respect to the corresponding experimental structures. in the cross-docking experiments we observed a strong dependence of the mean success ratio with respect to the protein structure used as reference. in situations we observed a mean success ratio % and % in cases among the possible ones. s. aida-hyugaji , h. nakagawa , j. nomura , m. sakurai , d. tokushima , t. takada , u. nagashima , t. ishikawa tokai university, japan, tokyo institute of technology, japan, nec corporation, japan, national institute of advanced industrial science and technology, japan irinotecan is a widely-used antitumor drug that inhibits mammalian dna topoisomerase i. however, overexpression of abcg can confer cancer cells resistance to sn- , that is, the active form of cpt- . in the present study to develop a platform for the molecular modeling to circumvent drug resistance associated with abcg , we have characterized a total of fourteen new sn- analogues by some typical properties, which were evaluated by molecular orbital (mo) calculations and neural network (nn) analysis. the nn was rst applied to estimate hydrophobic properties (logp) of the analogues. thereafter, the electrostatic potential (esp) and the solvation free energy ( g) were evaluated by mo calculation. these indexes were found to be well correlated with the drug resistance ratio experimentally observed in abcg -overexpressing cells. it is suggested that hydrophilic analogues carrying oh-or nh -groups are good substrates for abcg and therefore exported from cancer cells. in contrast, sn- analogues with cl or br atom at those positions have similar logp values and high af nities toward the putative active site of abcg , however they were not substrates of abcg . from these results, it is strongly suggested that hydrogen bond formation with oh-or nh -groups are critically involved in the transport mechanism of abcg . a. agopian , j. depollier , e. gros , g. aldrian-herrada , p. clayette , n. bosquet , g. divita crbm-cnrs, route de mende, montpellier, france., spi-bio-cea fontenais aux roses, france reverse transcriptase (rt) plays an essential role in the replication of hiv and constitutes the main target for the development of aids therapies. the biologically active form of hiv rt is a heterodimer of two subunits, p and p , each consisting of distinct subdomains: the ngers, the palm, the thumb, the connection and the rnase h subdomain, the latter only present in p . we have demonstrated that formation of fully active rt is a two-step process involving rapid association of the two subunits (dimerization) followed by a conformational change (maturation). thanks to the crystal structure of rt we have identi ed a new class of inhibitors based on short peptide motifs derived from the dimer interface. we rst identi ed a short mer peptide (pep- ) derived from the tryptophanrich motif of the connection subdomain that blocks dimerization of rt and ef ciently abolishes hiv- replication. pep- interacts preferentially in a pocket involving residues trp and phe on p . we then designed mer peptides derived from the thumb domain which inhibit rt maturation as well as viral replication when delivered into cells. taking into account these results we propose that dimerization of rt constitutes a potential target for the design of more speci c new antiviral drugs. . the success of gene therapy largely relies on the availability of vectors that would deliver the genetic material ef ciently to the target cells with a minimal toxicity. in this context, our purpose was to evaluate as possible vectors a series of newly synthesized low molecular weight ( kda) chitosan derivatives grafted with dodecenoyl (ddc) groups at different percentages ( , , and %). in the absence of dna, the critical micellar concentration (cmc) of these derivatives in mm mes buffer ph . was found to be strongly dependent on the percentage of ddc but not on ph or salt concentrations. this indicates that the ddc groups confer to the chitosan derivatives the potency to self-assemble probably in micellar structures: a property that may dictate the formation and the structure of their complexes with dna. next, we investigated by quasielastic light scattering the size and the surface charge of complexes of plasmid dna with these derivatives at different ph, salt concentrations and n/p ratios (expressed in charged units of chitosan amines to dna phosphates). we found the smallest and more positively charged complexes were obtained at ph . and n/p= in the absence of salt: a condition where the chitosan derivatives were fully protonated and in excess over the dna phosphate groups. biophysical and biological examination of dna/lipids complexes particles of virus-like structure designed for in vivo gene transfer d. durand , m. schmutz , b. lebleu , a. r. thierry lure, centre universitaire paris sud, orsay, france, institut henri sadron, strasbourg, france, laboratoire des défenses antivirales et antitumorale, umr , montpellier, france the structure of complexes made from dna and suitable lipids (lipoplexes lx) was examined by cryo electron microscopy. we observed a distinct concentric ring-like pattern with striated shells when using plasmid dna. these spherical multilamellar particles have a mean diameter of nm with repetitive spacing of . nm with striation of . nm width. small angle x-ray scattering (saxs) con rmed cryoem data and revealed repetitive ordering of . nm, suggesting a lamellar structure containing at least a dozen layers. this concentric and lamellar structure with different packing regimes was also observed by cryoem with linear dsdna, ssdna and oligodeoxynucleotides. for the rst time, dna chains could be visualized in dna/lipid complexes. such speci c supramolecular organization is the result of thermodynamic forces, which cause compaction to occur through concentric winding of dna in a liquid crystalline phase. cryoem of t phage dna packed either in t capsides or in lipidic particles showed similar patterns. saxs suggested an hexagonal phase in lx-t dna. thus, both lamellar and hexagonal phases may coexist in the same lx preparation or particle and transition between both phases may depend upon equilibrium in uenced by type and length of the dna used. organization of such nucleotidic supramolecular assemblies is relevant for prebiotic chemistry. engineering self-assembly peptides for targeted delivery of therapeutics and imaging agents s. s. dhadwar, m. sung, k. kawamura, j. gariépy department of medical biophysics, university of toronto, canada peptide-mediated delivery systems have recently emerged as a means to substitute or augment conventional drug and gene delivery technologies. these approaches are versatile and easily designed to incorporate a number of speci c attributes required for ef cient delivery of therapeutic and imaging agents. in particular, self-associating peptide domains can be utilized to construct stable and structurally well-de ned protein-like assemblies displaying a series of cell-routing functions. more speci cally, a peptide-based self-assembling intercellular delivery vehicle was designed by incorporating the -residue long tetramerization domain of the human tumor suppressor protein p (hp tet). the resulting peptide tetramer displays termini within its structure that allows for the simultaneous presentation of distinct cell targeting signal or functional domains. the fusion of polycationic sequences to the hp tet domain promotes the cellular import of the resulting constructs into eukaryotic cells. this internalization event was dramatically enhanced for such multivalent peptides in relation to their monomeric counterparts. peptides containing a nuclear localization sequence along with a polycationic sequence were found to shuttle reporter plasmids ef ciently to the nucleus of cells. these results have important implications in the design and construction of novel targeted delivery vehicles. mechanisms of non-covalent peptide mediated cellular delivery of therapeutics: a biophysical study s. deshayes , m. c. morris , a. heitz , p. charnet , g. divita , f. heitz crbm -cnrs fre montpellier france, cbs -cnrs umr -inserm u montpellier france two different cell-penetrating peptides mpg and pep- were shown to promote non-endosomal intracellular delivery of non-covalent bound cargos, namely nucleic acids and proteins; respectively. in order to identify the peptide mediated internalization pathway, we undertook conformational investigations of both peptides with and without associated cargos and checked the conformational consequences of the presence of phospholipids. from the conformational point of view, pep- behaves differently from mpg. cd analysis revealed a transition from a non-structured to a helical conformation upon increase of the concentration while mpg remained nonstructured. determination of the structure by nmr showed that in water, it's a-helical domain extends from residue to . cd and ftir indicated that pep- adopts a helical conformation in the presence of phospholipids while mpg is in a -sheet form. adsorption measurements performed at the air-water interface were consistent with the helical form. pep- did not undergo conformational changes upon formation of a particle with a cargo peptide. in contrast, we observed a partial conformational transition when the complex encountered phospholipids. for mpg, interactions with nucleic acids generated a partial folding into -sheet which was more pronounced in the presence of lipids. electrophysiological measurements showed that both peptides, whether associated or not with their cargo, can induce transmembrane pore-like structures. self-assembly of hydrolysed alpha-lactalbumin into nanotubes j. f. graveland-bikker , k. g. de kruif nizo food reseach, ede, netherlands, van´t hoff laboratory, netherlands nanotubes are formed by self-assembly of partially hydrolysedlactalbumin, a kda milk protein. there are several promising applications of these -lactalbumin tubes, in food, pharmacy and nanotechnology. we studied the mechanism of self-assembly, the structure and the properties of the nanotubes. limited proteolysis of the -lactalbumin (by a serine protease) makes the molecule prone to self-assembly. in the presence of ca tubular structures are formed. other divalent ions like mn and zn can also induce tubular self-assembly, while mg leads to random aggregation. light scattering showed that the self-assembly is reversible, which is of relevance for controlled release applications. on the other hand, we could also make stable tubes by cross linking, which would be a requisite for several other applications. from afm and saxs measurements, we obtained values for the outer diameter: nm; and the inner diameter: nm. afm and cryo-em revealed the helical structure of the tube wall; it is a right-handed helix. by performing nano indentations with afm we determined mechanical properties of the tubes. the tubes were shown to be relatively resilient upon small deformations; the elastic modulus is of the order of . gpa. targeted delivery of photosensitizers into the cancer cell nuclei enhances their cytotoxic ef cacy the search for new pharmaceuticals has raised interest in locallyacting drugs which act over short distances within the cell, and for which different cell compartments have different sensitivities, e.g. photosensitizers used in anticancer therapy should be transported to the most sensitive subcellular compartments where their action is most pronounced. earlier we have produced a number of modular recombinant transporters for locally-acting drugs comprising several functional modules for cell-speci c targeting, internalization, escape from intracellular acidic vesicle, and targeting to the nuclei of melanoma cells overexpressing melanocortin receptors. here we describe new transporters on the basis of epidermal growth factor which are speci c for a wide variety of cancers. these transporters possess all necessary functional activities and deliver photosensitizers into the nuclei of human carcinoma cells to result in photocytotoxic effects almost orders of magnitude greater than those of nonmodi ed photosensitizers. characterization of mixtures of dna and nonionic polymeric agents for gene delivery in muscle j. m. gau , j. lal , l. auvray laboratoire mpi -lrp, umr cnrs , université d´Évry val d´essonne, Évry cedex, france, argonne national laboratory, ipns, south cass avenue, illinois , usa a strategy to cure muscle disease is to introduce genes (dna) into the muscle cell to correct or to add genes. nonionic polymeric agents have emerged as an ef cient vector to deliver dna in the muscle. these polymers protect dna from extracellular nuclease degradation by allowing the dna diffusion throughout the muscle tissue. there is at present no understanding about how nonionic polymers enhance transfection in the muscle. the kind of interactions between these nonionic agents and dna, dna-nonionic polymeric agent mixtures and cell membrane are currently unknown. also the structure of dna-nonionic polymeric agent mixtures is not yet well de ned. more information is needed to improve this delivery system. neutron scattering (contrast variation) and light scattering were used to investigate the interaction between: dna and nonionic polymers (pvp, di-and triblock copolymers). furthermore, electrical measurements with the same polymer complexes and black lipid membrane were also performed. depending on the polymer type there is either direct interaction with dna or in other cases polymers exhibit strong interaction with the lipid membrane. an explanation for transfection ef ciency of these nonionic agents in gene delivery to muscle will be given. high throughput in-silico screening against exible protein receptors b virtual screening of chemical databases to targets of known threedimensional structure is developing into an increasingly reliable method for nding new lead candidates in drug development. based on the stochastic tunneling method (stun) we have developed flexscreen, a novel strategy for high-throughput in-silico screening of large ligand databases. each ligand of the database is docked against the receptor using an all-atom representation of both ligand and receptor. in the docking process both ligand and receptor can change their conformation. the ligands with the best evaluated af nity are selected as lead candidates for drug development. using the thymidine kinase inhibitors as a prototypical example we documented the shortcomings of rigid receptor screens in a realistic system. we demonstrate a gain in both overall binding energy and overall rank of the known substrates when two screens with a rigid and exible (up to sidechain dihedral angles) receptor are compared. we note that the stun suffers only a comparatively small loss of ef ciency when an increasing number of receptor degrees of freedom is considered. flexscreen thus offers a viable compromise between docking exibility and computational ef ciency to perform fully automated database screens on hundreds of thousands of ligands. maturation and inhibitor design of sars-cov cl protease based on a product-bound crystal structure severe acute respiratory syndrome (sars) is an emerging infectious disease caused by a novel human coronavirus. here we report that the cl pro containing n-and/or c-terminal additional in-frame sequences underwent autoactivation to cleave the tags and yielded the mature protease in vitro. the -d structure of the c a mutant protease shows that the active site of one protomer of the dimeric protease is bound with the c-terminal six amino acids of the protomer in another asymmetric unit, suggesting a possible mechanism for maturation. the crystal structure of this product-bound form shows that the active site has a p pocket that binds the gln side chain speci cally. in addition, the p and p sites are clustered together to accommodate large hydrophobic side chains. the tagged c a mutant protein served as a substrate for the wildtype protease and the n-terminus was rst digested ( -fold faster) followed by the c-terminal cleavage as shown by the sds-page analysis. the analysis of t analytical ultracentrifuge experiments reveals the remarkably tighter dimer formation for the mature enzyme (k d = . nm) than for the mutant (c a) containing the n-terminal (k d = . nm) or the c-terminal extra amino acids (k d = . nm). taken together, the study here provides insights to the design of our new structure-based inhibitors. nevertheless, a signi cant proportion of patients do not respond to this therapy, and adverse effects are common. here we report the delivery and expression of recombinant mycobacterial dna vaccines in vivo and demonstrate the ability of multicomponent dna vaccines to enhance th -polarized immune responses. splenocytes from immunized groups of mice were re-stimulated in vitro and examined for cytotoxicity against bladder tumour cells. we used four combined recombinant bcg dna vaccines (multi-rbcg) for electroporative immunotherapy in vivo, and found that tumour growth was signi cantly inhibited and mouse survival was prolonged. increased immune cell in ltration and induction of apoptosis were noted after treatment with multi-rbcg alone, with the interleukin- (il- ) vaccine alone, and-most signi cantly-with their combinations. thus, electroporation immunogene therapy using multi-rbcg plus il- may be an attractive regimen for the treatment of bladder cancer. this approach presents new possibilities for the treatment of bladder cancer using recombinant bcg dna vaccines and il- dna vaccine. the cell-penetrating peptide (cpp) pep- is capable of introducing large proteins into different cell lines, maintaining their biological activity. two mechanisms have been proposed to explain the entrance of other cpps in cells, endosomal-dependent and independent. we evaluated the molecular mechanisms of pep- mediated cellular uptake of -galactosidase ( -gal) from e. coli, in large unilamellar vesicles (luv) and hela cells. fluorescence spectroscopy and immuno uorescence microscopy were used to study the translocation. internalization of -gal into luv and protein functionality in hela cells were detected by enzymatic activity. -gal translocated into luv in a transmembrane potentialdependent manner. likewise, -gal incorporation was extensively decreased in depolarized cells. furthermore, -gal uptake efciency and kinetics were temperature-independent and -gal did not co-localize with endosomes, lysosomes or caveosomes. therefore, -gal translocation was not associated with the endosomal pathway moreover transmembrane pores were not detected. these results indicated that the protein uptake in vitro and in vivo was mainly, if not solely, dependent on a physical mechanism governed by electrostatic interactions between pep- (positively-charged) and membranes (negatively-charged). peptide the dramatic acceleration in the identi cation of new nucleic acidbased therapeutic molecules has provided new perspectives in pharmaceutical research. however, the development of nucleic acidand peptide-based therapeutics is limited by their poor cellular uptake and traf cking. with the aim of addressing these issues, we have designed a family of short amphipatic peptides for delivery of nucleic acids (mpg) and of peptide/pna (pep). these carriers consist of a hydrophobic moiety and a nls-derived hydrophilic domain. they form stable non-covalent complexes with peptides, proteins, sirna or pna without any requirement for prior covalent cross-linking. both mpg and pep carriers enter cells rapidly, in a process involving membrane disorganization, independently of the endosomal pathway. mpg ef ciently delivers short odns and sirna into a wide variety of mammalian cell lines, without interfering with their biological function. pep signi cantly improves delivery of pna and peptides. both carriers were used for the delivery of sirna or antisense pna targeting the cell cycle regulatory protein cyclin b in an animal model and were found to block tumor growth upon intravenous injection. we believe that mpg and pepbased technologies will contribute signi cantly to the development of basic and therapeutic applications. probing the bound conformation of acetylcholinesterase (ache) inhibitor at the binding site c. g. kim, x. zhao, s. goodall, a. watts department of biochemistry, university of oxford, south parks road, oxford, ox qu, uk acetylcholinesterases are the enzymes which preferentially hydrolyze acetyl esters (such as ach or acetyl-â-methylcholine), containing amino acid residues in the eeache form and arranged as a -stranded â-sheet surrounded by á-helices. the protein is ellipsoidal in shape, with approximate dimensions of Å by Å by Å. inhibitors of acetylcholinesterase are of commercial and medical interest as pesticides and as therapeutics in the treatment of alzheimer's disease. an understanding of the conformation of inhibitors in the binding site enables the rational design of novel inhibitors with increased potency and speci city. interaction between the ligand, amino- -methyl- -( tri uoroacetylbenzyl-oxymethyl)quinoline (r ), and ache inhibitor has been studied by advanced solid-state nmr through double-quantum chemical shift and distance measurements. combining solid-state nmr data and docking simulations, conformation of the ache inhibitor at the active site has been predicted. in vivo, heat shock proteins (hsps) being stress-inducible chaperones can attenuate detrimental consequences of ischemic insults, inammation, neurodegenerative diseases, etc. also, intracellular accumulation and chaperone activities of some hsps may contribute to improved cell survival following uv or ionizing radiation. in models of pathological states and their treatment, we used special virusbased vectors for overexpression of hsp or hsp in cell cultures to confer cytoprotection under simulated ischemia/reperfusion. in parallel, similar cytoprotection was achieved after pretreatments of the cells with a pharmacological hsp inducer, geranylgeranylacetone. the cytoprotective effects were manifested in the lesser extent of oxidative modi cation and aggregation of cellular proteins, better preservation of the cytoskeleton, faster restoration of energy metabolism and the improved post-stress cell survival. in the other model, we treated normal and tumor cells with an inhibitor of the chaperone activity of hsp , geldanamycin. only the drug-treated tumor cells became more sensitive to gamma-irradiation; such results characterize this drug as a potentially selective radiosensitizer of tumors. taken together our data demonstrate promising approaches to clinically bene cial manipulating the levels of expression and/or chaperone activity of hsp(s) by means of gene therapy or pharmacotherapy. characterizaton of proteins from human pleural uid r. jain, s. kumar, n. singh, s. sharma, t. p. singh all india institute of medical sciences, new delhi ,india the samples of human pleural uid were obtained from both healthy subjects and patients infected by tuberculosis. after the preliminary processing these samples were run in independent lanes of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the two lanes indicated variations in the intensities of a few bands and some new bands were also observed in the infected samples. these were characterized by determining their nterminal sequences. the new bands which had low density were carefully identi ed and cloned. some of the common bands that showed intensity variations were characterized. these were matrix metalloproteins, secretory phospholipasea , transferrin and ceruloplasmin. they were also studied with maldi-tof and their molecular weights have been determined. some of these proteins have been crystallized and their detailed crystal structure determinations are in progress. biophysical study of non-lethal stress response of cultured dc f cells stress factors may induce two kinds of responses in living cells: either cell death or adapting mechanisms. our aim was to search for non-lethal effects of various stress conditions on cultured hamster lung broblasts (dc f cells) as well as to assess the recovery time after stress removal. dc f cells were cultured in standard conditions and were submitted to stress, either by incubation with chemical substances (sodium arsenate, sodium nitroprusiate) and drugs (bleomicine and statins) either by irradiation (uv, he-ne laser). the doses and exposure times were chosen as to avoid cell death. after stress removal, cells were allowed to recover and the recovery time period was measured. structural and functional parameters were evaluated before and after stress, as well as during recovery. by now, experimental models for the in vitro study of non-lethal stress inducing factors have been set up. severe acute respiratory syndrome (sars) is an emerging infectious disease caused by a novel human coronavirus. the viral maturation requires a main protease ( cl pro ) to cleave the virus-encoded polyproteins. we report here that the cl pro containing n-and/or c-terminal additional in-frame sequences underwent autoactivation to cleave the tags and yielded the mature protease in vitro. the -d structure of the c a mutant protease shows that the active site of one protomer of the dimeric protease is bound with the cterminal six amino acids of the protomer in another asymmetric unit, suggesting a possible mechanism for maturation. the tagged c a mutant protein served as a substrate for the wild-type protease and the n-terminus was rst digested ( -fold faster) followed by the c-terminal cleavage as shown by the sds-page analysis. the analysis of the quaternary structures for the tagged and mature proteases by analytical ultracentrifuge experiments reveals the remarkably tighter dimer formation for the mature enzyme than for the mutant (c a) containing the n-terminal or the c-terminal extra amino acids. taken together, the study here provides insights to the design of our new structure-based inhibitors. characterisation of macromolecular transport in physiologically relevant mixed ecm based gels s. lelu, a. pluen school of pharmacy and pharmaceutical sciences, university of manchester (uk) the extracellular matrix (ecm) a complex gel made of hyaluronic acid, collagen and proteoglycans (pg) impedes the penetration of macromolecules especially in tumours, and may compromise the success of novel therapies. though recent in vivo investigations pointed out that, not only ha but brillar collagen its content and organisation and its interactions with pg were involved in macromolecular transport hindrance, transport mechanisms relating the macromolecular drug and these ecm components are unknown. in this study we seek to evaluate the determinants of passive transport mechanisms of biomacromolecules in complex gels made of ha, collagen using uorescence techniques (frap and confocal re ection microscopy (crm)) and rheology. focus was on conditions relevant to tumours and, initially, on low collagen and relatively high ha content. rheology experiments showed that mixed systems containing less than mg/ml of ha present higher elastic modulus ge than pure ha network or pure collagen gels. interestingly crm and frap studies revealed similarities for collagen and mixed gels: the organisation and spacing of the collagen bres did not change and the ratio of the diffusivities (d/d ) of dextran m and igg were not different but higher than those in ha networks. systems with higher collagen content are under investigation to complete the characterisation of transport. encapsulation of clone vector dna by cationic diblock copolymer vesicles for gene delivery a. v. korobko , j. r. van der maarel leiden university, the netherlands, national university of singapore, singapore we will discuss the design, control, and structural characterization of cationic copolymer vesicles loaded with dna. these vesicles serve as a model system for diverse applications such as gene delivery, micro-arraying techniques and packaging of dna in congested states. encapsulation of dna was achieved with a single emulsion technique. for this purpose, an aqueous puc or pegfp-n plasmid solution is emulsi ed in an organic solvent and stabilized by an amphiphilic diblock copolymer. the neutral block of the copolymer forms an interfacial brush, whereas the cationic block complexes with dna. a subsequent change of the quality of the organic solvent results in a collapse of the brush and the formation of a capsule. the capsules are subsequently dispersed in aqueous medium to form vesicles and stabilized with an osmotic agent in the external phase. inside the vesicles, the dna is compacted in a liquid-crystalline fashion as shown by the appearance of birefringent textures under crossed polarisers and the increase in uorescence of labeled dna. the compaction ef ciency and the size distribution of the vesicles were determined by light and electron microscopy, respectively, and the integrity of the dna after encapsulation and subsequent release was con rmed by gel electrophoresis. we demonstrate the gene transfer ability of this new carrier system by the transfection of encapsulated pegfp plasmid into hela cancer cells. cellular transduction of nucleotide kinases to improve the activation of nucleoside analog prodrugs m. konrad , c. monnerjahn , s. ort , a. lavie max-planck-institute for biophysical chemistry, goettingen, germany, university of illinois at chicago, chicago, usa the objective of our study is to improve therapeutic enzymeprodrug systems by generating catalytically superior nucleoside and nucleotide kinases that are essential for activation of nucleoside analogs. compounds, such as azt for the treatment of hiv infections, acv and gcv used against herpes virus, or the anticancer compounds arac and gemcitabine, can enter cells only in the unphosphorylated state (prodrug) and need to be transformed by different kinases to their pharmacologically active triphosphate state that interferes with dna replication. we have rst designed mutants of the human tmp kinase (htmpk) that phosphorylate aztmp up to -fold faster than wildtype. expression of this enzyme in human cells leads to -fold higher intracellular concentrations of azttp and to enhanced hiv inhibition. second, the prodrugs acv and gcv are not phosphorylated by human kinases, but are converted to their monophosphate forms by hsv -tk which is used in enzyme/prodrug-dependent cancer suicide gene therapy. we generated enzyme variants which show selective and ef cient phosphorylation of gcv. third, an engineered human dck variant catalyzes more ef ciently the activation of the prodrugs arac and gemcitabine. thus, the concept of a gene (or enzyme) therapeutic treatment involving expression (or direct intracellular transduction) of a catalytically improved human enzyme may pave the way to the development of novel strategies in nucleoside prodrug-dependent cancer chemotherapy. docking-molecular dynamics studies on the peroxidase site of prostaglandin endoperoxide h synthase prostaglandin endoperoxide h synthases- and (pghs- and ) catalyze the rst step in the biosynthesis of prostaglandins, prostacyclins and thromboxanes. arachidonic acid is transformed into prostaglandin g (pgg ) at the cyclooxygenase site of the enzyme and the -hydroperoxide oxygen-oxygen bond of pgg is subsequently cleaved by reaction with haem at the distinct peroxidase site (pox) to produce prostaglandin h (pgh ). herein we present a plausible productive conformation obtained by docking calculations for the binding of pgg to the pox site of pghs- . the enzyme-substrate complex stability was veri ed by a -ps molecular dynamics simulation. structural analysis unveils the requirements for enzyme-substrate recognition and binding: the pgg -hydroperoxide group is in the proximity of the haem iron and participates in a hydrogen bond network with the invariant his and gln and a water molecule, whereas the carboxylate group establishes salt bridges with the remote lysines and . the interaction of the peptide lah with anionic lipids during dna/rna delivery to eukaryotic cells a. j. mason , a. martinez , c. leborgne , a. kichler , b. bechinger faculté de chimie, université louis pasteur, strasbourg, france, généthon, evry, france the histidine rich amphipathic peptide lah has antibiotic and dna delivery capabilities. the peptide has a strong af nity for anionic lipids found in the outer membrane of bacterial membranes and has shown evidence of higher transfection activity against transformed over healthy tissue in culture. it has been proposed that anionic lipids can ip-op to reach the cytoplasmic monolayer. here they neutralise the cationic transfection complexes thereby causing release of oligonucleotides into the cytoplasm. we were, therefore, particularly interested to test for the role of the acidic lipid phosphatidylserine (ps) in mediating lah -mediated delivery of dna ef ciency. to understand the potential peptide-lipid interactions in more detail, solid-state nmr experiments on model membranes have been performed. p mas nmr on mixed phosphatidylcholine (pc)/ps and pc/phosphatidylglycerol (pg) membranes has been used to investigate speci c lah interactions with anionic lipids. by using deuterated lipids and wide-line h nmr when probing lipid chain order, it is demonstrated that lah preferentially interacts with ps over pc. lah thereby effectively disorders the anionic ps lipid fatty acyl chains. the lipid chain destabilising effect of lah and also lah analogues can then be compared with their transfection ef ciency for dna or sirna in cell culture to aid in rational peptide vector design. virtual screening is now widely accepted as a basis for drug discovery thanks to signi cant improvement and good hit rates [ ]. however, it is still highly cpu-consuming. at the same time, the number of protein-ligand complexes described at the atomic level is rising and the sequence similarity is used for structure and function predictions. new approaches are being developed to take advantage of the available structural data and the huge number of protein sequences in order to allow better tuned virtual screening. new web servers are being built to ease and to speed up the whole process (http://abcis.cbs.cnrs.fr/kindock/). integrating these servers into a pipeline dedicated to molecular modelling (http://abcis.cbs.cnrs.fr/atome/) shall allow both the re ned validation of modelled active sites as well as the oriented screening for the primary caracterization of potential ligands. an ideal drug delivery system should own the following characteristics, the rst is the targeting of therapeutic agent to the speci c site of their action. the second is the controlled delivery of a therapeutic molecule or protein in a pulsatile or staggered fashion. the third is the achieving sustained zero-order release of a therapeutic agent over a prolonged period of time. in this study, a new drug delivery system combined these characteristics was provided, which contains azobenzene derivatives (ab lipid) as an on-off switch incorporated into liposomes. the drastic release of calcein was observed on the rst uv irradiation of ab lipid to the cis isomer, while a suppressed release was observed when irradiated with the rst visible light. after that, the slope of release pro le became coincident. furthermore, calcein release was greatly increased after uv irradiation of ab lipid to the cis isomer and the drug release was greatly suppressed after vis irradiation of ab lipid to the trans isomer. we can control the release rate of calcein from ab lipid/egg pc mixed liposomes by uv or vis light irradiation. tryptophanase (trpase), a bacterial enzyme with no counterpart in eukaryotic cells, produces l-trp pyruvate ammonia and indole. it was suggested that indole is essential for bacteria multiplication and bio lm formation. bio lms destroy equipment and food and cause many illnesses. most synthesized quasi-substrates inhibit trpase at mm range. an optimal and speci c inhibitor of trpase may eliminate indole production and prevent bio lm formation. x-ray crystallography of the holo-wt e. coli trpase soaked with l-trp and the known mechanism of trpase activity should provide the information for the design and synthesis of active-sitespeci c quasi-analogs. utilizing the chromogenic substrate s-(onitrophenyl)-l-cysteine, the following michaelis-menten kinetics analyses determined the mode of trpase inhibition by trp and quinone based quasi-analogues and the corresponding ki values (in µm): dl- -alanyl- , anthraquinone, noncompetitively, ; trypthophan ethylester, competitively, ; acetyltryptophan, uncompetitively, . ; s-phenylbenzoquinone-l-tryptophan, uncompetitively, . plp-l-trp, inhibited irreversibly only the apo form of trpase and may serve for structure-determination purposes. further attempts are being made to synthesize improved trpase inhibitors, i.e., in the nm range. polyelectrolyte multilayer lms (pem) adsorbed on biomaterial surfaces are a new way to create a controlled release system. using biodegradable polymers, the lms can be degraded in vivo and release active molecules. in this work, we demonstrate the possibility of tuning the degradability of polysaccharide pem in vitro and in vivo. chitosan and hyaluronan pem (chi/ha) were either native or cross-linked (cl) using a water soluble carbodiimide (edc) at various concentrations in combination with nhydroxysulfosuccinimide. the in vitro degradation of the lms in contact with enzymes was followed by quartz crystal microbalance measurements and confocal laser scanning microscopy after lm labeling with chi fitc . whereas the native lms were subjected to degradation, the cl lms were more resistant to enzymatic degradation. films made of chitosan of medium molecular weight were indeed more resistant than lms made of chitosan-oligosaccharides. in addition, macrophages could degrade all types of lms and internalize the chitosan in vitro. the native lms implanted in vivo in mouse peritoneal cavity for a week showed an almost complete degradation whereas the cl lms were only partially degraded. these results suggests that the polysaccharides pem are of potential interest for in vivo applications as biodegradable coatings and that degradation can be tuned by controlling lm cross-linking. membrane electroporation -tool for therapeutic electrotransfer of drugs and gene dna e. neumann, s. kakorin physical and biophysical chemistry, faculty of chemistry, university of bielefeld, germany membrane electroporation (mep) is a new electrical high voltage scalpel, transiently opening the cell membranes of tissue for the penetration of foreign substances. due to the enormous complexity of cellular membranes, many fundamental problems of mep have to be studied at rst on model systems, such as the curved bilayer membranes of unilamellar lipid vesicles. electrooptical and conductometrical data of unilamellar liposomes indicate that electric eld pulses cause not only the formation of membrane electropores but also shape deformation of the liposomes, both processes mutually affecting each other. the primary eld effects of mep and cell deformation can trigger a cascade of numerous secondary phenomena, such as pore percolation and transport of small and large molecules across the electroporated membrane. the chemical mep theory represents a molecular physico-chemical approach to electrochemomechanical pore formation, yielding transport parameters, such as permeation coef cients, pore fractions and pore sizes. the pore concept is successfully applied to rationalize optimization strategies for biotechnological and medical applications of mep. in silico elucidation of xenobiotic processing loops k. nakata , y. tanaka , t. nakano , t. ishikawa , h. tanaka , t. kaminuma national institute of health sciences, tokyo, japan, tokyo medical and dental university, tokyo, japan, tokyo institute of technology, yokohama, japan, hiroshima university, hiroshima, japan one of the important challenges for drug designers is to predict and analyze how drugs are absorbed, distributed, metabolized and excreted (adme) in the body. these processes highly correlated with toxicity of drugs and are actively studied in pharmacology. two classes of proteins, the drug metabolizing enzymes such as cytochrome p s (cyps) and transporters, are the target of such adme/tox research. it was relatively recent that these two classes of proteins are synthesized by the genes that are the target genes of the nuclear receptors. nuclear receptors are ligand-activated transcription factors that form a superfamily. in case of humans there are nuclear receptors almost half of whose ligands are identied, leaving some as true orphans. thus it was now recognized that these nuclear receptors play the role of sensors of drugs and other xenobiotic substances including environmental chemical pollutants and nutritional ingredients, while the drug metabolizing enzymes and the transporters are the processors which carry the actual cleaning jobs. we have started to elucidate the feedback loops that are formed by the xenobiotic ligands, nuclear receptors, their target genes, their product proteins, and their feedback actions on the ligands. the work is being carried out on our background database on the ligands and their receptors called kibank, and search programs for target genes of nuclear receptors algorithmically. the most recent results will be presented at the presentation. the major route for drug entry into cells is permeation across lipid bilayers. due to methodological limitations there are only few studies on permeation of drug-like molecules across lipid bilayers. an assay developed in our lab allows the direct measurement of lipid bilayer permeation of aromatic carboxylic acids (acas). tb , which forms a uorescent complex with acas, is entrapped in liposomes and aca entry is determined from luminescence increase. lipid bilayer permeation was ph-dependent, following a henderson-hasselbalch function with a plateau for the neutral and the anionic species, respectively. in contrast to the expectations of the ph-partition hypothesis, permeation of the anionic species was only to magnitudes lower than that of the neutral species, leading to anion-controlled permeation at ph . , independently of bilayer state and lipid composition. permeation across bilayers with a biologically relevant lipid composition was signi cantly slower than across egg-phosphatidylcholine membranes. the in uence of single lipids, such as cholesterol, was dependent on the structure and ionization state of the permeant. permeation coef cients of the neutral species correlated better with the polar surface area (psa) than logp oct , therefore psa is a better predictor for bilayer permeation of the neutral species of small acas than logp oct . interplay between polymerized liposomes physicochemical properties and composition and citotoxicity this study was aimed at investigating whether there is an interplay between diacetylenic polymerized liposomes physicochemical properties and lipid composition affecting citotoxicity in vitro. unsaturated , -bis( , -tricosadiynoyl)-sn-glycero- -phosphocholine with saturated , -dimiristoyl-sn-glycero- phosphocholine in molar ratio : , were combined to give a chemically modi ed membrane by uv-polymerization. biophysical characterization was carried out determining the hydrophobic factor and hydrodynamic radius. citotoxicity was evaluated through haemolytic capacity on bovine red blood cells and indirectly by capacity of induction of lipid peroxidation on microsomes or mitochondrial membranes. the haemolysis percentage in presence of dc , pc/dmpc is less than that induce by polymers used in dentistry. the data obtain suggests that the polymerized lipids can not induce lipid peroxidation on natural membranes. the polymerized diacetylenic liposomes showed less interaction with serum proteins than non polymerized and lower citotoxicity as compared with natural lipids. also cell viability was determined in cell line nih t after exposure to lipids systems under study. the hydrophobic factor showed further augmentation for polymerized liposomes and is discussed in relation to in vitro stability. the above results suggest that polymerized and non-polymerized liposomes would serve as an effective delivery vehicle. s. sonar, s. d´souza, k. p. mishra radiation biology and health sciences division,bhabha atomic research centre,mumbai- , india liposomes offer new approaches for drug delivery through their encapsulation to alter pharmacodynamic properties of loaded drug leading to reduction in toxicities and/ or improved ef cacy. for prolonged systemic circulation, the liposomes size has been shown to be limited to nm or less. the ethanol injection method is an excellent technique for the formation of liposomes of < nm without the need of sonication or extrusion. the present study was aimed to produce liposomes encapsulating doxorubicin in minimum procedural steps. liposomes were prepared using distearoyl phosphatidylcholine and cholesterol, distearoyl phosphatidylcholine, cholesterol and oleic acid. the effects of different operational conditions for vesicle production and drug encapsulation were evaluated, with a view to achieve process cost to a minimum, suitable size and high encapsulation ef ciency. although high ef ciency of doxorubicin encapsulation was obtained by 'active' or 'remote' loading process in dspc/chol system, it was poor in one-step injection method. oleic acid was included to cut down the active loading by ph-gradient. dspc/chol/oa systems spontaneously loaded doxorubicin with encapsulation ef ciency of % and nal drug to lipid molar ratio upto . . the mean diameter of the vesicles was + nm. the method offers liposomes of small size with high loading. design of peptides with consecutive dehydro phenylalanine residues r in order to develop general rules for the design of peptide conformations with consecutive alpha, beta-dehydro phenylalanine-residues, peptides were synthesized, crystallized and crystal structures and molecular conformations were determined. following conclusions were drawn based on the structural data: -peptide unit sequences with two consecutive dehydro-phe residues at (i+ ) and (i+ ) positions adopt an unfolded s -shaped structure with dihedral angles phi-psi centred at , . -the peptides containing two consecutive dehydro-phe residues at (i+ ) and (i+ ) positions • form two overlapping type iii beta -turns (incipient -helix). • with branched beta -carbon residue only at (i+ ) position adopt a conformation with two overlapping types ii and iii beta -turns. • with branched beta-carbon residues such as val and ile at both (i+ ) and (i+ ) positions form two overlapping types ii and i beta -turns. the consistency in the formations of these conformations makes the design of peptides with alpha,beta ?-dehydro -residues a useful and highly predictable method for developing speci c ligands for various biological applications including drug design. binding of cationic porphyrin to isolated double-stranded dna and nucleoprotein complex k. zupán , l. herényi , k. tóth , z. majer , g. csík institute of biophysics and radiation biology, semmelweis univ., budapest, hungary" biophysics of macromolecules, german cancer research center, heidelberg, germany, department of organic chemistry, eötvös loránd univ., budapest hungary the complexation of tetrakis( -n-methylpyridyl)porphyrin (tmpyp) with free and encapsidated dna of t bacteriophage was investigated. to identify binding modes and relative concentrations of bound tmpyp forms, the porphyrin absorption spectra at various base pair/porphyrin ratios were analyzed. spectral decomposition, uorescent lifetime, and circular dichroism measurements proved the presence of two main binding types of tmpyp, e. g., external binding and intercalation both in free and in encapsidated dna. tmpyp binding does not in uence the protein structure and/or the protein -dna interaction. concentrations of tmpyp species were determined by comprehensive spectroscopic methods. our results facilitate a qualitative analysis of tmpyp binding process at various experimental conditions. we analyzed the effect of base pair composition of dna, the presence of protein capsid and the composition of buffer solution on the binding process. protein crystallization and structural study of upa protease domain with active site serine mutation the urokinase (upa) system is composed of upa, its receptor (upar), and inhibitor (pai). it plays important role in various physiologic processes, including brinolysis, cell adhesion, and signal transduction, and has been recognized as a target for intervention in tumor growth and tumor metastasis. we constructed an active site mutant of upa protease domain ( - ) with three mutations (c a, n q, and s a) and expressed it as secreted protein in pichia pastoria with ppicza vector. the secreted mutant was captured from culture medium by a cation exchange column and then further puri ed on a gel ltration column. the puri ed mutant was then crystallized by sitting drop vapor diffusion method with several precipitant conditions: ( ) . - . m ammonium sulphate, - % peg , mm sodium citrate ph . or mm sodium phosphate ph . , . % sodium azide. ( ) . m ammonium sulphate, . m lithium sulphate, mm sodium acetate at ph . , ( ) . - . m sodium formate, mm sodium acetate at ph . . ( ) . - . m sodium chloride, mm sodium acetate at ph . . the crystals were of varying quality but generally diffracted from . Å- . Å with inhouse x-ray source. the structure of this upa mutant and its complex with various inhibitors will provide a platform for rational upa inhibitor design. abstract in this work, the linear interaction energy (lie) method was used to calculate the binding free energies of hiv- integrase (in) and a series of dicaffeoyl -or digalloyl pyrroliding and furan derivatives inhibitors. the model of binding free energy prediction for homogeneous inhibitors of hiv- in has been obtained with a root-mean-square deviation (rmsd) of . kj/mol and estimated to be a precise model with good prediction capability. in addition, the probable binding mode of this series of inhibitors with hiv- in was proposed by using molecular docking and molecular dynamics (md) simulation methods. our results indicate that caffeoyl -or galloyl group of inhibitors have close interaction with a hiv- in conservative dde motif. a speci c non-competitive inhibitor of a small g protein/gef complex on the protective role of selenium and catechin in cadmium toxicity s. Özdemir , s. dursun , s. toplan , n. dariyerli , m. c. akyolcu istanbul university, cerrahpasa medical faculty, department of biophysics, turkey, istanbul university cerrahpasa medical faculty, department of physiology, turkey cadmium as heavy metal is toxic and carcinogenic for organisms. cadmium perform their effects on living organisms by accumulation in blood and various tissues. due to their accumulation in various tissues and in blood, tissue antioxidant enzyme systems are affected. the present study was planned to determine the possible protective roles of selenium and catechin against the toxic effects of considered heavy metals. the study has been performed in wistar albino type rats which divided into four groups as control and cadmium, cadmium+selenium, cadmium+catechin received groups. besides cadmium as heavy metal, selenium concentration determinations were performed in blood, liver and kidney tissues of each group of rats. in the same tissue samples besides lipid peroxidation measurements, glutathione, glutathione peroxidase and superokside dismutase enzyme activity determinations were also performed. the accumulation of heavy metals was determined in blood, liver and kidneys after cadmium administration during experimental period. in the tissue of experimental group animals there was an increased lipid peroxidation but decreased antioxidant enzyme activities were observed. while effects of selenium in decreased toxicity of cadmium have been detected, there was no statistically signicant effect of catechin observed. proposed that motors could dynamically cluster at the tip of tubes when they are individually attached to the membrane. we demonstrate, in a recently designed experimental system, the existence of an accumulation of motors allowing tube extraction. we determine the motor density along a tube by using uorescence intensity measurements. we also perform a theoretical analysis describing the dynamics of motors and tube growth. the only adjustable parameter is the motor binding rate onto microtubules, which we measure to be . +/- . s . in addition, we quantitatively determine, for a given membrane tension, the existence of a threshold in motor density on the vesicle above which nanotubes can be formed. we nd that the number of motors pulling a tube can range from four at threshold to a few tens away from it. the threshold in motor density (or in membrane tension at constant motor density) could be important for the understanding of membrane traf c regulation in cells. kinesin and dynein move a peroxisome in vivo: a tug-ofwar or coordinated movement? c. kural, p. r. selvin, k. hwajin, g. goshima, v. i. gelfand university of illinois at urbana-champaign, usa we have used fluorescence imaging with one nanometer accuracy (fiona) for analysis of organelle movement by conventional kinesin and cytoplasmic dynein in a cell. we can locate a green uorescence protein (gfp)-tagged peroxisome in cultured drosophila s cells to within . nanometer in . milliseconds, a -fold improvement in temporal resolution, suf cient to determine the average step size to be nanometers for both dynein and kinesin. furthermore, we nd that dynein and kinesin do not work against each other in vivo during peroxisome transport. rather, we nd that multiple kinesins or multiple dyneins work together, producing up to times the in vitro speed. engineering a bio-molecular walker h. jankevics, j. e. molloy division of physical biochemistry, mrc national institute of medical research, the ridgeway, mill hill nw aa, london, uk in this work we describe the design and development of a biomolecular walker based on the motile system found in certain ciliated protists. the motile system is driven by the binding of ca ions and in contrast to other commonly studied motor proteins is independent of atpase (amos et al., ) . the motor protein is a kda ca-binding protein called spasmin (maciejewski et al., ; itabashi et al., ) which belongs to the ef-hand family of calcium binding proteins called calmodulins. upon calcium binding, spasmin is thought to undergo a large conformational change as it binds its own target peptide and we wish to exploit this in order to create our own novel molecular walker.we have created a recombinant spasmin with sequence tags to enable speci c immobilization and various conjugate chemistries. cystein mutants have been introduced at speci c points in the protein to enable attachment of other small molecules, for example uorophores. we are now optimising protein expression and puri cation to maximise the yield of active protein on which we can perform the conjugate chemistries. we will characterize the structural changes using biophysical methods such as circular dichroism, analytical ultracentrifugation, electron microscopy, afm and total internal re ection uorescence spectroscopy on single molecules. the force generation in muscle arises from direct interaction of the two main protein components of the muscle, myosin and actin. the process is driven by the energy liberated from the hydrolysis of atp by myosin. the interaction is performed by cyclic interaction of myosin with atp and actin, and at least six intermediates are proposed for actomyosin atpase in solution. the powerful dsc technique allows the derivation of heat capacity of proteins as a function of temperature. from the deconvolution of the thermal unfolding patterns it is possible to characterize the structural domains of the motor protein. in this work we tried to approach the temperature-induced unfolding processes in different intermediate state of atp hydrolysis in striated muscle bres. we have extended the experiments to study the ber system prepared from psoas muscle of rabbit in rigor, strongly binding and weakly binding states of myosin to actin where the inorganic phosphate (p i ) was substituted by the phosphate analogue orthovanadate. the dsc transitions were analyzed in different buffer solutions (tris and mops) to get information about the temperature dependence of ph on the conformational changes. single kinesin motor proteins walking through the searchlight s. verbrugge, l. c. kapitein, e. j. peterman vrije universiteit, de boelelaan , hv, amsterdam, the netherlands the dimeric motor protein kinesin steps by a hand-over-hand mechanism. this means that the centre of mass moves with nm steps, while the two motor domains, the one after the other, move nm to the next binding site on the microtubule. the molecular details of what happens during a step are not fully understood, partly because of lack of time resolution in wide-eld, single-molecule uorescence experiments. we set out to develop an approach to study the motility of kinesin with a time resolution below a millisecond (a single step takes on the order of milliseconds). this approach allows us to look into the mechanochemistry and coupling of the two kinesin motor domains while they are stepping. our method is based on confocal microscopy and we study the uorescent properties of single labeled motors while they walk through the confocal laser spot. we present the experimental details of our approach and show our results on human kinesin constructs that are speci cally labeled in the tail. we show that our approach enables us to study the mechanism of kinesin with a much higher time resolution than what was achieved before with single-molecule uorescence experiments. movement of coupled single-headed kinesins analysed by a brownian-ratchet model . the analysis of this system is expected to provide insights into the mechanism underlying the motility of conventional double-headed kinesin, espetially the roles played by individual heads. we would like to clarify whether the experimentally observed behaviors that are supposed to be caused by a pair of single-headed kinesins can be explained by a simple brownianratchet model, which is successful in describing the motion of an unconventional single-headed kinesin kif a. our model consists of two brownian motors (ratchets) separated by a xed distance r. the velocity and other quantities of the coupled motors are calculated by solving the fokker-planck equation with various choices of r and other parameters. then, assuming a certain probability distribution of r associated with random attachment of kinesin heads on a bead in the experiment, the statistical properties of the motion of the coupled brownian motors are analysed. the force-velocity relation observed experimentally is found to be consistent with the present model with appropriate choices of the model parameters. the adequacy of the parameter choice needs to be con rmed by other experiments. optical trap with fast programmable feedback loop to study rotary molecular motors t. pilizota, f. bai, r. m. berry clarendon laboratory, univeristy of oxford an optical trap with back-focal plane detection and fast programmable feedback has been developed for the study of rotary molecular motors. a helium-neon laser ( nm) is used for position detection and a solid state bre laser ( nm, w cw) forms the trap. acousto-optic de ectors (aods) controlled by a digital signalling processing board are used to achive programmable feedback loops with exible control options and speeds up to khz. several modes of feedback are demonstrated, controlling both bead position (x,y) and angle (r, ). polystyrene beads or bead pairs can be held at set (x,y) or , and the set-point can be changed while the program is running. for example, feedback can be used to move a bead or a bead pair in a circle. results of using the system to study the bacterial agellar motor are presented. a dimeric -d lattice gas as model for molecular motors collective dynamics p. pierobon , t. franosch , e. frey ludwing-maximilian universitaet, münchen, germany, hahn meitner institut, berlin, germany, arnold sommerfeld center for theoretical physics, münchen, germany the transport of molecular motors along microtubules closely resemble the dynamics of a driven lattice gas of dimers without conservation of particles. the unidirectionality, asymmetry and stochasticity of the motion are encoded in the well studied totally asymmetric simple exclusion process (tasep). we extend the model to a more realistic one, including attachment and detachment kinetics and extended (dimeric) particles. we study the stationary phase diagram by means of monte carlo simulations combined with a continuum description (based on an extended mean eld theory). we also evaluate the domain wall theory nding out the effective potential con ning the phase interface into the bulk. a. e. wallin , j. lisal , r. tuma department of physical sciences, pobox , fin- , university of helsinki, finland, institute of biotechnology, university of helsinki, finland molecular motors often consist of two or more subunits that cooperate to convert chemical energy into mechanical motion. hexameric helicases and viral packaging atpases constitute a special class of molecular motors that translocate along nucleic acids. recent structural and spectroscopic characterization of these motors revealed that their enzymatic cooperativity does not result from cooperative binding [ - ]. in order to understand this new type of cooperativity we simulated the kinetics of a single hexameric motor by multiple coupled stochastic reactions using the gillespie algorithm [ ] . in contrast to analytical methods, our direct simulation allowed us to investigate the kinetics with an arbitrary model for cooperativity between the subunits. simulations on the kinetics of the hexameric rna packaging motor p from dsrna bacteriophage [ ] with different cooperativity mechanisms provided insight into the rnamediated cooperativity and yielded a sound theoretical basis for the interpretation of experimental results [ ]. the viral infectivity factor (vif) encoded by hiv- is a small basic protein that strongly modulates the viral replication and is required for pathogenicity. vif is packaged into hiv- particles through a strong interaction with genomic rna and is associated with viral nucleoprotein complexes. moreover vif acts during the early stages of the viral infection (capsid disassembly, reverse transcription) as well as during the late stages of virus replication (virus assembly and maturation of the virion). however the effect on early stages is probably a consequence of a defective assembly and / or virion maturation. understanding the rna-binding properties of vif would contribute to elucidate the role played by vif in the regulation of the genomic rna traf cking in the cytoplasm to unable ef cient packaging, and the prevention of cellular inhibitors from altering hiv- rna. in this context, we have characterised the interactions of recombinant vif with hiv- genomic rna by uorescence spectroscopy, and determined the af nity of the protein for synthetic rnas corresponding to various regions of hiv- genome. taken together our results demonstrate cooperative and speci c binding. in particular, we showed that vif has a high af nity for the 'untranslated region of hiv- genomic rna. s. bernacchi , e. ennifar , k. toth , p. walter , j. langowski , p. dumas cnrs upr -strasbourg (france), dkfz -heidelberg (germany) we have used the dimerization initiation site (dis) of hiv- genomic rna as a model to investigate hairpin-duplex interconversion by using a combination of uorescence, uv-melting, gel electrophoresis and x-ray crystallographic techniques. fluorescence studies with molecular beacons and crystallization experiments with -nucleotide dis fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on rna concentration, and not on cooling kinetics. with natural sequences able to form a loop-loop complex (or 'kissing complex'), concentrations as low as µm in strands are necessary to obtain a majority of the hairpin form. on the contrary, when kissing-complex formation was made impossible by mutation in the loop, a majority of hairpins was obtained even at µm in strands. this mutated sequence also showed that kissing-complex formation is not a prerequisite for an ef cient conversion to duplex in presence of salts. we proved that this happens through hairpins engaged in a cruciform intermediate, but not from free strands after hairpin melting. supporting this view, the very rst step of formation of such a cruciform intermediate could be trapped in a crystal structure. such a mechanism might be biologically signi cant beyond the strict eld of hiv- rna dynamics. generation of rna dimeric form of the human immunode ciency virus type (hiv- ) genome is important for the viral replication. the dimerization initiation site (dis) has been identi ed as a short sequence that can form a stem-loop structure with a selfcomplementary sequence in the loop and a bulge in the stem. a mer dis rna fragment, dis , spontaneously formed "loosedimer" and was converted into "tight-dimer" by supplement of nucleocapsid protein ncp . nmr chemical shift analysis for dis in the kissing-loop and extended-duplex dimers revealed that three dimensional structures of the stem-bulge-stem region were similar between the two types of dimers. therefore, we determined the solution structures of two shorter rna molecules corresponding to the loop-stem region and the stem-bulge-stem region of dis , and the solution structures of dis in the kissing-loop and extendedduplex dimers were determined by combining the parts of structures. the mechanism of conformational conversion will be discussed based on the solution structures and the molecular dynamics analysis. nmr and molecular modelling studies of an rna hairpin containing a g-rich hexaloop the mrna of the pgy /mdr gene encoding the transmembrane p-glycoprotein (p-gp) contains a hairpin that is the target of antisense oligonucleotides, suppressing the p-gp function of multidrug resistance. the solution conformation of this hairpin constituted by the '(gggaug) ' loop closed by a g-u mismatch containing stem is studied by nmr and molecular dynamics in explicit solvent. special attention is given on the sugar and the backbone conformations and the hexaloop intrinsic properties of these two components are carefully investigated. the stem structures obtained by molecular dynamics with and without nmr constraints converge to the same a-type double helix. the wobble g-u mismatch moderately perturbs the overall conformation, despite of c '-endo sugars and unusual backbone conformations located between the mismatch and the loop. in the hexaloop part, the sugar puckers are in majority in c '-endo conformations, probably to extend the strand with the help of unusual backbone angles conformations. the loop appears stabilized by one hydrogen bond and stacking interactions. thus, from the ' to the '-ends, the four purine bases ggga are stacked together, then a u-turn like is observed, and nally, u stacks on the last g that remains rather far from the stem. nmr and molecular modelling studies of an rna hairpin containing a g-rich hexaloop the mrna of the pgy /mdr gene encoding the transmembrane p-glycoprotein (p-gp) contains a hairpin that is the target of antisense oligonucleotides, suppressing the p-gp function of multidrug resistance. the solution conformation of this hairpin constituted by the '(gggaug) ' loop closed by a g-u mismatch containing stem is studied by nmr and molecular dynamics in explicit solvent. special attention is given on the sugar and the backbone conformations and the hexaloop intrinsic properties of these two components are carefully investigated. the stem structures obtained by molecular dynamics with and without nmr constraints converge to the same a-type double helix. the wobble g-u mismatch moderately perturbs the overall conformation, despite of c '-endo sugars and unusual backbone conformations located between the mismatch and the loop. in the hexaloop part, the sugar puckers are in majority in c '-endo conformations, probably to extend the strand with the help of unusual backbone angles conformations. the loop appears stabilized by one hydrogen bond and stacking interactions. thus, from the ' to the '-ends, the four purine bases ggga are stacked together, then a u-turn like is observed, and nally, u stacks on the last g that remains rather far from the stem. aminoglycoside binding to hiv- dis kissing-loop complex: from crystals to cells e. ennifar , j.-c. paillart , a. bodlenner , p. pale , r. marquet , p. dumas cnrs upr , strasbourg -france, cnrs/université louis pasteur strasbourg umr , strasbourg -france all retroviral genomes consist in two homologous single stranded rnas. dimerization is an essential step for viral replication. hiv- dimerization initiation site (dis) is a strongly conserved stem-loop in the ' leader region of the genomic rna. it was shown in vivo that alteration of the dis dramatically reduces viral infectivity. we have previously solved crystal structures of the dis kissing-loop complex. analysis of these structures revealed an unexpected resemblance between the dis kissing-loop and the s ribosomal aminoacyl-trna site (a-site), which is the target of aminoglycoside antibiotics. we have shown that some aminoglycosides specifically bind to the dis kissing-loop complex with an af nity and geometry similar to that observed in the a-site. in agreement with these previous results, we have now solved highresolution crystal structures of the dis kissing-loop complex bound to four aminoglycosides. these structures show that, as expected, two aminoglycosides are bound per kissing-loop complex. importantly, the binding is observed not only in vitro on large hiv- genomic rna fragments, but also on infected cells. moreover, we showed that some of these aminoglycosides stabilize the kissingloop rna dimer, which is consistent with the observation in crystal structures of numerous direct and water-mediated drug-rna contacts. these structures are currently used as starting points for designing potential new drugs targeted against the viral rna. modeling the long range entropy of rna: w. k. dawson , k. fujiwara , k. yamamoto , g. kawai chiba institute of technology, - - tsudanuma, narashino, chiba, japan, international medical center of japan, - - toyama, shinjuku-ku, tokyo, japan non-coding rna appears to make up a large part of the human genome. a reliable rna structure prediction program is needed to understand the structure of this non-coding rna. we recently developed a new way to model the long range entropy in rna and applied it to rna secondary structure prediction. in some of instances, the new approach is able to achieve far better predictions than the state of the art secondary structure programs even given exactly the same parameters. predictions using this method tend to show distributions that are funnel shaped. a new and important parameter in these calculations is the persistence length (a measure of the correlation and exibility of the rna). (url: http://www.rna.it-chiba.ac.jp/ vsfold/vsfold /) this new approach has now been extended to prediction of pseudoknots. the method is a heuristic wherein the hierarchical folding hypothesis is used to nd the pseudoknots as the rna secondary structure is folding, and corrections to that secondary structure are made to accommodate the pseudoknot. it is able to do these searches in roughly n^ time. the model is consistent with the hierarchical hypothesis and it is possible to estimate rna folding times that are of the correct order of magnitude using this model. with further adaptations to account for the size, shape and variablity of amino acid residues (hydrophobicity etc.), the model also appears to be transferable to protein folding problems. a. v. melkikh ural state technical university, ekaterinburg, russia a model of the genome as a gene network capable of receiving information about the environment and performing some operations on genes has been considered. the evolution rate of replicators for the mechanism of random mutations has been estimated. it was shown that the evolution rate under random actions is negligibly small for real dimensions of genomes of replicators [ ]. it was inferred that only a deterministic mechanism of the evolution can explain the known evolution rate of replicators. a deterministic model of the evolution has been proposed. the basic principles of this model include: ) information about the replicators evolution is encoded in the conformational states of proteins; ) the conformational language of proteins is translated into the language of nucleotide sequences during the evolution; ) the structure of genes is controlled such that the transition to a nearest free ecological niche takes a minimum time (at a preset restriction on the control). transfer rnas are synthesized as part of longer primary transcripts that require processing of both their ' and ' extremities in every living organism known. the ' side is matured by the quasiuniversally conserved endonucleolytic ribozyme, rnase p, while removal of the ' tails can be either exonucleolytic or endonucleolytic. the endonucleolytic pathway is catalysed by an enzyme known as rnase z. rnase z cleaves precursor trnas immediately after the discriminator base in most cases, yielding a trna primed for addition of the cca motif by nucleotidyl transferase. rnase z is found in the vast majority eukaryotes and archaea and in about half of the sequenced bacteria. it is often essential for growth and mutations in one of the two genes encoding rnase z (elac ) have been linked with prostate cancer in man. in this poster we present the crystal structure of bacillus subtilis rnase z at . Å resolution ( ) resolved by mad method and propose a model for trna recognition and cleavage. the structure explains the allosteric properties of the enzyme and also sheds light on the mechanisms of inhibition.it also highlights the extraordinary adaptability of the metallohydrolase domain of the b-lactamase family for the hydrolysis of covalent bonds. ( )i. most rnas undergo several steps of post-transcriptional modi cation before carrying out their assigned functions. one of the major modi cations is the splicing process, by which non-coding introns are removed from the coding exons. splicing can be performed by autocatalytic, self-splicing introns (e.g. group ii introns), i.e. catalytic rna or ribozymes. group ii introns, which occur in bacterial genomes and in organellar genes of plants, funghi and lower eukaryotes, consist of a conserved set of six domains. domain recognizes the '-exon through a - base pairing interaction formed by two regions within the intron (exon binding sites, ebs and ebs ) and the last - nucleotides of the '-exon (intron binding sites, ibs and ibs ). as the correct recognition of ibs by ebs is crucial for a successful splicing event we are investigating the structural and metal ion requirements of this part by various spectroscopic techniques, e.g. nmr. our data shows that the hairpin including ebs consists of a helical region followed by an unstructured single stranded part, which is ready for splice site recognition. the results of the structure analysis will be presented. financial support by boehringer ingelheim fonds (fellowship to d. k.) and the swiss national science foundation (snf-förderungsprofessur to r. k. o. s.) is gratefully acknowledged. structural basis for the antigene and antisense properties of modi ed dna:dna and rna:dna duplexes e. c. m. juan , t. kurihara , j. kondo , t. ito , y. ueno , a. matsuda , a. takenaka graduate school of bioscience and biotechnology, tokyo institute of technology, graduate school of pharmaceutical sciences, hokkaido university, faculty of engineering, gifu university oligonucleotides containing polyamines are currently being evaluated as potential antigene and antisense compounds. those with -(n-aminohexyl)carbamoyl- '-deoxyuridine ( n u) and its '-omethyl derivative ( n u m ) exhibit improved nuclease resistance and form stable duplexes with their dna and rna targets. x-ray structures of these duplexes have shown good correlation between the conformational changes and the observed chemotherapeutic properties. the amide groups of the modi ed uracil bases form six-membered rings through the intra-molecular nh-o hydrogen bonds, so that the aminohexyl chains protrude into the major grooves. some of the terminal ammonium groups are involved in intra-duplex interactions with phosphate oxygen anions, whereas the others interact with those of the adjacent duplex. such interactions contribute to the stability of duplex formation. the '-o-methyl modi cation in n u m shifts the ribose ring toward the c '-endo conformation and in uences duplex stability. observed changes in the dimensions of the minor grooves and in the hydration structures are also well correlated to nuclease resistance and duplex stability. group ii intron ribozymes catalyze selfsplicing in bacterial genomes as well as in organellar genes of lower eucaryotes. for correct structure and function these ribozymes need speci c concentrations of monovalent and divalent cations such as k and mg . most of these ions are used for charge screening, but some are also bound to distinct sites ful lling various speci c tasks. the conserved secondary structure of group ii intron ribozymes consists of six domains grouped around a central wheel. here the focus is set on domain (d ) of the yeast mitochondrial intron ai , a hairpin of nucleotides, which is crucial for catalytic activity. the -nucleotide bulge in d is known to be exible and acts as a metal ion binding platform. we have investigated the binding of different metal ions (mg , ca , mn , cd ) to this platform by uorescence spectroscopy. for this the bulge site adenosine in d was replaced by the uorescent nucleotide base analogue aminopurine ( ap). the binding data ts to an equation describing a binding to a single class of sites. the titration experiments not only reveal different dissociation constants for the tested metal ions but also indicate different effects on the bulge structure. financial support by the swiss national science foundation (snf-förderungsprofessur to r.k.o.s) is gratefully acknowledged. modi ed nucleosides and across the anticodon loop interactions in trna u. b. sonavane, k. d. sonawane, r. p. tiwari national chemical laboratory, pune , india in several interesting trna molecules, the ( th ) as well as the ( th ) nucleoside are hyper modi ed. as an example, unique hypermodi ed nucleosides mcm s u and ms t a are crucial in human trna lys , which acts as a primer in hiv replication. modi ed nucleosides may facilitate or hinder across the loop interactions. large substituents in th and th modi ed nucleosides if oriented suitably may also interact with each other. across the loop interactions may lead to unconventional anticodon loop structures also affecting exibility of the anticodon loop. this may restrict or enlarge synonymous codon choice and decoding during protein biosynthesis. except for trna asn (with interacting q and t a ), our studies show conventional 'open' loop structure -free of across the loop interactions, for a number of interesting trna anticodon loops with diverse hyper modi ed nucleosides at both of these locations. molecular dynamics simulations of hydrated anticodon arm of trna asn show persisting interaction involving the diol group of q and carbonyl group of ureido linkage in t a . additionally, the hoogsteen edge of th adenine base participates in hydrogen bonding with watson -crick edge of rd base and thus contributes to unique loop structure of trna asn . resulting suboptimal q:c base pairing leads to unbiased reading of u or c as the third codon letter. absence of queuosine modi cation, q happens to be also associated with uncontrolled rapid proliferation of cells and malignant growth. structural properties of ctg/cag repeats, and preliminary x-ray analysis of cug repeats y. sato, k. kimura, a. takénaka graduate school of bioscience and biotechnology, tokyo institute of technology, yokohama, japan. the human genome contains so many different types of repetitive sequences. some of them are tandem repeats of trinucleotides. their unusual expansions cause genetic diseases such as type myotonic dystrophy (dm ) and huntington's disease (hd), the unit sequences being ctg and cag, respectively. the numbers of repeats of the two complementary sequences change independently during dna replication or repair. the direct origin of dm is, however, the transcribed rna fragments with cug repeats, which forms a speci c structure and inhibits other protein syntheses. in the present study, structural versatilities of such dna and rna fragments has been examined. native pages of (cug) n show that the hairpin structure with even number is more stable than that with odd number. this difference might be ascribed to the structural difference at the hairpin head. the pages also show that duplex formation is dependent on coexisting cationic species and their concentration. crystal data of (cug) (a=b= . , c= . Å, and the space group r ) suggest that the asymmetric unit contains the rna fragment. an approximate crystal structure solved by molecular replacement techniques at . Å resolution shows that the rna fragments form a duplex similar to an a-form rna. context-dependent selection of promoter in a natural selection-type evolution reactor h. nagayasu, y. ageno, x. t. ma, y. husimi saitama university, saitama, japan using an isothermal ampli cation of hairpin dna/rna (developed by g.joyce), we drove a natural selection-type evolution reactor taking the speci c growth rate as the tness. we used hiv- rt and thermot rnap at c. starting from the random promoter pool, we selected the strongest promoter at c, which was separated by hamming distance from the strongest promoter at c. the latter was found to be identical to the natural t promoter. when we used a simple random pool, the selection process showed one-step convergence. when we used a random pool with a speci c short sequence at the upstream anking region of the random region, we observed an evolution process as convergencedivergence-convergence of the promoter sequence, driven by deletion of the speci c short sequence. this context-dependent selection was found to come through a neutral path, judged from the tness measurement. intronic sirna and mirna, and dna methylation gif sur yvette, france. abnormal activation of small g proteins is involved in several human diseases. small g proteins are activated by gdp/gtp exchange, which is stimulated by their guanine nucleotide exchange factors (gefs). thus, small g protein/gef complexes appear as emerging targets for interrupting signalling networks regulated by small g proteins in pathological contexts. the activation of small g proteins of the arf family is initiated by exchange factors which carry a sec catalytic domain stimulating the dissociation of the bound gdp nucleotide. the structure of the arf -gdp-arno reaction intermediate, trapped by a mutation of the catalytic glutamate, was recently solved by x-ray crystallography (pdb code: r s) acknowledgements: the work was supported by contract k- j. q. yin, f. chen, y. tan, t. gu institute of biophysics, chinese academic sciences, beijing, china sirna/mirna can ef ciently induce mrna cleavage or translational repression at the posttranscriptional level in a sequencespeci c manner. recently, it has been shown that these small rnas guide genome modi cation in mammalian cells. however, their ability to direct cognate dna methylation has been con rmed so far only in plants, and their biogenesis, functions, and modes of silencing genes are yet elusive. here, we report that small rnas derived from intron regions of some genes can target homologous dna sequence in promoter, 'utr or 'utr regions of genes in different human tumor cells. surprisingly, we also discovered that endogenous sirnas from introns of genes possessed a large number of target mrnas by using bioinformatics, and con rmed their existence in human cells with northern blot analysis. intronic small rnas generated by sliceosomes can form mature mirnas or sirnas through the processing of drosha and /or dicer. rt-pcr analysis indicated that vector-based small rna repressed expression of homologous genes at the transcriptional and/or translational levels. western blotting demonstrated that the expression of some proteins was greatly reduced or completely inhibited owing to promoter methylation. these ndings reveal that the expression of some genes can incredibly control cell activities at both protein and rna levels. our results also suggest that these small rnas may regulate gene expression in different modes of action. role of stacking in speci c recognition of capped rna by the cbc protein the stacking interaction involving -methylguanine moiety of mrna ' terminal cap (m g) and aromatic amino acid side chains is a common feature of all known cap-binding proteins. the crystal structures of the human cap binding complex (cbc) showed its induced folding upon m gpppg cap analogue binding. stabilization of the cbc-m gpppg complex by sandwich stacking of m g in between y and y is additionally enhanced by stacking of the second base of capped mrna with y . gibbs free energy of the association of various cbc mutants with the synthetic cap analogues, m gpppg, m gpppu, and m gtp, has been determined by uorimetric titration. preference of the wild type (wt) cbc for the dinucleotide analogues is also observed for the y a mutant, with the energy loss of . - . kcal/mol. however, all proteins with the mutated second stacking partner y prefer to bind m gtp compared with m gpppg. the binding of m gtp to the y a mutant is only . kcal/mol less favourable compared with the wt cbc. these divergences may be ascribed to smaller entropic costs of conformational rearrangement of cbc in the case of a smaller ligand, which can nd more favourable contacts when its second part is not ef ciently 'constrained' by stacking with y . supported by kbn p a annealing of the tar dna hairpin to a complementary tar rna hairpin, resulting in the formation of an extended duplex, is an essential step in the minus-strand transfer process of hiv- reverse transcription. in this work, we use gel-mobility-shift analysis to follow the kinetics of this reaction in the absence or presence of hiv- nc prepared by solid-phase peptide synthesis. to elucidate the reaction pathway, we use either the complete -nt tar hairpins or truncated -to -nt minihelices (mini-tar) derived from the top part (i.e. hairpin loop) of tar. assays were also carried out with mutant tar constructs. the annealing kinetics were studied systematically as a function of dna concentration and temperature. we show that the annealing initiates through a weak loop-loop kissing interaction, followed by a much slower conversion step, which results in formation of the extended duplex. nc facilitates both reaction steps, resulting in the overall -fold and -fold rate enhancement for mini-tar and tar annealing, respectively. we show that the kissing step is facilitated by the nc-induced nucleic acid aggregation, which is more pronounced for the longer tar hairpins. at the same time, the conversion steps in tar and mini-tar appear to be very similar and are similarly facilitated by nc - -fold. the later effect relays on the ability of nc to destabilize nucleic acid duplexes, and is equivalent to destabilization of a few base pairs required for the conversion initiation. linking of the n-terminus of a peptide to its encoding mrna s. ueno, h. arai, y. husimi department of functional materials science, saitama university, saitama, japanin evolutionary protein engineering, in vitro selection using a cellfree translation system has advantages of large library size and also of applicability to cytotoxic protein. although many in vitro protein selection techniques such as in vitro virus, ribosome display are developed, most of these are the techniques that link the genotype molecule to the peptide at its cterminus. we developed a method to link the genotype molecules to the nterminus of its encoding peptide. the mrna has dna-linker hybridize region, translation enhancer, initiation codon, single codon, amber stop codon, n-terminus sequence in gfp gene, his-tag and ochre stop codon. the mrna is also linked at '-terminus to sup trna via spacer and the speci c amino acid. the mrna is translated in the cell-free translation system. thus, c-terminus of the protein becomes free. moreover, in this system, the free protein of the full length is never generated. for this reason, the problem in the conventional technique, that is, the competition between the protein displayed on the genotype molecule and the free protein is eliminated. we succeeded to turn one round of the " life cycle " of the in vitro virus, judged by his-tag selection. key: cord- -yx oyv authors: amar, patrick title: pandæsim: an epidemic spreading stochastic simulator date: - - journal: biology (basel) doi: . /biology sha: doc_id: cord_uid: yx oyv simple summary: in order to study the efficiency of countermeasures used against the covid- pandemic at the scale of a country, we designed a model and developed an efficient simulation program based on a well known discrete stochastic simulation framework along with a standard, coarse grain, spatial localisation extension. our particular approach allows us also to implement deterministic continuous resolutions of the same model. we applied it to the covid- epidemic in france where lockdown countermeasures were used. with the stochastic discrete method, we found good correlations between the simulation results and the statistics gathered from hospitals. in contrast, the deterministic continuous approach lead to very different results. we proposed an explanation based on the fact that the effects of discretisation are high for small values, but low for large values. when we add stochasticity, it can explain the differences in behaviour of those two approaches. this system is one more tool to study different countermeasures to epidemics, from lockdowns to social distancing, and also the effects of mass vaccination. it could be improved by including the possibility of individual reinfection. abstract: many methods have been used to model epidemic spreading. they include ordinary differential equation systems for globally homogeneous environments and partial differential equation systems to take into account spatial localisation and inhomogeneity. stochastic differential equations systems have been used to model the inherent stochasticity of epidemic spreading processes. in our case study, we wanted to model the numbers of individuals in different states of the disease, and their locations in the country. among the many existing methods we used our own variant of the well known gillespie stochastic algorithm, along with the sub-volumes method to take into account the spatial localisation. our algorithm allows us to easily switch from stochastic discrete simulation to continuous deterministic resolution using mean values. we applied our approaches on the study of the covid- epidemic in france. the stochastic discrete version of pandæsim showed very good correlations between the simulation results and the statistics gathered from hospitals, both on day by day and on global numbers, including the effects of the lockdown. moreover, we have highlighted interesting differences in behaviour between the continuous and discrete methods that may arise in some particular conditions. france was hit by the sars-cov- epidemic probably at the beginning of january , the first case being reported on january [ ], and went into lockdown on march [ ] . in response to the expected reduction of the number of cases, the french government eased the lockdown restrictions on may and eased them again on may (except in the ile-de-france region, where the density of population is very high). these measures have been taken to stop the exponential growth of the number of cases, as observed earlier in china [ , ] . the basic reproduction number r tells us the average number of new infections caused by an infective individual and it describes the exponential growth of the epidemic [ ] . if r is greater than the epidemic will spread; otherwise, when r is less than , the disease will gradually fade out [ ] . compared to the r of h n ( . ) [ ] the reproduction number of covid- indicates awful potential transmission. the r was estimated as . [ ] , . [ ] and . [ , ] by many different research sources around the world. the world health organization (who) published an estimated r of . to . [ ] . many approaches have already been used to model the covid- epidemic using compartment models and deterministic ordinary differential equations (ode) [ , ] and also to estimate the effects of control measures on the dynamics of the epidemic [ ] . these particular approaches give good results, but they do not take into account the stochastic nature or the spatial aspects of the propagation mechanism. however, stochastic differential equations (sde) have been successfully used to tackle the stochastic aspects of epidemic propagation [ ] [ ] [ ] [ ] . more recently, multi-region epidemic models using discrete and continuous models, taking into account the effectiveness of movement control have been published [ , ] , as well as sde multi-region models [ ] . stochastic models based on economic epidemiology have been applied to the covid- epidemic, for example, in south korea, to determine the optimal vaccine stockpile and the effectiveness of social distancing [ ] . approaches using agent-based systems have also been used to model both the stochastic and spatial characteristics of epidemic propagation [ , ] . in agent-based methods the number of machine instructions needed for each timestep, relative to the size of the data (algorithmic complexity), is at best proportional to the number of agents. those using one agent per individual may need a high computing power when used on large populations. these approaches are often applied to smaller areas (towns mainly) than the entire country, and/or use one agent to model a set of individuals ( in [ ] ). population-centred methods have an algorithmic complexity that does not depend on the size of the population, but on the number of rules considered at each iteration (for example, the number of reactions for biochemistry systems). when used on large populations these methods are much more efficient than entity-centred methods, but they do not take into account the spatial localisation. we adopted here a hybrid model derived from the sub-volumes method that adds coarse-grained spatial localisation capabilities to the standard stochastic simulation algorithm (ssa) used, for example, in the domain of biochemistry. to increase the computing efficiency we also used an original variant [ ] of the gillespie algorithm with tau-leaping [ , ] that automatically adapts the proportion of randomness vs. average-calculation, at each timestep. our implementation allows us to easily switch from this stochastic variant of ssa to a deterministic continuous solver (dcs), and therefore compare the two methods. to test our approach we applied it to the sars-cov- epidemic in france where relevant data [ , ] have been made available throughout the duration of the epidemic. most of the simulation parameters we used have been obtained from statistics gathered in the literature, such as the proportion of cases that needed hospitalisation and the proportion of severe forms among them [ , ] that needed beds in icu (intensive care unit). the number of infectious individuals and their localisations at the beginning of the epidemic have been inferred from statistical data made available by the french government and from the literature [ ] [ ] [ ] . we used our simulation tool to ascertain the effects of control measures on the dynamics of the epidemic and compared the results to the real statistical data. we focused our study of the impacts of the epidemic only on the part of the population that moves on a daily basis: workers, pupils, students, retired people, etc. people in nursing homes were not taken into account since their environment and way of life are very different. starting from a known initial state, we wanted to compute a stochastic sample of the evolution in time of the number of people at each state of the disease. a transition between such states is often described by a set of probabilistic rules, or by a stochastic automaton. the epidemic spreading can be modeled as a markovian process in the sense that the number of people in each state at time t + ∆t depends only on the numbers at time t (and on other variables that do not depend on t). in most of the cases, it is not possible to find an analytic solution that gives those numbers as a function of time. hopefully, iterative numerical methods exist. one of them is the gillespie algorithm, frequently used to find the evolutions of the quantities of chemical species s(t) = {s (t), ..., s n (t)} that can react according to chemical rules r = {r , ..., r m } and their kinetics k = {k , ..., k m }. starting from the initial value s( ) of the n species, the algorithm computes the values at time t > by iterating the following process: . based on the quantities s(t), the rules and their kinetics, compute stochastically at what time each reaction is triggered {t , ..., t m }. . let r i being the next reaction: t i = in f {t , ..., t m }. . apply r i ; i.e., update the vector s(t i ) by decreasing the quantities of the substrates of r i and increasing the quantities of its products. . update the time: t ← t i . this algorithm gives an exact stochastic trajectory of the system, but can be slow when some reactions are quick. these quick reactions will often be triggered, so the time increment at each iteration will be small and the number of iterations per second high. to decrease the computing time, the tau-leaping method uses a fixed timestep, τ. at each iteration, the number of times each reaction is triggered during the time interval τ is stochastically estimated based on the quantities at time t. this method gives an approximation of the stochastic trajectory of the system, which is accurate as τ is small. the value of τ must be chosen to be large enough to minimise the number of iterations per second, but not too large to get good precision. the algorithm used in pandaesim, a variant of the tau-leaping gillespie method, is detailed at the end of this section. the population-centred methods such as those presented here share the same constraint: the entities evolving in the environment are considered homogeneously distributed in the environment. in other words, the spatial localisation is not taken into account. the entity-centred approaches, which compute the behaviour of each individual at each timestep, take into account the spatial localisation of each individual, but need much more computing power. to add coarse grained spatial localisation to our model, we partitioned the territory in sub-regions where one instance of a population-centred ssa is run. these instances use the same timestep and are synchronised. the interactions between sub-regions are modelled by taking stochastic samples of individuals that travel between sub-regions. this is done at a higher time scale since such travelling is less frequent than the travelling inside the original sub-region. most of the individuals that travel go back in their home sub-regions after a variable period of time. thus, the population of each sub-region remains approximately the same, although people enter and leave the sub-region. if this is not taken into account in the model, the population of each sub-region may tend to become the same as time goes on. we describe in the next section how this constraint is implemented in our model. the territory studied is partitioned in two levels of geographical organisation: region and sub-region. a region contains at least two sub-regions, a sub-region belongs to only one region and all the territory is covered (partition). in our case study, france, the first level is the administrative région, each one containing from two to a dozen départements. there are régions and départements in france. of course this can be applied to any partition of a territory. for example in england we could use the nine regions for the first level, and the ceremonial counties and greater london for the second level. the population is divided into four age slices: to years old, to years old, to years old and over years old [ ] [ ] [ ] . each of these four sub-populations has its own values for the population parameters (infection immunity, travelling rate, etc.). we used one instance of a population-centred simulation process for each sub-region, with a one hour timestep. the simulation of the upper level (region) uses a bigger timestep, one day, and mainly processes the people which are travelling to another sub-region. thus, the population distribution is supposed homogeneous inside each sub-region, but can be heterogeneous at the region level and therefore at the level of the entire territory. depending on the age, and except for ill or hospitalised people, each day, people have a probability to travel from their homeplace to some place else either belonging to the same sub-region (local travel) or to another region (remote travel). these probabilities are part of the population parameters mentioned earlier. of course, quarantine type control measures forbid any kind of local or remote travel; people must stay in their respective homes sub-regions. the number of people of each age slice leaving their home sub-regions is a stochastic sample (or averaged value for the deterministic continuous solver) of a percentage of the population of this sub-region. for local travel, they are scattered according to the relative population of each sub-region belonging to their region. the more populated sub-regions attract more of the travellers. for remote travel, people go from their home-regions to the most populated sub-regions of the other regions, where airports and train stations are. the same method is used to dispatch the travellers according to the relative populations of their destination sub-regions. this way of computing how many individuals travel and where they go is a simple way to maintain constant the density of population of each sub-region. the sub-region population-centred model is a variant of the widely used susceptible, exposed, infectious and removed model. we added two states: hospitalised and deceased. the exposed and infectious states have slightly different meanings in our model; they have been renamed to asymptomatic and ill ( figure ). unlike ill people, who show symptoms of the disease, recently infected people are asymptomatic hosts, but both of them are infective. hospitalised patients are also contagious, but to a lesser extent because they are confined inside the hospital. the three red dotted arrows in the figure indicate the potential sources and targets of the infection. we have assumed that people in recovered state are immune to the virus and therefore cannot be reinfected [ ] . an incubation period of approximately five to six days before the apparition of the first symptoms has been observed [ , ] . in consequence, in our model, asymptomatic people are subdivided into six subcategories according to the number of days since contamination. a large majority of cases, around %, present a mild form of the disease which is probably even not reported. the other cases need hospitalisation, and among them, from % [ ] to more than % [ ] present severe forms wherein patients need to be admitted in icu. the duration of the disease, after the incubation period, depends on the age of the patient an on the severity of the form of the disease. in our model it has been set to a maximum of days, and therefore we have subdivided the ill (resp. hospitalised) people into at most subcategories according to the number of days since the apparition of the first symptoms (resp. the date of the hospitalisation). people with mild infections will recover after a stochastically variable period of time ( to days) that depends on their age. the severe form of the disease is (stochastically) lethal according to a rate also varying with the age of the patient. the deterministic solver uses fixed average values. all these rates, probabilities and average durations are parameters of the model. their values came or were inferred from observed statistics of real cases. as mentioned before, the simulation algorithm uses a one hour timestep. it mainly computes in a stochastic way the state vector: i.e., the number of people that is in each state and subcategory, at each timestep. there are four state vectors, one for each age slice. of course these four vectors are not independent since whatever their age is, contagious people can infect susceptible people regardless of their own age. basically, from the value of the state vector at time t, the process computes the new value of the state vector at time t + τ (here τ = h). thus, starting from a known initial value of the state vector at time t = , we can obtain its value at any time (t = t end ) > by iterating this process until t end is reached, or until a specific value of the state vector is reached. pandaesim automatically stops the simulation when there are no more infective people. our model assumes that people have uniform daily routines. without specific measures, the daily schedule begins at o'clock in the morning for work (or school, university, etc.) with the use of public transportation for one hour. next comes staying at work three hours, followed by a two-hour midday break, four hours in the afternoon at work, another hour in public transportation to go back home and the remaining hours at home. we defined four possible environments, each one having its probability of contagion: home, public transportation, workplace and restaurant. these parameters have default values that reflect the local concentrations of people: very low at home, higher at work and restaurant and much higher in public transportation. to reduce the number of parameters we used the same value for the workplace and the restaurant. many kinds of measures can be used to slow down the propagation of the epidemic; we implemented two examples of such measures: . soft quarantine: people do not use public transportation at all and do not go to restaurants during the midday break. . full quarantine: this corresponds to what actually happened in france; people were confined at home except for a one hour stroll per day in low populated areas (public parks, forests, etc., were forbidden). again, to reduce the number of parameters, we assumed that the probability of contagion during the stroll was the same as at work. this also allowed us to take into account errands made to get food in more populated places such as groceries or supermarkets. starting from an initial state (number of contagious people in each sub-region), the simulation algorithm iterates the following process at each timestep until either the epidemic ends or the maximum duration of the simulation is reached (defaults to days). . first, the infection rate at time t, i rt (t), is computed as the product of the global daily rate of infection, g dri (t), by the infection factor of the current location (home, workplace, public transportation) l in f (t). this infection rate i rt (t) is used the same way the propensity is in the standard ssa. then, for each of the four age slices the deterministic continuous solver computes the average number of individuals of that age that will go from susceptible to asymptomatic state, avnew asympt , as the product of the population in that state and the infection rate at time t: the stochastic discrete solver (sds) computes stochastic integer numbers such that, on the long run, they will average to the same values as the continuous solver. even when the population is an integer number of individuals, this product, avnew asympt , is generally a floating point number because the infection rate is itself a floating point number. this number has an integral part (≥ ) and a fractional part (between and ). the (discrete) number of new asymptomatic hosts is then computed as the integer part of the average number, plus if a uniform random number taken into the interval [ . . . ] is below the fractional part: as the difference is . on the average, the higher the value is, the lower the relative impact of this stochastic discretisation becomes and the result is equivalent to a discrete averaged approach. conversely, the lower the value is, the more important the stochastic discretisation becomes. this mechanism allows the simulator to automatically choose the best strategy to adapt to the value range of the population [ ] . . finally, when the current time indicates the beginning of a new day, t ≡ (mod ), individuals in each state either remain in the same state but shifted by one day, or change to another state. all the states transitions are computed stochastically by the sds (or deterministically by the dcs) using the method described earlier. • the population in the asymptomatic state that has on average reached the / day limit is moved to the first day of the ill state. • according to the illness duration by age slice parameter, a proportion of the population in the ill state is moved to the hospitalised or to the recovered state. the others remaining in the ill state one more day. • according to the disease severity by age slice parameter, a proportion of the population in the hospitalised state is moved to the deceased or recovered state. the others remain in the hospitalised state one more day. the global daily rate of infection is then simply computed by multiplying the constant of propagation of the virus, k prop , by the proportion of the total contagious population: by fitting the simulation results after the beginning of the lockdown to the data gathered from hospital statistics, we empirically found a good estimation of k prop for the sars-cov- to . . we think that using pandaesim to model another type of epidemic, only this constant, along with the severity parameters, needs to be changed. we applied our simulation tool to the sars-cov- epidemic in france. we used the partitions of région and département in the country for the regions and sub-regions of our model. most of the parameters we used were gathered from the literature and statistical data made available by the french government. a few others were obtained empirically, mainly the number of contagious people in each région at the beginning of the simulations, and the constant of propagation of the sars-cov- . the per-age values of the percentage of lethality [ ] , illness duration and percentage of local and remote travellers are shown on table a , the various rates of contamination on table a , and the initial number of contagious people in each département on table a in appendix a. in order to test our population-centred algorithm, we first ran simulations without countermeasures and without any travel possibility, either local or remote. these simulations were run using successively the stochastic discrete solver and the deterministic continuous solver. when the initial number of contagious people was relatively high, for example, in the val-de-marne sub-region ( ), the results for both solvers were nearly identical: deaths for the average of stochastic runs and deaths for a deterministic run (figures and ) . the standard deviation for these runs went from ≈ at the beginning of the simulations (with a few tens of deaths) to ≈ at the peak of the infection (a few thousands of deaths), and then ≈ at the end. the same kinds of results appeared for the ill people with the maximum value of the standard deviation of ≈ reached on the th day, with , ill people. on the other hand, when the initial number of contagious people was low, as in loiret ( ), the dcs did not find any deaths, whereas runs of the sds showed two distinct behaviours; of these runs showed the same results as the dcs, no deaths at the end of the epidemic. the other runs took another direction leading to deaths on average with a standard deviation of ≈ ( figure ). the reasons for this apparent inconsistency will be explained in the discussion section. using the countermeasure applied in france (lockdown) the simulations showed us retrospectively that the probable date whereat there was a total of contagious people in france (beginning of the simulations) was approximately the end of january . this correlates with the period of time when the first deceased person was reported ( january). the view of the main window of pandaaesim shown on figure displays the real numbers of deceased people in each département. the map shown on figure displays the mean values of runs of a stochastic simulation. the overall results are very close, , for the real statistics and , for the mean value of the simulations. the département by département results are also fairly close, except for a few départements, but the orders of magnitude are more or less identical. to determine whether there is a form of convergence of stochastic trajectories to average values, we ran hundreds simulations and computed the mean value of the number of deaths (and of the other states) at each time step, in each département. the results showed no unique limit values, but the averages obtained with many runs stayed inside a range of values near the real statistics. we also ran pandaaesim using the deterministic continuous solver with the same parameters. the results were completely different: the epidemic ran only for days ( to weeks less) and reported deaths (figure ) , far from the , obtained with the stochastic simulations. the results département by département are also very different, with more than half the départements showing no deaths at all. again, probable reasons for this inconsistent behaviour are proposed in the next section. we developed a hybrid model and simulation programme derived from standard models and simulation techniques widely used in the fields of epidemic propagation and biochemistry. our approach used an original variant of the gillespie ssa with tau-leaping, where the inner algorithm can be easily switched from stochastic discrete to deterministic continuous. this allowed us to compare these two methods of simulation. to test our approach we applied it to the sars-cov- epidemic in france, for which relevant data were available. we also tested the consequences and the efficiency of the lockdown countermeasure applied in france for days. in order to gain spatial localisation but with an efficient population-centred algorithm where the population was supposedly being homogeneous, we partitioned the territory into relatively small units for which an instance of the population-centred simulation was run. the movements of populations between these units were taken into account at a higher scale, with a larger timestep. we first tested one instance of our population-centred algorithm, where no countermeasure was used. using each method (sds and dcs) with the same parameters values, we compared the results in two different situations: (i) with a moderately high number, and (ii) with a very low number of initially contagious people. when the numbers were relatively high, the results of both methods were very similar. this was not surprising because at each timestep the absolute value of the increment computed by each method must be significantly higher than , and the stochastic rounding to the inferior or superior integer cannot be relatively very far from the floating point value computed by the continuous method. however, when the numbers are low, the absolute value added at the next timestep is only a bit higher than , and therefore the stochastic rounding to or to drastically changes the future trajectory. this is particularly important in this very case where the populations experience an exponential growth. this may look like chaotic behaviour since a small difference in initial conditions can lead to very different futures, but when the numbers grow, the importance of this switch effect is dampened. we used many simulations batches with initially only two contagious individuals in the sub-region. the results of , , and simulations showed approximately the same proportions of cases, ≈ %, ending with no death at all, while the rest of the batch converged to approximately deaths. the same model using the dcs show no death at all. we think this behaviour is a consequence of a bifurcation due to the high non-linearity of the system. when the number of contagious individuals is below a certain threshold, the contagion tends to fade, but if this number goes over the threshold, there is a kind of positive feedback that increases it until a large enough part of the total population is removed. if we assume that the initial number of contagious individuals in our example ( ) is below the threshold, the result shown by the dcs is therefore correct. due to both its discrete increments and its stochastic behaviour, the sds can sometimes compute a trajectory that goes above the threshold and switches the other way. in order to deepen the study of this bifurcation phenomenon, we have tried to find the approximate value of the threshold. first we used the dcs with the initial number of contagious individuals varying from to . no deaths were found up to ; then deaths from to ; and deaths for and above. then we did the same tests with sds runs, counting the number of runs leading to zero deaths, and in the other case, the average number of deaths. with initially to contagious individuals, the number of runs leading to no deaths decreased from to ; with six and above initially contagious individuals no more simulations lead to zero deaths. for all the runs not leading to zero deaths, the average number of deaths was ≈ . the threshold for the sds is somewhere below . as expected, this value is very low. then we tested the whole simulator with all the population-centred processes, running independently for timesteps in each sub-region and then synchronised by exchanging a portion of each population either stochastically or deterministically. again, depending on the type of solver chosen and for the reasons mentioned earlier, the results were different but not by too much. with the number of people travelling from a given sub-region being a (small) fraction of the total population of this sub-region, the consequences in terms of infection spreading are very dependent on the value itself: less than , it is amplified by the stochastic processing, or else smoothed with the continuous calculation. both global results and sub-regions' local results were found to be very similar using the two methods. this can be explained by noticing that sub-regions with low initial contagious populations "benefit" from the migration of contagious people from more populated sub-regions, and as no countermeasure is applied, the number of contagious people grows rapidly over the threshold. the main difference appears in the shape of the nglobal curves: the deterministic solver showed a bigger dependency on the propagation effect ( figure ). since the dates sub-regions had their peaks of contamination were very different, the propagation effect was slower. although the global number of deaths is approximately the same ( , for the dcs, , for the sds) the slope of the curve obtained with the sds is steeper than the one obtained with the dcs (figure ). this can be explained by the relative sequentiality of the infection peaks showed by the continuous solver, whereas with the stochastic solver all the peaks are almost simultaneous and therefore the resultant is higher. for our last test, we set the simulator with the equivalent of the lockdown countermeasure used in france. the effect of this countermeasure was to decrease the number of contagious people, and while the sds gave results that correlate with the real statistics ( figure ), the dcs did not work well mainly because the initial number of contagious people was too low to be taken into account (figure ). more than half the départements did not show any death and therefore the total number of deaths was largely underestimated. we speculate that if we start from an initial state where there are enough contagious people in most sub-regions, it is very likely that the dcs will yield reliable results. this study gave us the opportunity to compare two different methods to get the trajectory of a complex system. at the beginning we were confident that they would yield very similar results, but facts proved us wrong. the reasons that caused the inconsistency of the behaviour of the stochastic discrete algorithm on the one hand and of the deterministic continuous algorithm on the other hand, lead us to be more confident in the stochastic approach for the simulation of this particular epidemic spreading model. more generally, with this type of model, an exponential growth phase is very sensitive to any variation, even small, in the initial values, and to artefacts, or calculation errors, and can therefore sometimes exhibit chaotic behaviours. nevertheless, this hybrid approach, a mix of an efficient population-centred process that plays the role of an agent in a multi-agent system, seems very promising. the stochastic simulations' results were very similar to the real statistics gathered from hospital data. future works could include improvements to the simulator such as the implementation of other types of countermeasures, the use more accurate methods to model the behaviour of individuals and the use different types of sub-regions to reflect their diversity. in this study we supposed no possible reinfection, so the epidemic effectively stopped after certain amount of time. although simplifying the model, this assumption forbids the possibility of modelling other waves of infection. recent publications discussed the consequences of different transmission scenarios, with and without permanent immunity, that can lead to multiple waves of infection [ ] . an interesting perspective would be to include in our model a probability of reinfection in order to test the effectiveness of countermeasures. funding: this research received no external funding. acknowledgments: many thanks to martin davy at sys diag, for the early version of the parameter dialog box, and the gathering of information about the sars-cov- . the authors declare no conflict of interest. the following abbreviations are used in this manuscript: in order to fit the simulation results to the real statistics, we estimated the number of asymptomatic hosts in each sub-region (départements) at the beginning of the simulations (table a ) . per-age values of the percentage of lethality (extrapolated from [ ] ), illness duration, and percentage of local and remote travellers (table a ). rates of contamination according to the location, percentage of hospitalised patients who can infect healing people, and proportion of severe form of the illness (table a ) . first cases of coronavirus disease (covid- ) in france: surveillance, investigations and control measures portant réglementation des déplacements dans le cadre de la lutte contre la propagation du virus covid- . legifrance the effect of human mobility and control measures on the covid- epidemic in china an investigation of transmission control measures during the first days of the covid- epidemic in china on the definition and the computation of the basic reproduction ratio r in models for infectious diseases in heterogeneous populations preliminary estimation of the basic reproduction number of novel coronavirus ( -ncov) in china, from to : a data-driven analysis in the early phase of the outbreak early estimation of the reproduction number in the presence of imported cases: pandemic influenza h n - in new zealand early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia novel coronavirus -ncov: early estimation of epidemiological parameters and epidemic predictions nowcasting and forecasting the potential domestic and international spread of the -ncov outbreak originating in wuhan, china: a modelling 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an emergency response cluster of covid- in northern france: a retrospective closed cohort study the french connection: the first large population-based contact survey in france relevant for the spread of infectious diseases cmmid covid-working group, estimating the infection and case fatality ratio for coronavirus disease (covid- ) using age-adjusted data from the outbreak on the diamond princess cruise ship estimating the asymptomatic proportion of coronavirus disease (covid- ) cases on board the diamond princess cruise ship reinfection could not occur in sars-cov- infected rhesus macaques the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application serial interval of covid- among publicly reported confirmed cases projecting the transmission dynamics of sars-cov- through the postpandemic period this article is an open access article distributed under the terms and conditions of the creative commons attribution key: cord- -lvdl puw authors: lardon, zélie; watier, laurence; brunet, audrey; bernède, claire; goudal, maryvonne; dacheux, laurent; rotivel, yolande; guillemot, didier; bourhy, hervé title: imported episodic rabies increases patient demand for and physician delivery of antirabies prophylaxis date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: lvdl puw background: imported cases threaten rabies reemergence in rabies-free areas. during – , five dog and one human rabies cases were imported into france, a rabies-free country since . the summer event led to unprecedented media warnings by the french public health director. we investigated medical practice evolution following the official elimination of rabies in ; impact of subsequent episodic rabies importations and national newspaper coverage on demand for and delivery of antirabies prophylaxis; regular transmission of epidemiological developments within the french antirabies medical center (armc) network; and armc discussions on indications of rabies post-exposure prophylaxis (rpep). methodology/principal findings: annual data collected by the national reference center for rabies nrcr ( – ) and the exhaustive database ( – ) of armc were analyzed. weekly numbers of patients consulting at armc and their rpep- and antirabies-immunoglobulin (arig) prescription rates were determined. autoregressive integrated moving-average modeling and regression with autocorrelated errors were applied to examine how – episodic rabies events and their related national newspaper coverage affected demand for and delivery of rpep. a slight, continuous decline of rabies-dedicated public health facility attendance was observed from to . then, during the summer event, patient consultations and rpep and arig prescriptions increased by %, . % and . %, respectively. moreover, elevated medical resource use persisted in , despite communication efforts, without any secondary human or animal case. conclusions: our findings demonstrated appropriate responsiveness to reemerging rabies cases and effective newspaper reporting, as no secondary case occurred. however, the ensuing demand on medical resources had immediate and long-lasting effects on rabies-related public health resources and expenses. henceforth, when facing such an event, decision-makers must anticipate the broad impact of their media communications to counter the emerging risk on maintaining an optimal public health organization and implement a post-crisis communication strategy. media-communicated health alerts are being used more-and-more frequently by public health decision-makers to prevent consequences of a sudden event, such as, emerging and episodic zoonotic diseases. the medical community must now consider these communications to be preventive intervention tools for public health officials [ ] [ ] [ ] . obviously, as during any effective health intervention, undesired effects may also occur, such as rapidly rising numbers of potential cases to treat, leading, in turn, to health-resource saturation, especially if the pathogen involved is rare [ , ] . rabies is a viral encephalitis [ ] that is considered to be a reemerging zoonosis throughout much of the world [ ] . in western europe, rabies in non-flying terrestrial mammals was a well-known illness that has now become a rare disease, because many countries have succeeded in eradicating it. the major risk of rabies is now due to translocation of infected animals, mainly dogs, from rabies-enzootic areas and humans with rabies infection acquired abroad [ ] . although untreated rabies is invariably fatal, death can be avoided by proper administration of rabies postexposure prophylaxis (rpep), e.g., antirabies vaccine, with or without antirabies immunoglobulins (arig), before disease onset [ ] . thus, rapid identification of individuals potentially exposed to rabies is critical and media alerts can be extremely useful to identify people who were in contact with the rabid animal. in france ( , , inhabitants, , km ), primary health-care management of patients seeking rpep is delivered through an official national network of antirabies medical centers (armc), which are distributed throughout the country. rpep is administered, predominantly according to the zagreb schedule, to people bitten by an animal suspected of being infected with rabies or exposed to its saliva. clinicians conduct a risk assessment for each exposed patient, and decide to administer rpep according to the general recommendations, epidemiological data and grade of the bite [ ] . the french network for rabies prophylaxis provides exhaustive national data collected by armc [ ] , and laboratory diagnoses of humans suspected of having rabies [ ] and animals suspected contaminating humans. from to , a period during which rabies was endemic in french foxes, more than , animals were diagnosed as rabid. in , france was declared free of rabies in non-flying terrestrial mammals based on world organisation for animal health (oie) criteria and, as a consequence, the number of rpep began to decline progressively. however, in summer , one imported rabid dog generated unprecedented media communications by the public health director, whose official press release, dated august , warned, ''at least, nine people are at risk of death and are actively and intensively being sought by the health authorities…'' during this episode, antirabies vaccine stocks in armc were almost exhausted, leading to a temporary marketing license for the multidose verorab vaccine (sanofi pasteur), which had not previously been authorized in france. that arig supplies were dangerously low is illustrated by the postponement of arig injections in some armc until day after starting rpep [ , ] for several patients. controlling rabies reintroduction and communicating the risk of rabies spread remain a challenge to public health officials in rabies-free areas. in this study, we analyzed why and how the french rabies-control organization became so oversaturated. in particular, we examined the impact of newspaper reports on the numbers of patients consulting at armc, and their rpep and arig prescriptions. this research has complied with the french national guidelines and institut pasteur policy. the analysis of data collected by the national reference center for rabies (nrcr) from the amrc was done anonymously and approved by the commission french veterinary and human authorities work in close collaboration to detect cases and organize the medical responses to rabies (figure ) , with a territorial network of veterinary services and armc disseminated throughout continental france, in ( figure ). on the one hand, each animal responsible for human exposure is confined under veterinary surveillance. if dead and for whatever the reason, diagnostic laboratory tests are conducted at the nrcr, institut pasteur, paris, france. on the other hand, armc are the only primary care centers allowed to prescribe rpep. for each patient, a standard case-report form (table s ) is systematically filled out describing important epidemiological features, such as geographic location, consultation date, type of exposure, animal species, contact date with the animal, medical decision concerning rpep. based on the data collected by armc, annual reports are written, which describe the patients visiting armc and those receiving rpep (http://www.pasteur.fr/sante/clre/cadrecnr/rage/rageactualites.html). our analysis of the behavior patterns of patients consulting armc, and the rpep and arig prescribed to them between and was based on those annual data. among the french armc, systematically entered their data into the nrcr database between and . the following statistical analysis is based on the exhaustive weekly information provided by these armc. the armc network also constitutes an effective communication infrastructure coordinated by the nrcr, including conference calls and regular exchanges of information via the internet. when rabies is suspected in a human, biological specimens are sent to the nrcr. articles on rabies-related news published in three major national daily newspapers, le monde, le figaro and libération, were retrieved from the french association for auditing media circulation: an on-line service: http://www.factiva.fr. weekly numbers of patients consulting at armc, as a function of the date each was in contact with a potentially rabid animal, were used to construct times series. autoregressive moving average (arma) [ ] modeling was used to determine the significance of event-associated modification of armc weekly patient numbers and its duration. because several known events could have affected the series, a step-by-step procedure was undertaken [ , ] . before the onset of event # , trend and/or seasonality were estimated and removed, so that the time series was obtained in a stationary mode and, autoregressive integrated moving-average (arima) modeling was done using box-jenkins procedure from sas/ets [ ] ). the model was then used to predict armc consultations and their % confidence intervals ( % ci). an event was considered to have an impact when the number of consultations during consecutive weeks exceeded the upper % ci. observed values were then replaced by forecasts, to obtain analyses of the subsequent weeks. similarly, consecutive weeks within the % ci defined the end of the event's impact period. relative differences between observed and predicted values were calculated. for impacting events, the number of cases attributed to the event (ncae) was estimated by subtracting the prediction from the observed data during the impact period. an increase rate rabies has been eliminated from a large part of the european union and, thus, any newly imported cases threaten its reemergence. the - data derived from the exhaustive surveillance system implemented in france was analyzed to evaluate the impact on demand for and delivery of antirabies prophylaxis following introduction of five rabies-infected dogs and one infected human into this rabies-free area. using these events, we were able to illustrate the difficulties encountered in reducing the demand for and prescription of postexposure rabies prophylaxis in this context of episodic importation. moreover, we highlighted the need for public health decision-makers to anticipate the broad spectrum of consequences of their media communications and to prepare appropriate responses (in terms of health resources) to maintain an optimally effective public health organization after importation of an exotic infectious agent or its emergence. these responses are particularly relevant in the context of limited availability of rabies post-exposure prophylaxis, especially antirabies immunoglobulin. (ir) was then calculated as the ratio of the ncae/number predicted for the impact period. with the aim of evaluating potential repercussions of an identified event impacting on rpep prescriptions, two other time series were investigated: the weekly rpep rate, defined as the number of rpep prescribed/the number of consulting armc patients, e.g. rabies vaccine with or without arig; and the weekly arig rate, corresponding to the ratio of the number of arig/the number of consulting armc patients. during the period associated with modified armc weekly numbers, weekly rpep and arig rates and mean numbers of consultations were analyzed using regression with autocorrelated errors to account for the regression residuals (arima procedure). to explore whether care provided by the armc might be influenced by experience in previous french endemic enzootic areas, we divided the country into three areas based on the french administrative regions: area , the former enzootic rabiesinfected-fox region from to ; area , a region that has always remained rabies-free, and area , the region where event # occurred ( figure ). all analyses were performed using r (www.r-project.org) and sas software. after the reintroduction of rabies into france in , the number of rabid animal cases increased to reach a maximum of , cases in [ ] , followed rapidly by a maximum of , rpep prescribed for , patients consulting at armc recorded in ( figure ). in , france was declared free rabies reemergence and antirabies prophylaxis www.plosntds.org of rabies in non-flying terrestrial mammals based on oie criteria [ ] and, as a consequence, the number of patients consulting armc and receiving rpep began to decline progressively to respective minima of , and , in ( figure ). however, the numbers of patients consulting at armc and given rpep suddenly rose in . therefore, - data were further investigated using arima modeling to describe in greater detail the trends observed. between january (week ) and december (week ) , five rabid dogs illegally imported from morocco and one rabies-infected human from gabon were detected in france. during the period examined, the first event # dog ( months old) was confirmed as being rabid in may (week ) and the second, event # dog ( months old) in september (week ); they entered france from morocco, months and weeks before their deaths, respectively. the human case (event # ) was a -year-old boy, who traveled from gabon and died months later, in october (week ) [ ] . event # , # and # dogs were diagnosed as being rabid, respectively, in february (week ), may (week ), and august (week ) [ ] . event # was a -month-old puppy, illegally imported by car from morocco to bordeaux, france, via spain, who died of rabies in august (week ); he was not officially vaccinated. between january and december , , rabiesexposed individuals in france (all patients exposed abroad were excluded from the analysis) consulted in an armc, among whom , had valid exposure dates and bite/contact locations. among them, , had valid consultation dates and , had valid treatment information (figure ). because the data presented -week seasonality, the time preceding event # was too short to be analyzed. in such a case, box and jenkins recommend using at least two seasonality periods to calibrate the model [ ] . data analyses concerning events # , # , # and # , corresponding to rabid dog importations, were simple and rapidly done, as these dogs had had no known contact with animals and humans other than their owners during their communicable risk periods. as a consequence, events # , # and # were not reported in the major national newspapers and were not associated with any significant increase of armc activity. in contrast, events # and # were reported in and published articles retained for this study, respectively, and significantly affected the numbers of patients consulting at an armc ( figure ). until event # (october ), the weekly number of patients consulting an armc declined significantly (slope = . ; p, . ), with -week seasonality that peaked during the summer ( figure ). in october , the weekly number of armc patients was significantly higher than the predicted number during the weeks surrounding event # (weeks - ), with an estimated ncae of (ir = . %, % ci = . - . ). furthermore, event # was followed by a significant flattening of the decreasing slope of armc activity ( . versus . ; p = . ). no rpep-or arig-rate modification associated with event # was observed. in the summer of (event # ), the weekly number of armc patients differed significantly from the predicted number during the weeks surrounding it (weeks - ). the total week number of additional armc patient load was estimated at , (ir = . %, % ci = . - . ) over the model predicted , ( figure ). during that period, the observed mean rpep and arig rates were significantly higher than those recorded during the period preceding event # , ir = . % and . %, respectively ( table ) . the slopes of the armc-consultation decline after week and before week were estimated at . and . , respectively; p, . . surprisingly, between weeks and , the mean rpep rate remained persistently and significantly higher rabies reemergence and antirabies prophylaxis www.plosntds.org than before the reference period, as did the arig rate, which was more than two-fold higher than before week ( table ). the increased number of patients consulting at an armc in response to the newspaper articles concerning event # peaked at the same time as the media coverage in the three different french areas defined according to their rabies experience ( figure a ). in area , the exposure dates reported by armc patients corresponded to the risk period coinciding with the dog's movements and infectivity, whereas in areas and , patients reported exposure dates more compatible with newspaper coverage than with the risk period ( figure b ). france progressively eliminated rabies in foxes and became rabies-free for indigenous non-flying terrestrial mammals in [ ] . consequently, use of public health facilities dedicated to the disease decreased steadily from until , suggesting a continuous impact of rabies elimination on related public health resources and expenses. however, the very mild decline of the - slope probably reflects the difficulties in convincing the public and adapting medical practice to the changing risk. although elimination of rabies in foxes reduced the number of rabid pets and other domestic animals, and thus exposure to rabies, pet bites continue. importation of rabid animals and infected travelers returning from abroad also regularly challenge the french public health organization of rabies control. therefore, the number of rpep prescriptions and the associated costs will not decline significantly until there is adequate assurance that the probability of a pet being rabid is sufficiently low that such therapy is not warranted, even when the pet's status cannot be verified [ , , ] . regardless of potential french specificities, public health decision-makers are obliged to consider such potential events and their ensuing demand on medical community resources when attempting to predict and maintain the efficacy of rabiescontrol policies even in rabies-free countries [ ] [ ] [ ] [ ] [ ] . among the six rabies events occurring during - in france, only two significantly affected armc activities and rpep rates. the human case imported from gabon in (event # ) was associated with enhanced armc activity during a brief period and also changed armc's declining activity, which had been observed since . the boy's demise was reported times in the newspapers, further confirming that ''death makes news'' for rare and acute diseases [ ] . in contrast, the illegally imported . rabies-exposure notifications to armc and numbers of rpep prescribed to exposed patients in france, france, - . these data are from the annual nrcr report (http://www.pasteur.fr/sante/clre/cadrecnr/rage/rage-actualites.html). doi: . /journal.pntd. .g rabies reemergence and antirabies prophylaxis www.plosntds.org rabid dog from morocco in august (event # ) had a significant and rapid impact on rabies public health resources. indeed, the critical shortage of prophylactic drugs resulted from the % ir of patients consulting at an armc with a . % rpep rate for those patients over weeks. this influx explains the bottleneck observed in armc. similarly, laboratory rabiesdiagnosis workload for animals increased by . % during the same period (data not shown). to comply with the threatened shortage of rpep and arig due to the cumulative effect of enhanced patient influx and their more frequent prescriptions, a specific communication strategy was established for the armc network to provide information concerning the evolution of the epidemiological situation and to recall the indications of rpep. this information was disseminated via the websites of the nrcr, the ministry of health (moh), the national institute for health surveillance and the ministry of agriculture, which were regularly updated as of august, fax on september, and phone conferences on and september. to complete this plan, temporary licensing of a multidose vaccine (verorab, sanofi pasteur) was accorded and arig injections were postponed, as necessary, in accordance with who guidelines [ ] . unfortunately, it was not feasible to quantitatively analyze the extent of that adaptation. however, rpep and arig never became completely unavailable. notably, the risk of a potential arig shortage in the event of an unplanned increase of demand or a limitation of supply is shared by many countries in europe and on other continents [ , ] . compared to similar events occurring during - in france, event # has several particularities. while only restricted contacts with humans (owners, neighbors…) were suspected for cases # , # and # , the event # dog traveled through southwestern france during the communicable risk period, and had been roaming unleashed at three large summer music festivals, each with at least , - , participants [ ] . according to immediate inquiries made by veterinary and medical services, this trajectory potentially led to extensive contacts between the rabid dog and humans and animals. therefore, the public health authorities' concern triggered extensive media alerts. first, the moh wanted to identify and contact each individual with confirmed contact with the event # dog. national and local authorities coordinated several news conferences and newspaper reports to inform the french rabies reemergence and antirabies prophylaxis www.plosntds.org population about the risk and recommendations concerning errant dogs in general, and how to react to potential exposure to a rabid dog. a european-wide alert was launched through the european warning and response system. second, beginning in early september , this intensive communication frenzy of newspaper articles heightened public awareness of the rabies risk. third, additional public concern might also have been heightened by controversies surrounding the crisis management. notably, event # occurred just before the annual opening of hunting season, in a strongly traditional hunting region. an initial decision was made to forbid hunting with dogs in the counties where the rabid dog had traveled during his infectious period. that restriction led to a passionate public debate, angering hunters and ending with hunting organizations successfully blocking the ban. fourth, public health authorities decided to eradicate freeroaming dogs. finally, press releases issued by the minister of rural affairs and the moh were contradictory concerning the implementation of mandatory antirabies vaccination of dogs and cats. the constant media attention drawn by these different players during event # may have contributed to enhancing the sense of rabies risk, thereby prompting people to associate dog bites with we only examined national newspaper stories available in factiva but not local newspaper reporting or television, radio and internet stories, and, thus, probably underestimated the global coverage of these episodes. in response to national newspaper coverage, people who are far from the event location can become concerned and start taking precautions as if they were in the affected area [ , , ] . this phenomenon is particularly well illustrated by event # , for which exposure dates reported by patients consulting at amrc in areas and corresponded to the period of newspaper coverage rather than to the risk-oftransmission period during the dog's movements. lastly, long-term modifications of armc activity and rpepand arig-prescription rates were observed. in particular, rpep and arig rates (arima study herein) and even those for had not yet returned to levels. this finding strongly suggests a persistent and unjustified heightened perception of the risk by individuals and physicians, even those specialized in rabies treatment, and this despite regular information provided by the nrcr to the armc network and a rapidly controlled situation with no recorded secondary animal and human cases during the following years. in conclusion, event # and its associated national newspaper coverage profoundly perturbed health services, with excessive consulting at armc and durably increased antirabies drug rates for several months, along with more animal diagnostic testing. this crisis highlighted a lack of experienced manpower and insufficient vaccine stocks. outbreaks of emerging and/or deadly infections, like severe acute respiratory syndrome [ ] [ ] [ ] [ ] [ ] , anthrax [ , ] and rabies (herein), have shown that media messages dramatically influence both the public's and health-care workers' perceptions of the risk with potential implications for health-care resources. our observations underscore to what extent, under such circumstances, public health decision-makers have to anticipate the depth and scope of potential consequences of emerging or reemerging infectious diseases and their related press communications, and the need to prepare appropriate responses to keep the public health organization effective. it also illustrated that, despite communication efforts implemented by the french public health authorities and messages released through the armc network, long-term modifications of armc activities and prescriptions were observed, further emphasizing that a post-crisis communication strategy is essential. table s case-report form for human exposure to rabies used in france. since , collection and dissemination of information are made by filling out questionnaires available at a centralized online site named voozanoo (http://www .voozanoo.net/tikiindex.php?page = what% s+voozanoo). found at: doi: . /journal.pntd. .s ( . mb doc) best practices in public health risk and crisis communication communicating the threat of 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application to s. bobimorbificans in france the theory and pratice of econometrics que penser de la rage en ? bulletin epidémiologique de la fox rabies in france la rage : une maladie encore présente en france! an imported case of canine rabies in aquitaine: investigation and management of the contacts at risk rabies postexposure prophylaxis in returned injured travelers from france, australia, and new zealand: a retrospective study rabies postexposure prophylaxis potential cost savings with terrestrial rabies control economics of human and canine rabies elimination: guidelines for programme orientation cost effectiveness of rabies post exposure prophylaxis in the united states rabies control in the republic of the philippines: benefits and costs of elimination rabies exposures, post-exposure prophylaxis and deaths in a region of endemic canine rabies death makes news: the social impact of disease on newspaper coverage is there a need for anti-rabies vaccine and immunoglobulins rationing in europe appropriateness of rabies postexposure prophylaxis treatment for animal exposures the power of the pen: medical journalism and public awareness what are the roles and responsibilities of the media in disseminating health information media effects on students during sars outbreak the impact of the sars epidemic on the utilization of medical services: sars and the fear of sars sars epidemic in the press responding to global infectious disease outbreaks: lessons from sars on the role of risk perception, communication and management representations of sars in the british newspapers anthrax-related panic is more dangerous than the disease anthrax : observations on the medical and public health response the authors thank all the armc personnel, who collected and send their data to the nrcr, for their contribution. we are grateful to janet jacobson for expert editing of the manuscript. conceived and designed the experiments: dg hb. performed the experiments: zl dg hb. analyzed the data: zl lw ab cb dg hb. contributed reagents/materials/analysis tools: lw mg ld yr dg hb. wrote the paper: zl lw ld dg hb. key: cord- - nqkej authors: lansiaux, Édouard; pébaÿ, philippe p.; picard, jean-laurent; son-forget, joachim title: covid- and vit-d: disease mortality negatively correlates with sunlight exposure date: - - journal: spat spatiotemporal epidemiol doi: . /j.sste. . sha: doc_id: cord_uid: nqkej the novel covid- disease is a contagious acute respiratory infectious disease whose causative agent has been demonstrated to be a new virus of the coronavirus family, sars-cov- . alike with other coronaviruses, some studies show a covid- neurotropism, inducing de-myelination lesions as encountered in guillain-barré syndrome. in particular, an italian report concluded that there is a significant vitamin d deficiency in covid- infected patients. in the current study, we applied a pearson correlation test to public health as well as weather data, in order to assess the linear relationship between covid- mortality rate and the sunlight exposure. for instance in continental metropolitan france, average annual sunlight hours are significantly (for a p-value of . × (− )) correlated to the covid- mortality rate, with a pearson coefficient of - . . this correlation hints at a protective effect of sunlight exposure against covid- mortality. this paper is proposed to foster academic discussion and its hypotheses and conclusions need to be confirmed by further research. the novel covid- disease is a contagious acute respiratory infectious disease whose causative agent has been demonstrated to be a new virus of the coronavirus family, sars-cov- . alike with other coronaviruses, some studies show a covid- neurotropism, inducing de-myelination lesions as encountered in guillain-barré syndrome. in particular, an italian report concluded that there is a significant vitamin d deficiency in infected patients. in the current study, we applied a pearson correlation test to public health as well as weather data, in order to assess the linear relationship between covid- mortality rate and the sunlight exposure. for instance in continental metropolitan france, average annual sunlight hours are significantly (for a p-value of . x - ) correlated to the covid- mortality rate, with a pearson coefficient of - . . this correlation hints at a protective effect of sunlight exposure against covid- mortality. this paper is proposed to foster academic discussion and its hypotheses and conclusions need to be confirmed by further research. keywords: covid- ; coronavirus; france; correlation; vitamin d; phototherapy; uv. la nouvelle infection au covid- est une maladie respiratoire infectieuse sévère dont l'agent causal a été identifié comme un nouveau virus de la famille des coronavirus , sars-cov- . comme les autres coronavirus, des études montrent un neurotropisme du covid- , induisant des lésions démyélinisantes comme dans le syndrome de guillain-barré. plus particulièrement, une note italienne conclue qu'il y a un déficit significatif en vitamine d chez les patients infectés par le covid- . . patients with the coronavirus pneumonia typically exhibit a fever, with temperature above degrees © and other symptoms such as dry cough, fatigue, dyspnea, difficulty breathing, and diarrhea . . furthermore, this diseases has a relatively high transmission rate as compared to other upper respiratory illnesses. as a result of this and other factors such as international travel and trade, the initial epidemic has turned into a pandemic in march , with hundreds thousands of individuals confirmed to be infected worldwide -and most likely millions of unreported cases . similar to other coronaviruses-caused illnesses , covid- infection has shown some amount of neurotropism [ ] [ ] [ ] , with lesions not unlike those of the guillain-barré demyelination or hemorrhagic necrotizing encephalopathy , . meanwhile, it has long been noted that in the case of guillain-barré syndrome, vitamin d deficiency, in relation with high latitude climates, is both a causal and a risk factor , . furthermore, a recent italian note has demonstrated a significant vitamin d deficiency in a cohort of covid- infected elderly women . therefore, it is important to assess the effect of vitamin d blood levels on covid- infection rate and disease course, as it may offer preventative and/or curative options in the context of the ongoing pandemic. specifically in the context of continental metropolitan france, the correlation between sunlight exposure and sars-cov- infection will be studied in this article, by using an adjusted pearson test applied to public health and weather data [ ] [ ] [ ] . we conducted a descriptive observational cross-sectional study in order to define a hypothetical relationship between sunlight exposure and sars-cov- infection. the source and targeted populations are the whole humanity in view of the ongoing covid- pandemic. the eligible population is constituted by the residents of metropolitan continental france. the study was conducted by a consortium of two data analysts, a md-phd specialized in radiology, and a medical student in clinical years. nexgen analytics had no role in making the decision to submit manuscript to the publication, nor did it receive any fee or compensation in the context of this work. the first author vouches for the data and analyses, as well as for the fidelity of this report to the study protocol. we gathered covid- -related data from various public health and social sources , . a parallel multiple group analysis was performed. we excluded the population from the non-metropolitan jurisdictions of france(guyane, mayotte, martinique, reunion, guadeloupe, etc.), due to ( ) the fact that their climates vastly differ from that of metropolitan france, and ( ) the substantially lower access to healthcare in these areas. moreover, albeit part of metropolitan france, the island of corsica was excluded from this study because of poorer access to healthcare there than on the continent. we chose to use covid- mortality rate as the primary variable to evaluate the role of sars-cov- infection in our hypothetical correlation. sunlight exposure was evaluated by using the average annual hours of sunshine exposure, as reported by that country's national weather service ("météo france") . our null hypothesis (h ) was the non-correlation between average sunlight hours at the locality (x) and covid- mortality rate (y). in order to assess the potential effect of confounding factors, we also considered ( ) finally, in order to further sustain our analysis, we also considered the confirmed covid- infection cases as well as the number of verified recovered covid- patients. we began by computing several descriptive statistics for each variable: arithmetic mean, sample variance, standard deviation and the corresponding confidence intervals (justified by having shapiro-wilk tested each of these variables). obviously unrelated to covid- mortality, the birth and death rates were kept off the analysis. furthermore, age was also eliminated from this analysis as the national statistics in this regard are provided in the form of age classes not directly usable in the context of pearson correlation analysis. all other variables were treated using the pearson correlation test, and the corresponding p-value are reported here in order to assess the statistical significance of these correlations. the this population was subsequently partitioned by region of residence (nb: "region" is the largest sub-national jurisdiction of france), as summarized in table . we note that none of the resulting subgroups was found to exhibit values significantly outside of their respective confidence intervals, per a manova-wilk test performed at the % significance level (table ) . the primary outcome of this analysis was the pearson coefficient between sunlight exposure and covid- mortality rate, for which we found a value of - . . with a corresponding p-value . * - , this allows us to reject the null hypothesis h ( we have shown via pearson correlation that sunlight exposure is significantly correlated (p-value: . * - ) covid- mortality rate in continental metropolitan france (table ) , which is the main outcome of this study. besides, we acknowledge an interesting secondary finding: namely, the protective effect of life expectancy (pearson r: . ; p-value: . * - ) and discuss it further as it appears counter-intuitive, as older age is already been broadly documented as being associated with worse covid- outcomes. however, we also note that in our sample life expectancy is strongly positively correlated with sunlight exposure (pearson r: . * (table ) , and possibly other unknown population confounding variables. nevertheless, our regression, linked with the hypothesized physiopathological mechanism , suggests a first order effect at least. we thus contend that the findings presented in our analysis should be taken into account, in order to envision possibly effective yet inexpensive diagnostic and therapeutic options against the novel covid- . our conclusions could easily be tested and further assessed by screening the prevalence of covid- infected among vitamin d deficient patients. in addition, in vitro cell studies and animal models could be of interest to test our statistical correlation and the physiopathological hypothesis. clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china clinical features of patients infected with novel coronavirus in wuhan, china. the lancet clinical characteristics of coronavirus disease in china epidemiologic and clinical characteristics of novel coronavirus infections involving patients outside wuhan, china hrct imaging features in representative imported cases of le potentiel neurotrope des coronavirus. médecine/sciences covid- -associated acute hemorragic necrotizing encephalopathy: ct and mri features. radiol guillain-barré syndrome associated with sars-cov- infection: causality or coincidence? the lancet neurologic manifestations of hospitalized patients with coronavirus disease pulmonary activation of vitamin d and preventive effect against interstitial pneumonia vitamin d defiency in patients with primary-immune mediated key: cord- - coxz l authors: souris, m.; gonzalez, j.-p. title: covid- : spatial analysis of hospital case-fatality rate in france date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: coxz l when the population risk factors and reporting systems are similar, the assessment of the case-fatality (or lethality) rate (ratio of cases to deaths) represents a perfect tool for analyzing, understanding and improving the overall efficiency of the health system. the objective of this article is to estimate the influence of the hospital care system on lethality in metropolitan france during the inception of the covid- epidemic, by analyzing the spatial variability of the hospital case-fatality rate between french districts. the results show that the higher case-fatality rates observed in certain districts are mostly related to the level of morbidity in the district, therefore to the overwhelming of the healthcare systems during the acute phases of the epidemic. however, the magnitude of this increase of case-fatality rate represents less than per cent of the average case-fatality rate and cannot explain the magnitude of the variations in case-fatality rate reported by country by international organizations or information sites. these differences can only be explained by the systems for reporting cases and deaths, which, indeed, vary greatly from country to country, and not attributed to the care or treatment of patients, even during hospital stress due to epidemic peaks. since the beginning of the epidemic, the case-fatality rate of covid- and the differences between countries have been the subject of many questions about national pandemic response policies and patient treatment. most studies on the lethality of the case-fatality rate (or lethality rate) is the ratio between the number of closed cases (i.e. recovered or dead) and the number of deaths due to the disease, it is estimated by the healthcare system based on the reporting of these two values. the case-fatality rate should not be confused with the mortality rate, which is the ratio of the number of deaths to the total population, or also with the morbidity rate, which is the ratio of the number of cases to the total population. mortality and morbidity rates depend on the extent of disease in a population, unlike case-fatality rates, which are normally calculated independently of the number of infected persons [por ]. the case-fatality rate of a disease in a population is an index of severity of the disease in that population, and of the capacity of the healthcare system to reduce mortality. in principle, this allows to compare the effectiveness of healthcare systems across regions or countries. the aim of this article is to analyze the effectiveness of the healthcare system in france in the context of the covid- epidemic. based on spatial differences in lethality, this study ultimately show that the case-fatality rates published by the international agency by country (may ) do not allow to compared the country one to the others. lethality depends on the intrinsic virulence of the virus but, unlike morbidity, it does not depend on its contagiousness. virulence comes from the reproductive capacity of the virus in the cell, its capacity for cellular degradation, and its ability to induce or not an innate or specific immune response. virulence is of purely biological origin and once the virus has entered the target cell where it will cause its pathogenic effect does no longer depends on environmental conditions outside the host. virulence is independent of the host population, but may change over time and space if there is a risk of natural mutation/selection of the pathogen. contagiousness characterizes the biological capacity of the virus to reach the target cell system of its host, and the ability to be transmitted from one individual to another. the efficiency of transmission depends largely on environmental conditions (e.g., climate, urbanization, population density, mobility), which can vary greatly from one country to another. in addition to the virulence of the virus, the case-fatality rate depends on biological risk factors and on population vulnerability (age structure, genetic factors, prevalence of co-morbidities, healthcare accessibility, etc.) as well as other factors related to the health system (equipment, capacity, staff, management, care of patients, effectiveness of therapies, patient management in a critical phase of the disease), and factors related to the detection and registration system for cases and deaths (clinical cases definition, detection, surveillance systems, case and death reporting). the evaluation of the case- fatality rate normally requires the detection and counting of all infected persons, irrespective of their level of symptoms (i.e. disease severity). when the population risk factors and reporting systems are identical, case-fatality rate evaluation represents an excellent tool for analyzing, understanding and improving the overall performance of the health system, particularly at the level of hospital units. studying the magnitude of differences in case-fatality rates between units also makes it possible to assess the impact of the quality of the health system on case-fatality. there are large differences in the case-fatality rates of covid- published by country (table ) or calculated directly from who data. these rates vary considerably, from less than . (thailand, australia, chile) to more than . (france, belgium, uk), with a mean at . and a standard deviation of . (who, may , , figure cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . in europe, the characteristics of populations (in terms of risk factor for covid- ) and health systems are quite similar, but the definition, detection and reporting of cases and causes of death can differ greatly from one country to another. some countries conducted significantly more detection tests and hospitalizations than others (table ) , resulting in differences in the protocols for patient management. the rate of testing performed (policy) and mortality rates (reporting) vary mainly according to the geographical extent of the epidemic within each country. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . the virulence of the covid- pathogen (sars-cov- virus) is assumed to be identical in all countries. in order to compare case-fatality rates across regions or countries (and thus analyze the effectiveness of healthcare systems), it is necessary, when calculating rates, to standardize population-related risk factors and to use the same definitions and enumeration methods to record cases and deaths. this is not the case for the current pandemic and discrepancies exist among the country systems. the objective of this article is not to estimate the actual lethality of covid- in france based on the rates published by the health authorities, but to estimate the influence of the healthcare system on lethality by analyzing the spatial variability of the hospital case-fatality rate (confirmed hospitalized cases and hospital deaths) in metropolitan france between districts (i.e. french départements). this analysis, limited to metropolitan france, makes it possible while it remains within the framework of the same system for defining and counting cases and deaths. we thus assume that this system of definition and enumeration was identical throughout france during the period ( march to may) corresponding to the first wave (inception) of the covid- epidemic in france. therefore the study focuses on the extent of spatial differences in the case-fatality rate in metropolitan france, and enable to highlight the relative differences between districts, as well as to analyze the causes independently of the system of definition and enumeration of cases and deaths, and also independently of the main biological risk factor of severity (age) after standardization on this factor. estimating the variability of the case-fatality rate attributable exclusively to hospital care of patients will then allow us to compare the case-fatality rate observed in metropolitan france with the one calculated for other countries. it will allow us to estimate whether the variability of the case-fatality rate due to the management of . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint patients in the acute epidemic phase can exclusively explain the significant differences in case-fatality rates observed between countries. this study is based on daily hospitalization and death declaration data by district in france and is accessible on the "santé publique france" website. (www.data.gouv.fr/fr/datasets/donnees-hospitalieres-relatives-a-lepidemie-de-covid- ) from march to may , , corresponding to days lockdown (i.e. quarantine) and the spread of the covid- epidemic in france. we also obtain demographic data by districts (source: population by age, insee, ), as well as data on the distribution of hospitalized cases according to age group ( -year age group) (santé publique france). this analysis was carried out on the districts of metropolitan france (figure ), while the french overseas districts and territories were excluded from the analysis for reasons of spatial analysis and mapping. the data were integrated into a geographic information system (savgis, ww.savgis.org) for analysis and mapping. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . severe and asymptomatic forms (which a fortiori do not cause deaths) -it is estimated that only . % of infected persons were hospitalized [sal ] -this overall lethality is necessarily much lower than hospital lethality, but it will be accurately calculated only at the end of the epidemic when the total number of positive cases (i.e. seroprevalence survey) will be available and the total number of deaths outside hospital due to covid- will be accurately assessed. all identified and hospitalized cases were tested positive (by rtpcr). all deaths counted were covid- associated. as of may , , not all hospitalized cases are closed since the epidemic is still ongoing: deaths counted at the beginning of the study period correspond to cases hospitalized but were not included in the study, and cases counted at the end of the period were not closed and no deaths from these cases were included in the study. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . belfort) (figure ) . the hospital mortality rate (not age-standardized) has the same spatial distribution. it varies from . per , (tarn-et-garonne) to . per , (territoire de belfort), with a mean of . (median . ) and a standard deviation of . . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . from the calculation of age-specific case-fatality rates, the slr is between . and . , with the mean at . and the median at (figure ) . in the following, we will consider only the slrs calculated with age-specific case-fatality rates that do not take into account the ile-de-france and grand-est regions. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . the spatial distribution of standardized morbidity rate (hospitalized cases) shows a significant spatial autocorrelation (moran index: . , p-value < - ), and this is expected for an infectious disease. the case-fatality rate shows also significant spatial autocorrelation (moran index: . , p-value < . ), and this is no expected. the analysis of the clusters clearly shows a clustering of high case-fatality rate values in regions of high morbidity (particularly the grand-est), and shows some cases of districts with high case-fatality rate values isolated in areas with low rates. the breslow & day significance test shows districts where the slr is statistically significantly different from , corresponding to districts with abnormally high (slr > , red) or abnormally low (slr < , green) case-fatality rates. the individual significance threshold is set at . , and for all districts at . to account for multi-testing ( figure ). . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. there is a correlation between the standardized hospitalization rate and the standardized case-fatality rate (bravais-pearson index= . ) (figure ), a correlation which increases ( . ) if we limit the calculation to districts whose slr is significantly different from (p-value < . ). to illustrate the increase of case-fatality rate with hospitalization rate, table gives the mean of the standardized case-fatality rate over the districts according to their standardized hospitalization rate. the average case-fatality rate varies from . for . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . the mapping of the hospitalization rate and the hospital mortality rate with the slr shows the spatial correspondence of these values ( figure ) . a typology combining hospitalization rates and case-fatality rates is proposed: low rates (values below the mean by less than one standard deviation), high rates (values . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . above the mean by more than one standard deviation), so as to represent four classes (low-low, low-high, high-low, high-high). the hatched areas represent those for which the slr is not significant (p-value > . ) (figure ). figure . combination of standardized hospitalization and case-fatality rates in four classes. the ratio between the rate of patients in intensive care and the rate of hospitalization gives in principle an indication of the severity of the patients in hospital. this hospitalization rate and severity rate show a weak negative correlation (r=- . ), indicating a decrease in the intensive care rate when the hospitalization rate is high. this trend may be due to the saturation of intensive care units. the relationship between hospitalization and severity could also be interpreted as a decrease in less severe hospitalizations in order to be able to manage more severe cases when the healthcare system is overloaded, which would result in an increase in lethality. nevertheless, in both cases, there is no correlation between the severity rate and the case-fatality rate (r=- . ), indicating that globally, the intensity of reanimation does not impact the case-fatality rate. finally, the severity rate does not have a spatial distribution corresponding to the increase in the hospitalization rate ( figure ) . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the standardized case-fatality rates (slr) of the districts in france ( . for tarn- et-garonne to . for the vosges district) remain in a ratio of . to . compared with the national average of . , calculated by excluding districts under stress in order not to take account of possible saturation of the care systems. the relationships between morbidity rates and standardized case-fatality rates in france show a correlation between these two indices, the average case-fatality rate for all districts being about % higher than the average rate calculated in the % of districts with the lowest hospitalization rates. it is therefore very likely that the increase in hospital tension over the period under consideration has increased the hospital case-fatality rate: for the districts with the highest hospitalization rates (essentially located in the grand-est and ile-de-france regions), the average case-fatality rate is per cent higher than the average for all districts, and per cent higher than the average for all other districts alone. it can be concluded that hospital case-fatality rates have increased the national average case-fatality rate by district from . to . . it can therefore be estimated that , deaths (out of the , deaths due to covid- in hospital in france from march to may , i.e. % of the total number of deaths) are due to the saturation of the health system in the grand-est and ile-de-france regions. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint there are clearly two risk profiles: / the districts where a high rate of hospitalization is coupled with a high case-fatality rate, and / the districts where a low rate of hospitalization is coupled with a high case-fatality rate. the first category probably results from an increase of lethality due to saturation of the health care system. the second category is probably linked to the opposite phenomenon: a low hospital case-fatality rate which would have led to an increase in lethality due to a local lack of healthcare access (e.g. medical deserts, poor hospital lethality preparation). it has also been noted that all these late districts are located in essentially rural areas. some districts in the south of france have both a very low rate of hospitalization and a very low case-fatality rate (gironde, dordogne, gers, pyrénées orientales), as a result of the low circulation of the virus and the effective response of the health system. another particular case, is the one of the bouches-du-rhône, which appears with a high hospitalization rate ( french average is therefore very significantly higher than the world average (p-value < - ). even if we consider only the average case-fatality rate calculated only for the french districts with the lowest hospitalization rates (thus not causing saturation of the health care system), this average is still very significantly higher than the international average (and the rates of most european countries, such as spain, . , greece, . , germany, . , etc.) (table ) . taking into account the quality of the healthcare system in france (table ) , it can be concluded that the difference between the case- . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . fatality rate calculated for france and the case-fatality rates presented using international who data is highly probably the result of a difference in the registration of cases and/or deaths and not due to the quality of health care. these differences in the counting of cases and/or deaths may be due to the hospitalization and screening policy specific to each country as well as the ability or willingness to hospitalize more non-severe forms, to the differences in case definition, or to insufficient quality of the system for detecting and reporting cases and deaths. this study shows that the higher case-fatality rates observed in france in certain districts during the first wave of the covid- epidemic (data from march to may ) are mostly linked to the level of morbidity in the district, and therefore to the congestion of the healthcare systems during the acute phases of the epidemic. when the hospitalization rate is low, high case-fatality rates concern rural districts and could be linked to health care access in these districts. however, the increase in the standardized case-fatality rate due to exceptional situations during epidemic peaks represents less than % of the average case-fatality rate per district in france, and the hospital case-fatality rate without these districts would be reduced from . to . . this increase cannot therefore explain the extent of the difference observed between the average case-fatality rate in france and the average of the rates reported for all countries by international organizations or information sites (who, wordometer, etc.). these differences probably stem from the reporting of cases and deaths, which is uneven from one country to another, and not from the care or treatment of patients during hospital stress due to epidemic peaks. real estimates of mortality following covid- infection [mor ] morteza abdullatif khafaie, fakher rahim. cross-country comparison of case fatality rates of covid- /sars-cov- . osong public health and research por ] porta m. a dictionary of epidemiology. th ed an empirical estimate of the infection fatality rate of covid- from the first italian outbreak using early data to estimate the actual infection fatality ratio from covid- in france estimating the burden of sars-cov- in france key: cord- -ao df q authors: chire saire, j. e.; oblitas cruz, j. f. title: study of coronavirus impact on parisian population from april to june using twitter and text mining approach date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: ao df q the fast spreading of coronavirus name covid , generated the actual pandemic forcing to change daily activities. health councils of each country promote health policies, close borders and start a partial or total lockdown. one of the first countries in europe with high impact was italy. besides at the end of april, one country with a shared border was on the top of countries with more total cases, then france started with its own battle to beat coronavirus. this paper studies the impact of coronavirus in the poopulation of paris, france from april to june , using text mining approach, processing data collected from social network and using trends related of searching. first finding is a decreasing pattern of publications/interest, and second is related to health crisis and economical impact generated by coronavirus. officially declared as a global pandemic by the world health organization (who) on march , , covid- outbreak (coronavirus disease) has evolved at an unprecedented rate. the covid- pandemic has resulted in over million confirmed cases and over , deaths globally. it has also sparked fears of an impending economic crisis and recession [ ] . social distancing, self-isolation and travel restrictions have lead to a reduced workforce across all economic sectors and caused many jobs to be lost. schools have closed down, and the need for commodities and manufactured products has decreased. in contrast, the need for medical supplies has significantly increased. all countries that have been affected by covid have followed a similar pandemic growth curve, where the number of cases of sars-cov- coronavirus infection continues to grow, and, as time goes by, together with prevention policies, the rate of contagion will start to decrease progressively until the situation is controlled. this has been observed in realities such as those of european countries, which shows that there is certain universality in the temporary evolution of covid- . this is demonstrated by the time lag graphs of infected populations confirmed in countries such as france, china and italy, which follow the same power law on average [ ] . in order to help public health and to make better decisions regarding public health and to help with their monitoring, twitter has demonstrated to be an important information source related to health on the internet, due to the volume of information shared by citizens and official sources. twitter provides researchers an information source on public health, in real time and globally. thus, it could be very important for public health research [ ] within the context of covid , users from all over the world may use it to identify quickly the main thoughts, attitudes, feelings and matters in their minds regarding this pandemic. this may help those in charge to make policies, health professionals and public in general to identify the main problems that concern everybody and deal with them more properly [ ] particularly, focusing on a densely populated region of france, we document evidence that the highest economic "indicators of precariousness," such as unemployment and poverty rates, lack of formal education and housing, are important factors in determining mortality rates for covid- . therefore, measuring what happens after having the pandemic under control is essential, and the economic issue is important to be monitored, since it goes hand in hand with public health policies for the containment of the pandemic [ ] , so that our study will help to show changes in issues that concern the french population at this stage. the actual paper uses data mining approach to perform an exploratory analysis of the dataset of brazilian patients of sao paulo state. the methodology to explore data is presented in section , the experiments and results in section . conclusion states in section , final recommendations and future work are presenten in section , . the conducted work follows a methodology inspired in crisp-dm [ ] . this methodology is explained in the next subsections, from collecting data, processing and visualization to support the study. twitter is a social network, where users can post/share ideas, opinions, thoughts about any topic. then, it is possible to collect text using twitter api(aplication programming interface). the parameters for accesing the data are: terms: covid , coronavirus -date collection: / / - / / -geolocalization: paris, france -language: french -radius: kilometers . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint a cleaning process is necessary to avoid characters with no meaning for the scope of this analysis. first, convert text to lowercase, remove french accents, remove non alphanumerical values. later, delete stopwords, i.e. articles, pronouns, etc. the scope of this paper is to analyze how reacted french population during the range of date: final week of may until third week of june. besides, know how was the perception around economy situation in paris, france. this step is important to know what kind of graphics will be useful to answer the questions related to the study. the collected date is textual then filtering, organizing it to show proper graphics that support analysis and let a better understanding about the situation in paris, france. cloud of words are useful to get a general overview, bar plots for frequency or histograms, and filtering process removing some terms can help to get a better view of terms. the present paper presents the description of dataset and results in the next subsections . , . . the results presents the interest of parisian inhabitants about health crisis originated by coronavirus and concern around econonomy topic. the collected data has the next features: all countries, including france, in response to 'flattening the curve', generated policies and rules for actions, such as border closures, travel restrictions and quarantine, which is a serious blow to one of europe's largest economies. these actions gave results, achieving a control of the epidemic, evidenced in the figure , where it is clearly observed that since may a constant control of this one was achieved. [ ] . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint a general ovierview of the collected is presented in figure , after fig. presents the hourly distribution of downloaded twitter posts. it is possible to appreciate that the process of downloading data recovered data from march to june , where clearly the interest evidenced in the web begins to decrease with respect to topics related to fear of the disease, which was very high in previous periods. this assessment is based on the discussion about fear of covid on twitter and the period in which the code to download the data was executed. the issues of fear of this pandemic have been related to issues of quarantine exhaustion, anxiety, depression and fear [ ] . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint it is necessary to remark there is a clear pattern of publication in population of paris from april to june(see ). people start interaction at h, continues during noon passing afternoon and decreases from - h. besides, considering image , duplicating number of june to have an estimation of the total number for this month. there is a decreasing pattern of publications. by the other hand, a small valley is starting to appear around - h, on may and june. helping the visualisation a cloud of words is presented in fig. , and , it can be seen the regions including words related to "corp lutter", "tue comment", "plus parisien", "plus personnes", "trump", these tweets reflect the early interesting around the coronavirus health global crisis on this social network. all the collected data were searched using the keyword "coronavirus". prior to the outbreak of covid- , people already relied on social media to gather information . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint and news, and since the outbreak in january , people in many countries have relied on social media like twitter to obtain information about the virus. april april may may may but along with this, the emergence of new causes of anxiety, as detected in the words associated "crise", "ouverture", "pleine crise", "criseéconomique" and "avant crise prix" (fig. ) , with this analysis, is evident, being the main finding the fear of an imminent economic crisis and recession in france. the covid- pandemic has had an unprecedented impact on the global economy as well as individuals' economic well-being [ ] [ ] the shock of the coronavirus pandemic and shutdown measures to contain it have plunged the global economy into a severe contraction in countries where the pandemic has been the most severe and where there is heavy reliance on global trade, tourism, commodity exports, and external financing. according to world bank forecasts, the global economy will shrink by . percent this year. [ ] this is reasonable as france's leading newspapers already talked about the impact of covid on french economy and made efforts to try to understand the effect it would have, focusing on one of the most important sectors, which is tourism industry with all associated services [ ] , including impacts on both the supply and demand of travel [ ] . as a direct consequence of covid- , the . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august , . world travel and tourism council warned that million jobs in the global travel and tourism sector may be at risk [ ] . the economic recessions are estimated to affect significantly on the people mental health and wellbeing by magnitude the relative and attributable risks. research [ ] indicates a significant adverse effect of job loss and unemployment on mental health sufferings like depression, stress, etc. with this we can show that monitoring and using text mining techniques can detect changes in concerns and fears in the evolution of a population during and after the health emergency by covid . this, along with the widespread popularity of social media that will provide the public with a fast platform to measure trends [ ] , makes this technique an important public health tool, as it measures in a short time the continuous evolution of communication strategies generated by government institutions. the information analysis on twitter indicated by the detected rates can help to monitor the evolution of the interests of a population like that of france, within the phase of control of the outbreak of the current covid- pandemic, showing that public interest in fear of health issues decreased and new fears arose, such as the issue of economic crisis, which is relevant information to generate effective communication policies meeting the needs of a population within the framework of public health. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint for researchers interested to work with this approach, consider: -select a topic to study and check if social networks are a source for your work. every country has different number of active users and preferences about social networks. -consider to use some tool to get an overview of geographical zone before of using a data collection of some city/state. -remember languages has patterns about how writing(grammar) besides slang or common phrases are dependant of the location, then if you can find one collaborator from the zone of study, this will be very valuable to support the analysis. -involve more people to avoid bias for your own thoughts/ideas and of course, invite specialists around the topic of analysis, they will give you the key terms, intuition about what is useful or not and enforce the project. covid- , sars and mers: a neurological perspective analysis and forecast of covid- spreading in china, italy and france association of the covid- pandemic with internet search volumes: a google trendstm analysis top concerns of tweeters during the covid- pandemic: infoveillance study the role of economic structural factors in determining pandemic mortality rates: evidence from the covid- outbreak in france the crisp-dm model: the new blueprint for data mining coronavirus en francia: , casos y frontline nurses' burnout, anxiety, depression, and fear statuses and their associated factors during the covid- outbreak in wuhan, china: a large-scale cross-sectional study intersecting ethnic and native-migrant inequalities in the economic impact of the covid- pandemic in the uk e-commerce y su importancia enépocas de covid- en la zona norte del perú covid- to plunge global economy into worst recession since world war ii tourism and covid- : impacts and implications for advancing and resetting industry and research the socio-economic implications of the coronavirus pandemic (covid- ): a review this is how coronavirus could affect the travel and tourism industry covid- suicides in pakistan, dying off not covid- fear but poverty? -the forthcoming economic challenges for a developing country shindo, and scientific and technical advisory group for infectious hazards who. covid- : what is next for public health? the lancet perform a deeper analysis about topics related to main sectors: economy, social, health, education. invite collaborators, i.e. economist, sociologist, physicists, teachers to do a global analysis, how one sector can impact/influence to anthers as chain effect. key: cord- -jop rx authors: vignais, pierre v.; vignais, paulette m. title: challenges for experimentation on living beings at the dawn of the (st) century date: - - journal: discovering life, manufacturing life doi: . / - - - - _ sha: doc_id: cord_uid: jop rx “we can talk endlessly about moral progress, about social progress, about poetic progress, about progress made in happiness; nevertheless, there is a type of progress that defies any discussion, and that is scientific progress, as soon as we judge it within the hierarchy of knowledge, from a specifically intellectual point of view.” introduction of new exploratory methods such as biocomputing or bioinformatics and high-throughput screening, which involves the simultaneous processing of hundreds or even thousands of samples. this approach is in contrast with traditional biology, in which the research strategy is based upon the observation of effects obtained as a function of experimental parameters that are modified one by one. another aspect of modern times is that, with the irresistible trend in genetic manipulation towards a focus on human beings, certain areas of fundamental research are finding themselves locked into philosophical dilemmas that are matter for ethical and sociocultural consideration, and the subjects of fierce debate. instead of setting out to discover unknown mechanisms by analyzing effects that are dependent on specific causes, with some uncertainty as to the possible success of the enterprise being undertaken, which is the foundation stone of the bernardian paradigm of the experimental method, many current research projects give themselves achievable and programmable objectives that depend upon the means available to them: sequencing of genomes with a view to comparing them, recognition of sequence similarities in proteins coded for by genes belonging to different species, with the aim of putting together phylogenetic trees, synthesis of interesting proteins in transgenic animals and plants, analysis of the three-dimensional structure of proteins, in order to find sites that are likely to fix medicinal substances, and synthesis of molecular species able to recognize pathogenic targets. the facilities that are called into play include instruments that are often sophisticated, the performance of which, in terms of miniaturization, computerization and robotization, is far beyond that of apparatus that was in use a few decades ago. these facilities, applied to research into living beings, have entered the framework of a methodology that has been given the label biotechnology. proceeding handin-hand with applications that have become more and more meaningful in the domains of medicine, pharmacology, agronomy and animal husbandry, the biotechnological process has come to the fore as a new paradigm for the experimental method as applied to living beings. in addition to new discoveries, the driving forces behind biotechnologies are related to economic imperatives as well as the interest and support they receive from the political powers-that-be. the academic spirit that presides over fundamental science gives way to the entrepreneurial spirit that implements a rational programming of facilities and an efficient organization of scientific collaborations. as an example, the sequencing of the human genome, which includes three billion nucleotide base pairs, required the coordination of several dozen scientific teams around the world and the matching of several tens of thousands of results. research on dna provides a typical illustration of the way in which research has become divided, over the last few decades, between an approach and an interest that had previously been purely academic, and the increasing role of technology, which can be justified by the results that arise in the life of society at large, but which, because of these results, also gives rise to questions concerning how wellfounded some of these results are, particularly in the health domain. the experimental method, which had been confined to the laboratory, is now a matter for public debate. before it won acclaim, dna, which was isolated under the name of nuclein by johann friedrich miescher ( - ), at the end of the th century, had to undergo a series of structural evaluation tests that were spread out over the first five decades of the th century. an overall conclusion then came to the fore. dna is a polydeoxyribonucleotide that carries four cyclic bases, adenine, thymine, cytosine and guanine. each base is involved in the structure of a mononucleotide where it is itself associated with a sugar, deoxyribose, which is associated with a phosphate residue. dna was compared to a ladder, the rungs of which (mononucleotides) were linked by ester bonds between an acid group of a phosphate residue of a nucleotide and the free hydroxyl group of the deoxyribose of the following nucleotide. research committed them to this path. it was the curiosity of each, a new way of considering old problems, that led a few men and women to solve the problems of heredity." the middle of the th century saw an accumulation of experimental evidence showing that dna carries genetic information, and because of this, that it controls the transmission of hereditary characteristics: the proof provided in by oswald avery ( - ) , colin macleod ( - and maclyn mccarthy ( - of the transforming power of dna in pneumococcus, the highlighting by alfred hershey ( - ) and martha chase ( - , in , of the role played by bacteriophage dna as an infectious agent for bacteria, the revelation by erwin chargaff at the beginning of the s of the equivalence of molar concentrations of adenine (a) and thymine (t), on the one hand, and of cytosine (c) and guanine (g), on the other hand, in dnas arising from a multitude of sources, animal, plant and microbial, thus suggesting a complementary pairing of adenine and thymine, and cytosine and guanine. based on the pairing of a/t and c/g bases, the model of the double helix structure of dna, formulated in by james watson and francis crick, made it possible to understand the identical synthesis of double strands of dna by replication during cell division (figure iv. ) and, as a consequence, the conservation of hereditary characteristics in descendants. afterwards, it was found that the information contained in the dna base sequence determines the amino acid sequence in proteins. then the roles played by messenger rna and transfer rnas were elucidated, the former acting as a carrier of information between dna and the proteins being synthesized and the latter acting as double-headed adaptors, able to recognize nucleotide triplets (codons) in messenger rna and to specifically fix amino acids in order to position them on the ribosomes, the final result being the synthesis of a protein chain. in , the genetic code was deciphered. the veil of mystery that had covered the mechanism of the synthesis of proteins was lifted, and the decisive role played by nucleic acids in this synthesis was shown. later on, there were a few adjustments. although, in bacteria, proteins are coded for by a continuous sequence of nucleotide triplets in dna, in the s the surprising discovery was made that in eukaryotic organisms, genes are discontinuous and made up of coding dna sequences (exons) interrupted by non-coding sequences (introns). from the end of the s, françois jacob and jacques monod had postulated the existence of a dual determinism for protein synthesis and shown that, next to structural genes expressed as proteins, there are regulatory genes able to control the expression of the structural genes. the importance of the differential regulation of gene expression in cell differentiation in higher organisms was quickly recognized. from this point on it was possible to explain why a particular species of protein is more specifically expressed in a given tissue and another species of protein is more particularly expressed in another tissue, each type of tissue finding its specificity in its molecular components. this fantastic framework of knowledge, which was built up over a couple of decades, has been used as the foundation stone for the so-called central dogma of molecular biology, which explains the transcription of dna sequences into messenger rnas and the translation of messenger rnas into proteins and which, with only a few variants, is the same throughout the living world (figure iv. ) . the fascinating history of molecular biology was well described by james d. watson in molecular biology of the gene ( rd edition ). there are now many works concerning this subject and how it has progressed, including two well-documented books in french: the secrets of the gene by françois gros ( ) and histoire de la biologie moléculaire by michel morange ( ) . new strand a -double helix structure of dna and simplified representation of its self-replication. each strand of the parent molecule of dna acts as a matrix for the synthesis of a daughter molecule of complementary dna, in conformity with the rules of pairing: adenine (a) with thymine (t) and guanine (g) with cytosine (c). the double strands that appear are identical to each other and identical to the parent dna molecule. b -transcription of dna into messenger rna and its translation into an amino acid chain. diagram of gene expression in a eukaryotic cell. one of the strands of dna (the coding strand) has coding sequences (exons) and non-coding sequences (introns). it is said that the gene is split. the transcription of the exons, accompanied by their splicing leads to the formation of a messenger rna, the codons (nucleotide triplets) of which are translated into amino acids that are linked to each other by covalent bonds in order to form a protein chain. in prokaryotic cells (bacteria), the genes do not contain introns and are not split. a: adenine; c: cytosine; g: guanine; t: thymine; u: uracil. met: methionine; his: histidine; tyr: tyrosine; gly: glycine; phe: phenylalanine. interlinked with the epic rise of molecular biology, there was a succession of technical innovations that led to the synthesis of dna by chemical or enzymatic means, and to its being cleaved at specific locations, with the pieces that were obtained being joined together again . in , in geneva, werner arber (b. ) and daisy dussoix highlighted the restriction phenomenon, which involves the degradation of bacteriophage dna by a recipient bacterium. they discovered that an extract of e. coli has a restriction activity, and that this activity is of an enzymatic nature, caused by a nuclease that breaks the phosphodiester bonds in dna. in , the americans hamilton smith (b. ) and kent wilcox purified the first restriction enzyme from a strain of haemophilus influenzae. in , daniel nathans ( - and kathleen danna (b. ) at johns hopkins university in baltimore (usa) drew up the first restriction map based on the circular dna of the monkey sv virus, using a restriction enzyme that was named hindiii and a follow-up of the sequential appearance of shorter and shorter fragments resulting from the partial digestion of dna. in the following years, dozens of restriction enzymes were isolated, all of them endowed with a surprising specificity with respect to specific base sequences in dna ( figure iv . ). these enzymes were to be indispensable tools in genetic recombination experiments. the transformation of rna back into dna was observed by howard temin ( - ) and s. mizutani , in experiments on the rous sarcoma virus, a virus with rna that, when it proliferates in host cells, is able to synthesize a dna that is complementary to its rna. the enzyme responsible, reverse transcriptase, was purified by both h. temin and david baltimore (b. ) . starting with a determined messenger rna, it then became possible to work back to the dna, i.e., to the gene, by a simple enzymatic reverse transcription operation. dna that has been synthesized in this way is called complementary dna (cdna). in eukaryotic organisms, reverse transcription has proved to be all the more useful as a technique in that all cdna is coding, unlike the situation in vivo in which the genes are divided up into portions that are coding (exons) and portions that are non-coding (introns). the ability to cleave dna and to join together the fragments obtained in a deliberately chosen order, or, in other words, to manufacture previously unseen dna sequences by making new combinations, led to the dawning of recombinant dna technology and caused scientists to come to the sudden realization that the pandora's box that contains the secrets of life had been opened, that uncontrollable catastrophes might arise from this, and that there was a potential danger of causing tumorigenic viruses to reproduce in commensal bacteria such as the enterobacterium escherichia coli. in , around a hundred molecular biologists gathered together at the asilomar conference center near monterey in california, in order to discuss the dangers of the new dna technology. they proposed strict regulation to govern genetic manipulation. time and experience have shown that the risks being run were very low. in , dna sequencing methods were published. one of them made use of chemical techniques , the other made use of enzymatic techniques . applications were not slow in appearing. from , the team led by frederick sanger (b. ) in cambridge (uk) determined the first sequence of a genome, that of bacteriophage phix , which is nucleotides long. this was the beginning of an audacious adventure, the apparently senseless challenge being met with unbelievable rapidity thanks to the innovative methods of bioengineering, resulting, during the first years of the st century, in the sequencing of the human genome. analysis of the human dna sequence involved the participation of two rival groups, one of them being academic, coordinated by francis collins (b. ) , and bringing together dozens of laboratories around the world, and the other being a private californian company directed by craig venter (b. ) . at the beginning of the s, when everyone was persuaded that the rnas could be placed into three well defined categories, messenger rnas, transfer rnas and ribosomal rnas, it was with great surprise that it was learned that there were rnas that have catalytic properties (see thomas cech [b. ] ). these rnas, called ribozymes, have, in keeping with enzymatic proteins, structured catalytic sites that are able to catalyze rna or dna cleavage or ligation reactions. recently, engineering techniques have been used to obtain artificial ribozymes that have been found to be able to catalyze reactions as varied as oxidations or the synthesis of peptides and nucleotides, thus opening up wide-ranging possibilities of applications in molecular therapeutics, and, in addition, reinforcing the famous theory of the "world of rna" at the beginning of the appearance of life on earth . another discovery of the s was the role of methylation of dna bases, cytosine and adenine, and its deregulation in a certain number of pathologies: fragile x syndrome, scapulohumeral dystrophy, certain forms of cancer … in the past decade or so, basic proteins known as histones that are associated with the nuclear dna of eukaryotes in the form of a complex called chromatin and which had previously been assigned a structural role, have now acquired the status of functional partners. thanks to specific modifications of certain amino acids (acetylation, methylation, phosphorylation), histones control the state of condensation of the chromatin and the efficacy of transcription of dna contained in the chromatin, to such an extent that we now speak of the "histone code". the development of our understanding of histones is a good illustration of the complexification of a concept, the dna code, into an entity that comes closer to living reality, the dna code in partnership with the histone code. there has also been the discovery of interfering micrornas, small polymers made up of around twenty nucleotide units, the role of which is to control protein synthesis (chapter iv- . . ). methylation of dna, structural modifications of the chromatin histones, and blocking of transcriptional activity by interfering micrornas are a few of the major areas of research in a scientific domain that is in full expansion, epigenetics, which could be said to have "pipped the science of genetics at the post," and which explains the plasticity of the functions of living beings. with the arrival of restriction enzymes and reverse transcription, the foundation stones of genetic engineering have now been laid, and are ready to be used, this being all the easier in that synthetic chemistry is now able to manufacture dna chains that are several hundreds of nucleotides long, and progress in robotics and computing techniques made it possible for chemists to avoid carrying out tedious routine tasks by using completely-programmable machines. the hope that it would be possible to experiment on living beings by means of the manipulation of dna became a reality when the american researchers paul in the first work carried out on the expression of foreign genes, the use of plasmids as vectors was preferred, particularly that of plasmid pbr , because of its considerable replication capacity. in , a first success was obtained by herbert boyer and his co-workers, with the expression of the gene for somatostatin, a peptide hormone comprising twelve amino acids that negatively regulates the secretion of growth hormone, in the bacterium e. coli. because of its small size, the somatostatin gene was synthesized by chemical means. the expression of somatostatin in e. coli was verified using immunological and physiological criteria, thus demonstrating the validity of the procedure that was used. the following year, human insulin was produced in e. coli. fairly soon, yeast was substituted for this bacterium because, as a eukaryotic organism, it has enzyme systems that are able to carry out chemical finishing operations on neosynthesized proteins that bacteria are unable to do, for example the formation of disulfide bridges in insulin. genetic recombination is used in order to cause bacteria to manufacture a foreign protein of animal or plant origin. this involves the insertion of the fragment of animal or plant dna that codes for this foreign protein into a plasmid. the plasmid, a small ring of bacterial dna, acts as a vector for the foreign dna. in order to carry out insertion, plasmid dna is cleaved by an appropriate restriction enzyme. the foreign dna is obtained by reverse transcription from a useful messenger rna. its duplication is catalyzed by a dna polymerase. the s nuclease makes it possible to break the covalent bond between two strands of dna. in the following step, a terminal transferase is used to add four nucleotides for which the base is a cytosine (c) to each of the two dna strands. the same lengthening operation is carried out on the bacterial plasmid, but, in this case, the addition involves a sequence of four nucleotides for which the base is a guanine (g) (complementary to the cytosine c). the bacterial plasmid is hybridized in vitro with foreign animal or plant dna and then introduced into the bacterium which, using its own machinery, perfects the junction between the integrated dna and the plasmid dna. a very small volume of dna ( . - ml) is injected under the microscope into eukaryotic cells (hela cells in inset) using a micropipette with a very fine end that pierces the cell membrane. the swelling of the cells at the moment of injection can be seen (inset). supported by these successes, genetic engineering started to come to the fore as an application-oriented discipline. levels of performance that would never have been imagined half a century before were achieved, such as the production of growth hormone, interferons, blood coagulation factors and vaccines. in the final decades of the th century, phenotype transformations using genetic modifications that had previously been carried out in bacteria and yeasts were successfully attempted in animals and plants. it was observed that a mutated dna integrated into a plasmid and introduced into a fertilized mouse egg (by micromanipulation) modifies the mouse's genetic inheritance, which affects first the embryo and then the adult mouse with phenotype modifications. such mice, which are said to be transgenic because of the stable integration of a foreign dna into their genome, are now widely used as animal models in studies that aim to understand the mechanisms involved in high-incidence human pathologies such as cancer, diabetes, and rheumatoid conditions. in , two american researchers , ralph brinster (b. ) and richard palmiter (b. ) carried out a spectacular transgenesis experiment in mice. using microinjection, they introduced the growth hormone gene (obtained from the rat) into oocytes of mice from a "little" germ line. once the transgeneic mice had reached adulthood, they were giants. at present, the transgenesis technique is being applied both to the animal kingdom and to the plant kingdom. in the s, kary mullis (b. ) perfected an ingenious technique, the polymerase chain reaction (pcr), which makes it possible to produce several tens of thousands of copies of a fragment of dna. using this technique, it is possible to detect traces of a fragment of dna of a given sequence down to an attomolar concentration, i.e., one billion billion ( - ) times smaller than molar concentration. by the end of the th century, genetic engineering had become well-established and wide-spread, thanks to a mastery of techniques involving the manipulation of dna such as the accurate cleavage of a gene into fragments using commercially available restriction enzymes, the covalent assembly of two fragments of dna by ligases, the automated chemical synthesis of fragments of dna of more than one hundred nucleotides and the possibility of manufacturing a complementary dna (cdna) from a messenger rna by using a reverse transcriptase and automated dna sequencing. given this particularly well-equipped toolbox, the molecular biologist is now able to manipulate dna, that is to say, the chemical material that contains the information that is central to the functioning of living structures (microorganisms, plant and animal organisms), and thus to modify, at will, the genotype of these structures that the selective pressure of evolution has previously favored. genomics has produced enormous quantities of data that are stored in databanks. automated procedures have been invented to make the information contained in these data intelligible, these procedures forming the basis for a new discipline, biocomputing, or bioinformatics, which develops programs, or algorithm-based strategies, that are able to solve specific problems, of which the annotation of genomes, i.e., the identification of coding and non-coding sequences. while the annotation of the prokaryotic genomes is relatively easy, because of the absence of introns, that of the eukaryotic genomes is considerably more difficult because of the alternating exons and introns and the small proportion of coding exons (fewer than % in the case of the human genome). this explains why, at the time of writing, several hundred genomes of prokaryotes (around at the beginning of ) have been sequenced, as opposed to only a few dozen genomes of eukaryotes. annotation was carried out manually at first, but it has become automated and it is now possible to analyze thousands of items of genomic data. the comparison of nucleotide base sequences in dnas and of amino acids in proteins of different origins involves biocomputing. the identification of similar or identical regions that provide information about functional similarities and phylogenetic proximity involves the use of alignment methods. one of these methods, which is in current use, is called blast (base local alignment search tool). comparison of protein sequences has been particularly instructive in the science of evolution. it has highlighted evolutive processes in the phylogenesis of proteins and linked these processes to precise functions. it has been possible to deduce that, over time, different families of proteins with similar functions appeared independently and evolved along different routes. this is the case for membrane proteins whose polypeptide chain crosses the thickness of the membrane six or twelve times; thus, the mitochondrial membrane proteins that transport metabolites are formed by triplication of an element with two transmembrane segments, while proteins located in other membranes of the cell are derived from duplication of an element with three transmembrane segments. from this academic context arose the study of paleogenetics, a new discipline that compares dna sequences extracted from fossils and amplified by pcr with dna sequences of current species. in addition to being of immense interest to fundamental biology, genetic bioengineering has led to innumerable industrial applications making use of genetically modified microorganisms that are able to synthesize molecules with a high added value that can also be used in xenobiotic depollution operations. so much data has already been deposited, equivalent to the sequencing of more than one hundred billion nucleotides, that it is inevitable that there have been errors, some of which might prove prejudicial for future use (comparison of sequences, screening of drugs…). nevertheless, the ever-increasing numbers of genome sequencing projects for animal, plant and microbe species show the interest that is shown in understanding the genetic information present in different types of cells, in order to be able to exploit their potential. dna chips appeared in the last decade of the th century, and came to the fore as part of a new technical revolution, the "high throughput" revolution ( figure iv. ). an article written by the group headed by ronald davis and patrick brown (b. ) at the university of stanford gives a precise description of the hybridization technique used in dna chips. thus, around a hundred short dna strands, corresponding to portions of genes of the plant arabidopsis thaliana, commonly known as mouse-ear cress, a small plant in the brassicaceae (formerly cruciferae) family, are synthesized. a robot is used to deposit microquantities of these dnas in solution in a dot pattern on a small glass slide coated with poly-l-lysine, thus comprising a "chip" on which the covalently fixed dnas act as probes for specific molecules. a later step involves both the use of reverse transcription to produce complementary dnas (cdnas) from messenger rnas arising from the expression of genes in the same plant and the labeling of these cdnas with fluorescent ligands for use in screening. once these fluorescent cdnas have been denatured, i.e., after separation into single strands, they are brought into contact with the dna chip. the unhybridized molecules are removed by washing. a -the term dna chip corresponds to a small, chemically-treated glass (or sometimes silicon) plate on which a robot has deposited dna strands of known sequence in a pre-determined order. b -the dna chip may be used for different types of diagnostic procedures. in the differential diagnosis experiment represented here, messenger rnas are prepared from two cell samples that have been treated in parallel, the control sample (normal cell) and the experimental sample (pathological cell). these messenger rnas are reverse transcribed into complementary dnas (cdnas) by means of a reverse transcriptase. each of these two types of cdna, corresponding to the two types of messenger rna, is labeled by a chemical reaction with a specific fluorescent ligand (cy , which emits at nm and cy , which emits at nm). they are then hybridized with chip dna strands. after hybridization and then washing, the fluorescence emitted at nm and nm under laser irradiation are analyzed using an appropriate detection system. the differential expression of the genes in the control cell sample and the experimental sample (shown by a color difference) can thus be analyzed. hybridization between the fluorescent dnas called targets and the complementary nucleotide probes fixed to the dna chip is detected by means of an automated fluorescence detection system. at the beginning of the s, stephen fodor and his group, who were working at the affymax research institute (palo alto, california), developed an ingenious procedure for microphotolithography that led to the synthesis of a network of a thousand peptides on chemically pre-treated glass microscope slides. the resolution of the network was shown by epifluorescence microscopy after fixation of specific antibodies labeled with fluorescent probes. soon after this, microphotolithography was used for the manufacture of dna molecule networks on solid supports. from then on, two competing techniques for the preparation of dna chips became well-established, either the depositing on a solid support of cdna obtained by gene amplification (technique used by davis and brown), or the synthesis in situ of oligonucleotides carried out directly on a solid support (technique used by the american affymetrix company, arising from affymax). one considerable advantage of dna chip technology is that it provides information on the level of transcription of thousands of genes into messenger rnas (mrnas), in a simultaneous manner and in a relatively short lapse of time. experiments that previously required weeks, months or even years to be completed can now be carried out in a matter of hours. we therefore have a sort of instantaneous, precise, freeze-frame picture of the state of a cell at a given moment, with a great number of parameters explored in a semi-quantitative manner. dna chips are a typical example of the application of high throughput technology to the study of living beings. the panoply of mrnas produced by the transcription of dna is called the transcriptome. the method by which transcriptomes are obtained is called transcriptomics. it should be added here that there is not necessarily a correlation between the abundance of a mrna, evaluated on a dna chip, and the functionality of the corresponding protein, which depends on multiple factors that particularly involve post-translational modifications: phosphorylation, glycosylation, hydroxylation, and so on. at the end of the s, dna chips were being used extensively in the research programs of many biology laboratories. they are used in a variety of domains: human pathology, to differentiate between forms of cancer linked to multiform mutations; microbiology, to identify pathogenic germs; comparative genomics, to look at model eukaryotic organisms that have in common a certain number of genes; or even in populations genetics, to detect polymorphisms linked to a change in a single base in a dna sequence. as a complement to the dna chip technique, the fish (fluorescent in situ hybridization) method holds a key position in the study of cytogenetics. this method is based on hybridization between fluorescent nuclear probes of known nuclear sequence and of complementary motifs located in the dna of the chromosomes. it allows the detection of chromosomal modifications with a gain or loss of genetic material, such as those that are found in certain tumors. it is used in prenatal diagnostics to diagnose such modifications. protein chips, which are used to characterize the reactivity of proteins with respect to specific ligands, are another example of the application of high throughput technology. dozens of proteins of different types (antibodies, for example), as well as derivatives of nucleic acids or even molecules capable of being ligands of proteins that might arise from combinatorial chemistry (chapter iv- . ), are arranged in a network on small glass plates that are chemically treated to act as hooks to entrap specific proteins present in a tissue extract or serum ( figure iv . a). this procedure, which is essentially analytical in nature, is complemented by a functional study in which proteins that have been isolated in their native form, i.e., those that are capable of expressing the same functions that they posses in vivo, are deposited on a glass microplate. this type of biochip makes it possible to analyze the reactivity of the proteins that are fixed to it with respect to a multitude of targets (proteins, nucleic acids or pharmaceutical substances) (figure iv. b). antigens peptides a -biochip for the identification of proteins. different types of ligands, antibodies, antigens, dna or rna, small molecules with a high affinity and specificity, are deposited on a reactive surface. these biochips can be used to determine the level of expression of proteins and the type of proteins expressed in cell extracts. they can be used for clinical diagnostics. b -biochip for the functional study of proteins. native proteins or peptides are arranged in micronetworks on a reactive medium. biochips produced in this way are used to analyze the activities of proteins and their affinities as a function of posttranslational modifications. they are useful for identifying drug targets. analytical proteomics, which was still in its infancy in the last decades of the th century (chapter iii- . . ) has become a vigorous discipline. the association of liquid nanochromatography and of mass spectrometry allows the identification of peptides obtained by the trypsin hydrolysis of samples of proteins of around one picomole in size. applied to fundamental and pathological cell biology, the aim of analytical proteomics is not only to decipher the list of proteins on the scale of the cell, but also to highlight variations in the abundance of synthesized proteins as a function of environmental conditions. it also aims to determine the posttranslational modifications that are undergone by the proteins inside the cells, which, for a large part, control the specificity of their operation. in parallel with the study of proteomics, the study of peptidomics, or the study of all of the peptides (peptidome) present in animal and plant cells and in the fluids that bathe these cells, has developed. for example, several hundred different peptides have been found in the cerebrospinal fluid. structural proteomics, which deals with the three-dimensional structure of proteins, has also become a domain in which the activity has been increasingly dominated by high-throughput techniques. this is partly due to the fact that the pharmaceutical industry has given considerable, sustained attention to the understanding of the structure of proteins that could play the role of therapeutic targets. this is the case for protein kinases that catalyze, via atp, the phosphorylation of endocellular proteins, of proteases involved in hydrolysis reactions, or even of cell surface receptors that are able to combine with ligands such as hormones. the use of automated crystallization systems has become common in structural biology. from the classical technique of the hanging drop, in plates comprising wells, we have moved on to plates with , and, recently, wells. obviously, this increase in dimensions requires the use of an automated system that includes a robot in charge of transferring microaliquots of the protein solution into the wells and adding media that differ according to their ph, ionic force and molecular composition to these wells. the crystallization process is followed by an automated microscopic examination coupled with video photography. making use of the recent development of genomics and of proteomics, and a detailed inventory of the structures and functions of the different protein species of living beings, contemporary biology is now able to sketch out a scheme of molecular systematics, including a classification into phylla, families, and classes that echoes those of the zoological and botanical systematics of the th and th centuries. however, modern systematics does not tell us how protein macromolecules interact within dynamic networks. there is still an enormous amount of work to be done in order to achieve an understanding of the meaning of the dialogue between macromolecules in a normal or pathological cell context. this work will require a detailed analysis of metabolic pathways and of how they are controlled, and their evaluation in kinetic and thermodynamic terms. it will be accompanied by modeling (chapter iv- ). there is no doubt that it will be successful. making use of subtle differences in the qualitative and quantitative expression of genes, it will become possible to understand the molecular principles that modulate differences in morphology and in function between neighboring animal, plant or microbial species. the science of evolution should benefit from this. in medicine, the forecasting of predispositions for certain diseases should be made easier (chapter iv- . ), opening up the perspective of prevention strategies. using recombinant dna technology, metabolic engineering applied to microorganisms and plants should make it possible to improve the production of molecules that are of economic interest or can be used for drugs. the diversity of bacteria is amazing, much greater than might be supposed by looking at the number of bacterial species identified by culturing on appropriate media. in fact, the number of bacterial species that can be cultivated only represents % of the total number of existing bacterial species on the surface of the earth. there are two major reasons for this: we do not know the appropriate conditions for culturing these bacteria; a certain number of environmental bacteria live in symbiosis, acting as commensal organisms that benefit from the products secreted by other organisms. nevertheless, the study of the bacterial genome, without any clonal culture, has been carried out, and comprises a branch of genomics known as metagenomics. instead of looking at an isolated, well-identified bacterial species, in order to analyze the sequence of its dna, as has been done traditionally, researchers look at a heterogeneous bacterial sample from which the dna is extracted, amplified, and then sequenced by high throughput methods. computer processing of the data provides information about individual germs. craig venter, who had already gained notoriety with the sequencing of the human genome, recently applied "metagenomic" procedures to the study of the sequence of the "metagenome" of the bacterial species of the sargasso sea . he came up with nucleotide sequences corresponding to approximately million kilobases of non-redundant nucleotides, attributable to more than two thousand different genomes. the challenge to be met by metagenomics is to connect a function to its phylogenetic source and to extend this information to specific species within a bacterial community. group . in human biology, a metagenomics approach has been applied to the study of the population of bacteriophages present in the intestinal flora. approximately genotypes have been identified, a number that greatly exceeds the bacterial species of this flora . this result leads us to think that the luxuriant community of bacteriophages which cohabits with that of the intestinal bacteria may influence the diversity of the latter by selective bacterial lysis and also by promoting the exchange of genes between bacteria. a rapid overview of the history of the exploration of genomic dna over the last fifty years shows the rapidity with which a traditional experimental paradigm can move thanks to modern computing and robotics procedures. in less than twenty years, we have moved from the manual sequencing of dna that was developed at the end of the s to automated high throughput sequencing. at the turn of the st century, the sequencing of communities of genomes (metagenomics) has been substituted for the sequencing of individual genomes. dna and protein chips have become objects of everyday use in fundamental and applied biology. transgenesis is widely practiced. dna, a molecule that remained mysterious for a long time after it was discovered, delivered some of its secrets during the second half of the th century. the purpose of the first experiments on dna was to understand how dna, detector of the genetic code, transmitted its message. after having questioned dna, researchers moved on to manipulating it. the current aim is to use oligonucleotides to build nanoscale constructions with original and, if possible, useful, properties. in addition, the possibility that has recently become available of being able to interfere with the expression of the genome in living cells, with the intervention of small rna molecules, allows the programmed manipulation of the genome. another challenge, the extending of the coding power of the genetic code, now appears to be achievable. from the fact that each of the strands of the double helix overhangs in one direction, and in the other the strand with which it is paired, thus leaving a few bases free ( figure iv. ) . if two strands of dna with sticky ends are brought into contact, when the bases of these ends are complementary, a branched structure will appear spontaneously. using this principle as a basis, cube-shaped nanometric constructions that make it possible to encage molecules of interest have been built. the opening of the cage by appropriate devices liberates the encaged molecules, which can act as substrates in specific reactions. the cutting of a double strand of dna using restriction enzymes able to create fragments with cohesive ends (a) has been used to "build" an artificial construction (b), which, in this case, is a cube (c), but which could be an object of a different geometrical type. a dna nanomachine that is capable of movement is becoming a reality. one dna nanomachine, which is admittedly still rudimentary, has been put together based on the structural difference that exists between b-dna, the classical double helix that twists to the right, and z-dna, a double helix that twists to the left. a propensity to adopt the z-form is triggered when there is an alternating sequence of cytosine (c) and guanine (g) (cg sequence) in the dna. the experiment illustrated in figure iv. makes use of a duplex formed of b-double helices. the dna nanomachine constructed by n.c. seeman comprised a duplex of double strands of dna. one of the double strands, of the classical b-form of dna (right-hand twist), has been cleaved in such a way as to fix fluorescent molecular probes onto the cleavage zones. facing this cleavage zone, a short nucleotide sequence, in which the cytosine (c) guanine (g) motif is repeated, can be found in the other dna double strand, which is also of b-form. the addition of cobaltihexammine induces the transition of the cg segment from a right-hand twist (b-dna) to a left-hand twist (z-dna), which leads to a rotation of this segment and to a rotation of the assembly, which can be detected using fret (fluorescence resonance energy transfer) spectroscopy. one of the double helices has a short cg segment. facing the cg segment, the other double helix is interrupted, and its ends, where the interruption is, carry fluorescent molecular probes. the simple fact of adding a cationic substance such as cobaltihexammine, which neutralizes the negative charges of the phosphate groups, triggers a conformational transition, with the cg segment taking the z-form, causing a rotational movement of the assembly that is detected by the movement of the probes. there is no doubt that the use of dna strands in order to build nanomolecular constructions that are capable of programmed movement marks the beginning of an adventure that we may imagine will be rich in outlets for domains such as computer technology, nanomechanics and even the life sciences. in addition, the discovery that dna conducts electrical current gives rise to dreams of a revolutionary technology in which dna may be used in the design of electrical circuits, in competition with classical electronics . interfering rnas are non-coding rnas of around twenty nucleotides that control gene expression at post-transcriptional level. as with many discoveries, that of rna interference was the result of serendipity. it began during the s with observations made by two american research groups, that of victor ambros now at darmouth medical school, hanover, and that of gary ruvkun (b. ) at boston's massachusetts general hospital, that a gene named lin- , which is involved in the post-embryonic development of the nematode c. elegans, did not code for a protein, but for a small size rna that played an antisense role. this odd discovery was supported and made more explicit a few years later by the research groups of andrew fire (b. ) at baltimore's carnegie institute and craig c. mello (b. ) at the university of massachusetts in worcester . in order to block the production of certain proteins in the nematode c. elegans, the researchers used synthetic antisense rnas. the control involved the use of sense rnas according to a classical protocol. unexpectedly, protein synthesis was blocked in both cases, suggesting that a contaminant was present in the sense and antisense rna preparations. this contaminant was identified as a double strand rna (dsrna -double strand) that is, an rna that is folded back on itself in a "hairpin" loop because of the pairing of complementary bases (adenine vs uracil and guanine vs cytosine). in order to verify the mechanism by which the translation of messenger rnas into proteins is silenced, the nematode c. elegans was injected with a synthetic dsrna, part of the sequence of which was complementary to that of the gene unc- , known to code for a protein involved in muscular contraction. within a few hours, the worm was making disordered movements, suggesting that the dsrna interferes with the production of proteins in the process of muscular contraction. the mechanism of action of dsrna was quickly unraveled: dsrna gives rise to two single strand rnas after cleavage by a specific enzymatic mechanism. one of the single strand rnas (sirna -small interfering rna) is paired thanks to a complementarity of bases with a short sequence of messenger rna transcribed from the gene unc- . the result is a blockage of the translation of messenger rna into a protein, followed by the destruction of messenger rna. this phenomenon was named rna interference ( figure iv the dicer cleavage enzyme, which has a ribonuclease activity, cuts the double strand rna into two strands. in the presence of the risc (rna-induced silencing complex) protein complex, one of the rna strands finds a complementary nucleotide sequence in a messenger rna (mrna) and associates itself with this rna, making it unable to be translated into protein. we now know that eukaryotic cells from animals and plants produce and host interfering rnas that are said to be "natural" . natural interfering rnas, of around twenty nucleotides, are called micrornas (mirnas). although a few details differ between the modes of formation and action of natural interfering rnas and those of synthetic interfering rnas, in particular the fact that messenger rnas are not destroyed by mirnas but blocked in their translation, the effect of negative regulation on the production of specific proteins comes to the same thing ( figure iv. ). there is a far from negligible number of genes that code for mirnas. already, several hundred mirnas have been identified in the genomes of plants and animals. the amount of interest that they arouse, and the feverishness of the research being carried out on them, are in keeping with the major mechanisms that they control: embryogenesis, hematopoiesis, neuronal differentiation, etc. given an understanding of the genome sequence in man, the rat and the mouse, trials have begun that aim to achieve an understanding of how the expression of mammal genes of known sequence might be manipulated by the interplay of interfering rnas (chapter iv- . ). the treatment of viral infections such as aids or hepatitis b, which are worrying public health problems, could benefit from this new technology. it appears that interfering rnas have much more to give to us in the near future than they have taught us up until now. the deciphering of the genetic code in the middle of the s was the end of a first step in elucidating the mechanism by which a sequence of nucleotides in dna is translated into a sequence of amino acids in a protein (chapter iv- . ). during the years that followed, the subtleties of the transcription of dna into messenger rna and of the translation of messenger rna into protein via transfer rnas were explored in hundreds of laboratories around the world. particular attention was paid to the understanding of how a given amino acid is activated and bound to a transfer rna (trna) after being picked up by an aminoacyl-trna synthetase. nevertheless, the idea remained of a code in which triplets of purine and pyrimidine bases of messenger rnas are translated into natural amino acids. recently, methods have been developed that give more flexibility to the action of the aminoacyl-trna synthetases, or, in other terms, relax their specificity . synthetases that have been manipulated in this way are able to recognize non-natural amino acids and to incorporate them into proteins by working together with the ribosomal machinery. it is in this way that, at the time of writing, around thirty non-natural amino acids, obtained by insertion of different types of chemical residue (photoactivable, fluorescent or radioactive residues capable of acting as probes for structural and functional analyses) (figure iv. ) have been incorporated into protein structures. with such an innovation, an unexpected field of exploration has opened up to research in domains as far apart as pharmacology and the science of evolution, giving rise to burning questions: could such non-natural proteins have therapeutic properties? could they give a selective advantage to the organisms that host them? with the addition of non-natural amino acids to the genetic code and the demonstration that proteins containing such amino acids can function in living cells, in sum, with the transgression of the potentialities of the natural genetic code, the experimental method appears to challenge the order of living beings. the triumph of genetic engineering via the study of dna is not unique to biology. many other sectors are undergoing changes in their type of experimental approach, dictated by the technosciences and making use of computer sciences, robotics and high-throughput screening. however, given the many questions that its operation continues to raise, and its central position at the heart of scientific ethics, the study of dna remains a typical example of the way in which the experimental life sciences and the techniques that underlie them are evolving nowadays. "i perfectly agree that when physiology is sufficiently advanced, the physiologist will be able to make new animals or plants, as the chemists produces substances that have potential, but do not exist in the natural order of things." more than a century after claude bernard predicted a genetically manipulated world, it has come to pass. the molecular biologist, having original, highperformance methods for "tinkering" with dna, has moved on to the application and use of his technical expertise for utilitarian ends. during the s, with transgenesis, research on bacteria (chapter iv- . . ) opened up a new biological domain, that of genetically modified organisms (gmos). results led to predictions that it would be possible to transfer a fragment of dna corresponding to a gene of a certain species into the genome of another species and have this foreign gene express itself as a protein in the host cell. in , the successful trans-genesis of a gene for resistance to an antibiotic, kanamycin, in tobacco plants, signaled the beginning of the technology of the first plant-type genetically modified organisms, still called gmps (genetically modified plants). in , the birth of dolly the ewe unveiled the era of reproductive cloning in mammals, i.e., the identical reproduction of an already-existing organism. an additional step was taken with the first tests on the differentiation of embryonic stem cells towards different types of lines that are characteristic of well-defined tissues, such as nerve tissue, cardiac tissue or the hepatic parenchyma, thus opening up promising perspectives in regenerative medicine. the frontiers of the experimental method continue to be pushed back to the limit of what is feasible and sometimes into the realm of fiction, as in immunology, for example, with the idea of xenotransplantation, using "humanized" animal organs. given the universality of the genetic code, any gene that is introduced into the genome of a plant, whether that gene is of animal, plant or microbial origin, is able to replicate itself and be expressed as a specific protein. thus plant gmos or genetically modified plants (gmps) are able to express specific foreign proteins from another plant, a bacterial microorganism or an animal organism. in the s, a short time after fundamental research had revealed the feasibility of plant transgenesis, the first transgenic plant, the flavr savr tomato, was marketed in the usa. since this time, numerous other plant gmos have been cultivated on a large scale and become available on the world market, including corn, soya, rice, cotton and the poplar. one of the desired aims is to produce modified plants that are able to resist destruction by the herbicides that are commonly used to eliminate weeds, while another is to prevent predation by harmful insects. in the first case, transgenesis involves the insertion of a herbicide-resistant gene, and in the second case, the inserted gene codes for an insecticidal toxin. recently, plant gmos that produce proteins with a therapeutic effect have appeared, ranging from antibiotic peptides to antibodies or proteins as unexpected as human hemoglobin. current projects aim to create plants that are resistant to adverse conditions such as the dryness of arid climate zones. the preferred procedure for producing a plant gmo is to use a bacterium, agrobacterium tumefaciens, a microorganism that is able to insert fragments of its own dna into plant cells ( figure iv . ). the useful gene that we wish to transfer into the plant may be a gene for resistance to a pesticide such as glyphosate, marketed under the name of roundup, phosphinothricin (basta) or glufosinate (liberty). the plasmid is reintegrated into a. tumefaciens. during infection of a plant cell by a. tumefaciens the t-dna carrying the gene of interest is inserted into one of the chromosomes of this cell. the ti plasmid is isolated and cut using a restriction enzyme. a foreign gene, known as a gene of interest, is inserted into the t-dna of the plasmid. a plant is generated from a modified clone. all of its cells carry the foreign gene. transgene of interest in the case of the fight against insect predators, the useful gene is carried by a fragment of dna contained in the genome of the bacterium bacillus thuringiensis. this gene, called bt, expresses a toxin responsible for the insecticidal capability of b. thuringiensis. a current application involves the protection of bt corn with respect to the corn-borer, a devastating insect whose caterpillars are particularly destructive. another, more direct, gene transfer method, known as biolistics, involves bombarding plant cells with tungsten microbeads covered with modified dna. with the implementation of large-surface-area experimental fields and the first marketing of gm soya, in , the question of whether or not the advantages achieved with respect to crop yields are counter-balanced by risks for the environment and for consumers came to the fore. food risks could arise from the toxicity or allergenic power of artificially synthesized proteins. at the time of writing, this question remains unanswered, due to the lack of epidemiological studies carried out rationally over several years. when the first creations of gmos took place, the transfer of the gene of interest was carried out by means of the co-transfer of an antibiotic resistance gene. the transformed cells were selected according to the criterion of their resistance to this antibiotic, which involved a risk of dissemination of the resistance gene. this selection technique has been abandoned. in practice, it is difficult to evaluate the theoretical ecological risk of wild plants being invaded by genes that have been inserted artificially into gmos. as a precaution, zones used for experimentation of plant gmos are now surrounded by refuge zones, i.e., fields in which the same species of plants, in non-gmo form, are cultivated. there has been a much fiercer and completely legitimate debate concerning the presence of the terminator gene in seed from the first gmos marketed by the monsanto company in the usa. the terminator gene blocked germination of the seed from the cultivated plant, so it was necessary for the farmer to buy more seed from the company each season, thus creating a state of dependency. this technique is no longer in use, but the fact remains that most transgenic seed is patented, and therefore farmers who use such seed are dependent on the companies that posses this genetic know-how. the culture of plant gmos has spread around the world, covering more than a billion hectares of our planet, more than half of which are in the united states of america. this type of culture is used on a large scale for soya and in a less extensive way for corn, rape seed and cotton, but there are many other applications of plant transgenesis. among the countries that are actively involved we may mention argentina, brazil, canada and china, and more recently india, paraguay and south africa. while the policies of these countries are based on the fact that gmo products do not differ fundamentally from non-gmo products with respect to checks carried out a posteriori, and that there is thus no reason to prohibit them, european policy has taken refuge behind a principle of precaution, and it remains basically restrictive. although the moratorium on the culture and marketing of plant gmos that was put in place in was lifted in , mandatory labeling for any consumable product containing more than . % gmo remains dissuasive. the united states of america has refused to use such labeling. the worries that are aroused by plant transgenesis, which are often exacerbated by the diktats of ecology groups, must be analyzed in a reasoned manner. common sense and lucid thought dictate that the debate should be situated within a scientific perspective in which the main role is played by the experimental method in long-term applications. simple reflection leads us to think that with time parasites and self-propagating plants will develop a resistance to the most drastic treatments, as was the case for bacteria confronted with antibiotics. the perspective of an acquisition of uncontrolled resistances, which gives rise to so much passionate debate, is, in fact, only the first stage of a technology with promising applications. the mastery of plant transgenesis that was acquired through the first experimentations should, in fact, allow the emergence of plant gmos that are assigned to the production of molecules with therapeutic effects (drugs, vaccines, human proteins, vitamins…). in this domain, there have already been creations that include golden rice, which carries β-carotene, the precursor of vitamin a, banana plants that express a vaccine against hepatitis b and tobacco that produces human lactotransferrin and hemoglobin. if we just look at the production of golden rice as a palliative for vitamin a deficiency, it should be remembered that, in certain countries of our planet, this deficiency affects people's sight and is a frequent cause of blindness, that it generates problems with development and the immune response to infections, that it affects more than a hundred million children around the world and that it is responsible for the death of three million of them each year. if these plants are considered to be a material of choice for the production of proteins with a therapeutic effect, this is partly due to the yield of such crops over large surface areas, and also partly due to the low risk of transmission of viral pathogens to man, because of the species barrier, a risk that is less negligible when animal productions are involved. genetically modified plants are also potential factories for the manufacture of chemical products with an industrial impact, for example lubricants, perfumes and aromas. given the unpredictable outlets that plant gmos may have in human medicine and the different domains of the economy, plant gmo technology should be considered in a manner that is free of any pressure or passion, and, as far as the political authorities are concerned, it should be subject to appropriate measures to surround and protect certain strategic experiments. when looking at the worries being expressed by the european society, it should be remembered that the genetic inheritance of plants has never ceased changing, not only in the most of natural of manners, over millions of years, particularly with the mobility of transposable elements located in the genome, but also artificially, at the hands of farmers from ancient times onwards, with their methods of hybridization and selection. the nervousness of european authorities, showing an ignorance of basic scientific ideas, with the pretext of a principle of precaution, and sometimes political compromises that are exemplified by fluctuating and contradictory positions, runs the risk, in the short term, of causing their countries to lag disadvantageously behind the united states of america, which holds the majority of plant biotechnology patents. the principle of gene therapy is simple: the introduction of an appropriate gene into the cells of a patient who carries a mutation can correct the phenotypical consequences of this mutation, or, in other terms, cure the disease affecting the patient, or at least slow down its evolution. the technical difficulty involved in gene therapy is that of finding an appropriate vehicle or vector for the transfer of the gene and addressing it to an appropriate location in the genome of the host cell. the most commonly used vectors in human gene therapy are viral. a certain number of criteria are necessary for a transfer to be efficacious, including a high concentration of viral particles carrying the gene to be transferred (more than a billion viral particles per milliliter) and a good capability on the part of the foreign gene to be integrated into the host's genome. the patient's immune response remains a major worry in the use of viral vectors: at cell level it often leads to a proliferation of cytotoxic lymphocytes and, especially at humoral level, to the synthesis of antibodies directed against the viral proteins. in order to minimize its immune response, the genetic material of the viral vectors is modified. for ethical reasons, gene therapy is currently only applied to somatic cells, germinal gene therapy being rejected. somatic gene therapy has been experimented in the treatment of hereditary illnesses linked to hematopoiesis. one of the technical reasons for this choice is easy access to the progenitor cells of the bone marrow, with the aim of transfection. it was with this in mind that mouse gene therapy models were developed a few years ago. the sickle cell mouse is one of these models. human drepanocytosis (sickle cell anemia) is a serious disease that is caused by a mutation in the β protein chain of normal human hemoglobin a. the molecules of sickle cell hemoglobin s tend to aggregate and form fibers that obstruct the blood capillaries of the microcirculation. somatic gene therapy has been applied to these sickle cell mice. this involves an autograft of bone marrow hematopoietic cells transfected with a retrovirus hosting the gene coding for the β subunit of normal hemoglobin. encouraging results have shown the validity of this approach. in , a gene therapy protocol that had been applied with success to man was described by the group of alain fischer (b. ) and marina cavazzana-calvo at the necker hospital in paris (science, vol. , pp. - ). the purpose of this therapy was to bring about a long term remission in the case of an immune disease known as scid-x (severe combined immunodeficiency linked to a mutation on the x chromosome). because of their susceptibility to microbial and viral infections, babies who are affected can only survive in sterile rooms. they are known as bubble babies. in this illness, the hematopoietic progenitor cells of the bone marrow are unable to differentiate into t and nk (natural killer) lymphocytes because of a mutation that affects a cytokine receptor. previous experiments carried out on model mice show that scid can be corrected by in vivo transfer of the cytokine receptor gene into hematopoietic progenitors. the transfer of the gene of interest paired with a retroviral vector was carried out first in march , in two babies, one of them eleven months and the other two months old. progenitor cells from their own bone marrow, cultured and modified genetically, were injected into them. these were therefore autografts, without any risk of immune rejection. a remission of symptoms over a period of nearly a year, shown by the almost normal behavior of the babies' immune cells, encouraged the application of the same therapy to other babies. in total, ten babies were given this therapy. the enthusiasm that greeted the successes that were recorded was nevertheless tempered by fact that in the spring of , and again in the following year, a child who had undergone the gene therapy developed a leukemia characterized by an anarchical proliferation of lymphocytes, necessitating chemotherapy. these two occurrences were explained by the random character of the insertion of the gene of interest into the patients' genomes: insertion into a site close to a proto-oncogene had led to activation of this proto-oncogene and the proliferation of the lymphocytes. while the trial carried out at the necker hospital gave rise to great hopes, it nevertheless showed that there is still a long way to go before we achieve a targeted transfection of genes so that no undesirable consequences follow. here we have a typical example of the limits of an experimental method that is based on an in-depth technological know-how, but also on a still imperfect understanding of the complex arcana of the mechanisms that regulate the positioning and interaction of genes in the chromosomes of eukaryotic cells. this example highlights a harrowing ethical dilemma: should we not treat a patient whose illness is likely to be fatal, or attempt a therapy that may save the patient, without having any formal assurance of its success? an experimental medicine that has the power to modify the human organism via its genetic material is now able to take over from the experimental method that up until now operated on animals and plants. we can easily understand, given the progress that has already been accomplished and that which is to come in the domain of gene therapy, that the temptation will be great, in the future, to consider manipulations of the human germ cell genome as being licit, insofar as such manipulations make it possible to eradicate a handicapping defect in our descendents. at present, the idea of any attack on the germinal genetic inheritance has been rejected unconditionally on the basis of ethical considerations. nevertheless, the history of science shows that prohibitions that were once considered to be untouchable finish by being contravened. this was the case for abortion. in a text entitled why genetic engineering should continue its battle , james watson writes of his confusion when faced with a choice that is likely to become more and more insistent over the years: "dare we be entrusted with improving on the results of several million years of darwinian natural selection? or do the human germ cells represent on the contrary rubicons that geneticists will never dare to cross?" a mastery of the differentiation of stem cells and of cloning are two essential weapons in the biotechnological arsenal, the use of which for utilitarian ends, particularly in human medicine, gives rise to hope and disquiet, agreement and disapproval. at the beginning of the s, experiments carried out by the canadian biologists ernest mcculloch (b. ) and james till (b. ) attracted attention to the particular properties of cells in the bone marrow, the stem cells, which would subsequently be found in other tissues . the experimental protocol is simple. bone marrow cells from a mouse are injected into another mouse that has previously been irradiated in order to destroy its stem cells. the injected cells go to the spleen where they divide and form colonies that take the form of nodules of different sizes. the researchers realized that the cells of these nodules present differences in their potential for renewal, which is more or less rapid. they reinjected the nodule cells into mice from a second batch. the reinjected cells showed themselves capable of multiplying and generating several types of blood line. these observations suggest the presence in the nodules of progenitor cells that have a strong potential for self-renewal and self-differentiation. in the following years, these observations were confirmed and explained by two characteristic criteria of stem cells; self-renewal and differentiation into multiples cell lines with specific characteristics. from this point on, it was possible to understand the enigma of the amputated hydra in the experiments carried out by trembley, two centuries beforehand (chapter ii- . . ). we now understand why, like the hydra, organisms like the flatworm, the salamander, the starfish and the zebrafish are able to recreate an amputated or damaged part of their bodies. the hydra mobilizes stem cells that it has preserved since its birth. in the case of the salamander, regeneration involves the reprogramming of cells that have already been differentiated. like all stem cells, embryonic stem cells (or es cells) are able to self-renew and differentiate into the different types of known adult cell line, giving rise to different types of cell such as neurons, cardiac cells that are able to contract, or hepatocytes ( figure iv . ). this potential has led to the hope that es cells could be used in regenerative medicine. in a fertilized egg that has developed to the blastocyst stage, it is possible to distinguish a cell mass (inner cell mass, icm) which protrudes inside the blastocyst. the icm cells are removed and placed on a mat of irradiated (and thus unable to divide) fibroblasts that provide them with a support and nutrients (steps and ) so that they can proliferate. the stem cells arising from the icm cells, placed in a medium that has been specifically conditioned to provide cytokines and other biomolecules, are able to differentiate into various cell types (step ). at what stage of embryo development is it possible to remove es cells for experimental purposes? after fertilization by a sperm cell, the ovum undergoes a series of divisions that give rise to a microstructure, the blastocyst, the cells of which are called blastomeres. each isolated blastomere remains capable of producing an entire organism of fetus and placenta, by division and differentiation. at this stage, blastomeres are totipotent. five days after fertilization, the embryo has the form of a hollow sphere. an external layer of cells, the trophoectoderm, surrounds a cavity, the blastocele, inside which a small mass of cells, the inner cell mass, protrudes. from the beginning of the implantation of the blastocyt in the uterus, the trophoectoderm evolves to form the placenta. the cells of the inner cell mass take part in the process of differentiation that generates all of the tissues of the future adult organism. these are called embryonic stem cells (es cells). es cells are said to be pluripotent. isolated, they have lost their ability to give rise to a complete individual, but they have maintained the possibility of differentiating, according to their environment, into any of the two hundred cell types that make up animal tissues. during their division, es cells evolve from a stage of being pluripotent to a stage of being unipotent, passing through a stage of multipotency beyond a hundred cells. a state of multipotency characterizes cells that give rise to a restricted number of cell lines in the tissues in which they nest. this is the case for of the hematopoietic stem cells of the bone marrow that form the red blood cells and the white blood cells. the term unipotent refers to the progenitors, which give rise to a single type of cell, for example the hepatocyte of the liver or the cardiomyocyte of the heart. when es cells are cultivated for to days in a conventional nutritive medium, they multiply and aggregate. if the culture medium is supplemented with certain biomolecules such as insulin, retinoic acid, transferrin or fibronectin, the differentiation of the es cells is oriented towards cells of different types, such as neuron cells, glial cells or muscle cells. there are many publications about experiments concerning the grafting of differentiated stem cells in the mouse or the rat. for example, neuron precursors derived from the spinal cord or the brain are grafted into rats whose spinal chords have been injured. five weeks after the grafts are carried out, the transplanted cells have filled the area of the injury and differentiated into oligodendrocytes, astrocytes and neurons. what is more, after about twelve weeks, locomotive function has been partially restoredirradiated . other experiments involving the grafting of differentiated stem cells have been carried out on rats in which the dopaminergic neurons of the "substantia nigra" of the brain that secrete the neurotransmitter dopamine have been selectively destroyed by injection of -hydroxydopamine. the problems found in the rat as a result of this neuronal degenerescence mimic those found in man in patients suffering from parkinson's disease. dopaminergic neurons obtained by the differentiation of mouse es cells are grafted into the striatum of each of these rats, a region of the brain whose neurons communicate with those of the substantia nigra and play a fundamental role in the control of movement. this results in a significant improvement in the motor deficit, coupled with the establishment of functional synapses between the injected neurons and those of the host , . a recent publication bringing together the results of two french research teams, that of michel pucÉat (b. ) in montpellier and that of philippe menaschÉ (b. ) in paris, provides interesting information about how mouse embryonic stem cells, grafted into sheep cardiac tissue where an infarctus has been artificially induced, are able to colonize the infarct zone and regenerate cardiac contraction in a functional manner. moving from the mouse to the sheep constitutes a considerable species leap, and the absence of any immune rejection leads us to say that embryonic stem cells have an "immune privilege" . the use of es cells in regenerative medicine necessarily requires that their differentiation be regulated in an exhaustive manner into well-defined pathways, in order to produce homogeneous cell lines with a view to implanting them in damaged tissues. in fact, contamination with non-differentiated es cells is likely to cause tumors (teratomas) over the long term. the mastering of the use of es cell culture and differentiation, as well as of cloning, in such a way as to overcome problems of histocompatibility, is still in its infancy. for a long time, the mouse was the preferred animal model for experimental studies on the differentiation of es cells. in , the first es cells from mouse blastocysts were isolated and successfully cultured by two groups of researchers in great britain and the usa. it was only in that human embryonic stem cells (hes) were isolated for the first time and held in culture, on a nutritive layer of fibroblasts from irradiated mice. this delay with respect to the ability to culture animal es cells can be explained by the fact that the molecular machinery that activates replication and cell differentiation programs is not completely identical in man and the mouse . for example, a cytokine called lif (leukemia inhibitory factor), which is indispensable for the renewal of es cells in an undifferentiated state in the mouse, has no effect on human es cells. there are several other differences concerning the control of proliferation and differentiation in human and murine es cells by growth factors. briefly, the conclusions obtained from experiments carried while there is a highly promising future for the use of es cells, this future is littered with obstacles, and rigorous checks and balances need to be put in place. nevertheless, research on such cells is mandatory if we wish to move on to a regenerative medicine that aims to be a new frontier in the art of healing. after specific differentiation, hes cells could provide unlimited quantities of the tissues needed to replace damaged tissues responsible for handicapping illnesses (dopaminergic neurons in parkinson's disease, cardiomyocytes in myocardial infarction, pancreatic islets of langerhans cells in diabetes, fibroblasts in skin grafts, chondrocytes in rhumatoid arthritis). in addition, metabolic analysis of hes cells carrying defective genes whose phenotypical expression is known in human pathology should improve our understanding of the perturbed mechanisms, and could lead to pharmacological advances. as well as the technical difficulties involved, which have not yet been adequately overcome, the handling of hes cells is subject to much ethical debate in many countries, with those who object to it holding to their prejudices, which are linked to religious or cultural traditions. this is the case in france, where, nevertheless, a few timid dispensations had begun to appear at the time of writing. in contrast, in great britain, the law authorizes the isolation of hes cells for therapeutic purposes, using embryos of less than one hundred cells, produced by in vitro fertilization, and surplus to requirements. the british response to the burning question of whether an isolated hes cell may be considered as a potential human embryo is clearly "no", for, in order to be able to develop in utero, such hes cells would need to have the placental progenitor cells. an alternative to the use of es cells is to make use of adult stem cells. however, the proliferation capacity of adult stem cells is considerably lower than that of their embryonic homologues. the hematopoietic stem cell is the paradigm of the adult stem cell. it can differentiate into all known types of cells. in the last decade of the th century, several publications concerning the plasticity of the adult stem cell awakened a hope that these cells could transform the treatment of degenerative illnesses. certain of these publications stated that adult bone marrow stem cells, implanted into different types of tissues, differentiate into hepatocytes, cardiomyocytes or neurons, depending on the specific environment. careful re-examination of the techniques used revealed that, in certain cases, interpretation of the results as showing cell transdifferentiation was an erroneous one, and that the fusion of the bone marrow stem cells with cells from other tissues was a more plausible explanation. in any case, while not ignoring the use of adult stem cells, experimentation on hes cells remains a judicious choice, given our current state of understanding. in france, the law application decree that was issued on the th of february , revising the restrictive bioethical standards of , opens up the possibility of using human embryonic stem cells for scientific purposes, with certain ethical reserves being maintained. one of the obstacles to the stabilization over time of a stem cell graft in a receiver involves the phenomenon of rejection for reasons of histocompatibility. considered to be foreign by the receiver (host), grafted stem cells coming from a donor are rejected. this obstacle could be overcome by using the technique of cloning. based on experiments on several animal species, it is now accepted that the transfer of the nucleus of an adult somatic cell from a host into an enucleated oocyte makes it possible to obtain from this oocyte, which is once again nucleated, and which is the equivalent of a zygote and able to divide, es cells whose genome is identical to that of the host. because of this, the es cells are immunologically compatible with the tissues of the host. in man, such cells could be directed by differentiation towards stable cell lines creating well-defined tissues and organs (liver, muscle…) that could be used in regenerative medicine. this is the principle of therapeutic cloning. in march , korean veterinary researcher woo suk hwang (b. ) and his co-workers, who were recognized experts in animal cloning, announced in the american review science that they had succeeded for the first time in obtaining around thirty human blastocysts by cloning, i.e., by the transfer of nuclei of somatic cells into enucleated ova. this first experiment involved autologous cloning (ovum nuclei and enucleated somatic cells taken from the same woman). hwang and his team used ova, and the yield from the experiment was close to that obtained at that time for the cloning of mammals. using the inner cell mass of one of the blastocysts, they isolated a line of embryonic stem cells able to maintain a normal karyotype after several dozen divisions. this publication, which appeared in a highly prestigious scientific review, triggered an enthusiasm in the media that was in keeping with the spectacular nature of the team's exploit, tempered here and there by a few comments that were mainly linked to questions of medical ethics. in , there were numerous other articles by the same team on the same subject, reinforcing the first results with a heterologous cloning technique (ovum nuclei and enucleated somatic cells taken from different people), thus giving rise to great hopes that the era of regenerative medicine was near. at the beginning of , professor hwang's retractation of all his work, and a public confession of a spectacular fraud, were even more dramatic, offering certain media an occasion for a disproportionate level of fury against therapeutic cloning. however, despite such rear-guard combats, it is obvious that one day these technical difficulties will be overcome. human cloning, in order to obtain stem cells for therapeutic purposes, cannot escape the future. once this aim has been achieved, it will be spoken of as the outcome of a long story. the adventure of animal reproductive cloning began in . in developmental biology , two american researchers, robert briggs ( - ) and thomas king ( - described experiments involving the transfer of cell nuclei of embryos from a frog (rana pipiens), at the blastula and gastrula stages, into enucleated eggs of the same species. a high percentage of the clones obtained in this way were able to reach the tadpole stage when the transferred nuclei came from the early blastula stage, but only mediocre success was achieved when the nuclei came from the later gastrula stage. these experiments emphasized both the totipotency of the embryo somatic cells and the equivalency of the somatic cell nucleus and the nucleus of the fertilized egg in cell division and differentiation. briggs and king's publication did not arouse any particular interest. it is true that the s were dominated by the saga of molecular biology, which would reach its culmination in the deciphering of the genetic code. from the s onward, the first attempts to clone mammals (rat, mouse, pig) began. moving from the amphibian egg, which was a millimeter wide, to a mammal egg that was one hundred times smaller, presented a technical difficulty that would be overcome by a technique of cell-to-cell electrofusion. cloned embryos were thus obtained by nuclear transfer and then implanted into the uterus of a surrogate female. however, in all cases, the nucleus came from embryo cells. in february , the announcement made by ian wilmut (b. ), keith h. campbell (b. ) and their collaborators at edinburgh's roslin institute of the birth of the cloned lamb dolly had an immediate effect in the media. in fact, this was not only the cloning of a higher mammal, but, above all, the cloning by insertion of an adult somatic cell, in this case a mammary tissue cell, into an enucleated oocyte. this went far further than the experiment carried out by briggs and king, which essentially involved the transfer of embryo cell nuclei into enucleated frog eggs. the trick that gave wilmut and campbell their success was to bring the cells providing the nuclei to a quiescent state corresponding to the interphase stage of the cell cycle, by impoverishing their culture medium, before electrofusion with enucleated oocytes. although we should be aware that attempts were made before a positive result was achieved, this does not make it any less astonishing that the nucleus of a cell in its adult state, i.e., completely differentiated, was able to behave as if it were totipotent. despite being committed to a program of differentiation that is considered to be more-or-less irreversible, and which will give it a specific identity, the nucleus of an adult cell can be reprogrammed and become totipotent. since dolly, many other mammals have been cloned from nuclei of adult cells; mice, cows, goats, pigs, rabbits, cats, dogs, rats and horses. as far as ethical discussion about cloning is concerned (chapter iv. ), it is essential to note that the demarcation line between reproductive cloning and therapeutic cloning is situated where decisions are made concerning the destiny of the cloned blastocyst ( figure iv . ). the transfer of the nucleus of a somatic cell (liver, epidermis, muscle) containing n chromosomes into an enucleated oocyte gives rise to an egg ( n chromosomes) that is able to divide and to produce a blastocyst. the cells of the blastocyst inner cell mass (icm) can be used as stem cells that can differentiate into different types of cell line (therapeutic cloning). on the other hand, if the whole blastocyst is implanted into a uterus, it will produce an embryo which, after birth, will grow into an adult animal (reproductive cloning). reproductive cloning and therapeutic cloning therefore differ because of the fact that in reproductive cloning, the whole blastocyst is used, while in therapeutic cloning, only certain cells, corresponding to the inner cell mass (icm) of the blastocyst, are used. the structural and functional identity of the cells of a given tissue in an adult organism involves a basic mechanism: while each cell has the same set of genes, only some of the genes are expressed as proteins and the genes that are expressed differ according to the tissue involved. the key to the mechanism responsible is in the epigenetic type chemical modifications of cell dna, for example methylations, which repress the expression of certain genes without altering the expression of others. these modifications of the dna, which control cellular specificity (muscle, liver, brain…) are not very reversible, but, in certain circumstances, they can become so. this is what happens from time to time when the nucleus of an adult cell is inserted into an enucleated oocyte. we are thus able to assume that in the molecular arsenal of the oocyte cytoplasm there are substances that can cancel the epigenetic modifications of the dna present in the nucleus of an adult somatic cell and recreate a state of pluripotency in this nucleus, or, in other words, provoke the reprogramming of the somatic cell nucleus. in the long term, it is to be hoped that biochemical technology will be able to find and purify the molecules responsible for the nuclear reprogramming of somatic cells. the use of human oocytes for the purpose of therapeutic cloning is still subject to severe criticism. certain groups wish it to be prohibited, because of a fear of a drift towards reproductive cloning. to obviate this risk, the idea has been to make use not of human oocytes but of those of animals, transferring the nuclei of human somatic cells into them. even supposing that the technical difficulties involved could be overcome, the cells that would result, a sort of man-animal chimera, would also be the subject of an ethical debate, even if the purpose of this type of cloning were to be solely therapeutic. some japanese researchers have succeeded in creating mice according to a parthenogenetic process that involves adding the nucleus of an oocyte that is haploid ( n chromosomes) to another haploid oocyte, the result being the equivalent of a fertilized egg ( n chromosomes). this exploit is achieved by the invalidation of one of the genes (h ) involved in the control of the parental imprint. it is known that sexual reproduction in mammals involves a phenomenon called the parental imprint, which, by means of the methylation of dna and perhaps also of histones, allows the expressing or silencing of certain genes in male and female gametes. a single copy of a given gene, originating either from the oocyte or from the sperm cell, is therefore expressed, while the other is inactive. in the japanese experiment, if the mouse h gene had not been invalidated, the result would have been an anarchical development of the responsible genes involved in the parental imprint with overexpression in the case of some and an absence of expression in the case of others. these disturbances would have been incompatible with the viability of the embryo. however limited its application might be, the manipulation of the germinal genome poses the problem of the mechanism by which the parental imprint intervenes in the viability of the egg, a parameter that at the time of writing is still not completely understood, but is being actively explored. in boston, massachusetts, in , a kidney was transplanted from a healthy boy into his twin brother, who was suffering from a fatal renal anomaly. the success of this graft ushered in the era of transplantations of such organs as the heart, liver and kidney in man. in order to try to prevent the rejection of grafts, caused by an immune incompatibility between the receiver and the graft from the donor, different immunosuppressing treatments were tried, one by one, involving corticosteroids or cytostatic agents such as -mercaptopurine. in the s, a decisive step forward was made with the fortuitous discovery of the powerful immunosuppressive effect of the cyclosporin a, a cyclic polypeptide isolated from the mold tolypocladium inflatum. each year, human organ transplants into patients make it possible to save many lives. however, for some time now, organ transplantation has been suffering from penury of donors. one alternative to the homograft is the grafting of animal organs, or the xenograft, and, at the dawn of the st century, this type of graft has entered an active, promising phase, with the creation of pigs that have been partially "humanized" and are thus, as a consequence, immunocompatible. for reasons of genetic and physiological similarity, the first choice for such grafts was to use apes or monkeys. however, this idea was quickly abandoned, for several reasons; a non-negligible risk of viral infection due to the phylogenetic kinship of human and simian species; a slow growth rate; a low reproduction rate and, finally, laws that protect primates. these disadvantages are not found, or are at least minimized, in the pig: the risk of a viral infection passing from the pig to man should be low because of the species barrier (but nevertheless, it ought to be evaluated), the pig growth rate is relatively rapid, pig litters are large and pig organs are of a size close to those of man. hyperacute rejection of grafts is the critical obstacle that must be overcome before it is possible even to envisage the feasibility of xenotransplantation. hyperacute rejection is caused by the presence in man of natural antibodies (xenoantibodies) that accumulate throughout a lifetime and are directed against antigenic motifs carried by the products of the digestion of food or dust that is breathed in. xenoantibodies are mobilized when a xenograft occurs, and when they combine with xenoantigens brought by the graft this activates immune proteins such as the complement proteins. the catastrophic effect of this xenoantibody/xenoantigen combination is a vascular thrombosis followed by necrosis and rejection of the graft. the pig xenoantigen that is considered to be the one mainly responsible for the phenomenon of rejection in man is a sugar molecule, galactose α- , -galactose, located on the plasma membrane of endothelial cells. synthesis of this molecule requires the enzyme α- , -galactosyltransferase, which is present in most mammals, but absent in man and the primates. this enzyme disappeared in man around twenty million years ago, following a double mutation of the gene. in , cloning by nuclear transfer, associated with the invalidation of the gene coding for galactosyltransferase, made it possible to create pigs without galactose α- , -galactose . this performance shows that the xenotransplantation objective, although it can only be envisaged over the long term, is not based on false hopes. plant gmos, gene therapy, embryonic stem cells, therapeutic cloning, and xenotransplantation are a few of the many examples that show how far experimentation on living beings has progressed in just a few decades, from inquiries into the operating mechanism of an organ or a cell, in the interests of pure understanding, to a programmed process, planned with an objective in mind, the chances of success of this objective being analyzed and counted in terms of impact and cost-effectiveness. during the renaissance, ecclesiastical authorities, worried by the libertarian forces that were assailing them, applied the brakes to audacious questioning of dogma such as the circumterrestrial revolution of the sun that had, since ancient times, placed man at the center of the universe. nowadays, civil authorities, conscious of the potential but also of the possible misuses of genetic manipulation, insist on having the right to oversee such procedures. in truth, since the th century, governments have been interesting themselves in research on living beings and encouraging it, as long as its applications have allowed improvements in human health. this has been the case for vaccinations against infectious diseases or for prevention of microbial infections by means of aseptic or antiseptic methods. with the breakthroughs made in genetic manipulation at the end of the th century, it was more than just the results of experiments on living beings that attracted the attention of the political authorities, it was, above all, the manner in which the experimental method, with all its hazards, made use of living material, sometimes of human origin, in order to unlock mysteries. conscious of the social impact of emerging discoveries that are subject to considerable media coverage and are sensationalized in both the written and audiovisual media, the state, with the help of researchers and philosophers, has laid down a code of bioethics, applied through strict or even restrictive legislation. it remains to be seen whether the rules of this code will continue to be an inviolable absolute or will be modified according to the evolution of the moral codes and the cultures of nations. university teaching and the education of a society must now take into account not only the content of successive discoveries, but also the fallout of these discoveries, insofar as they concern man, and even the ethical justification of the methods that have allowed these discoveries. in "remaking" living beings according to imposed norms, and in scheduling, in a certain fashion, the manufacture of life according to new codes, certain questions move from the "how" to the "why", i.e., from the scientific domain that is accessible to human thought to the metaphysical sphere, with its problems of the limit of what is surmountable and tolerable in terms of ethics. in his birth of predictive medicine, jacques ruffiÉ ( - ) reminds us that medicine has evolved through three stages over the course of time: curative medicine, which has been practiced since ancient times and is still being practiced; preventive medicine, which is more recent, and is designed to prevent people from falling ill, either by vaccinating them, in the case of infectious diseases, or by recommending an appropriate diet and medication in the case of metabolic disorders such as diabetes or arterial hypertension that have been detected by means of systematic examination; and, finally, predictive medicine, a branch of medicine that is still in its early phases, and which is based on modern technology and is able to predict situations of risk because of anomalies detected in the genetic inheritance or because of exposure to environments that are reputed to be dangerous (for example, carcinogenic smoke, asbestos). about one and a half centuries ago, the publication of the introduction to the study of experimental medicine ( ) provided proof, based on scientific arguments, that the time had come to transfer the experience that had been acquired through the experimental method practiced on animal models to the ill person. after claude bernard, attentive to the progress made by ideas and techniques in the physical and chemical sciences, and making use of its own advances in the understanding of the living cell, both normal and pathological, experimental medicine was to live through a development that was without precedent in the history of humanity. to understand the causes of epidemics, nutritional deficiencies, metabolic deviations of hereditary origin and degenerative illnesses, and then to translate these causes in cellular and molecular terms, this was the process undertaken by medicine once it began to use the experimental method. in fact, for several decades, from the beginning of the th century, medicine had already undergone some major revisions of outdated practices and had inaugurated a new era in diagnosis. for example, the differential diagnosis of pulmonary ailments became possible because of the invention of the stethoscope by rené laennec ( - ) and the practice of auscultation and percussion by joseph skoda ( - ), the uncontested master of the vienna school. in france, pierre louis ( - ) used statistical methods to evaluate the efficacy of different treatments. armand trousseau ( - ), a pupil of pierre bretonneau ( - ) wrote a famous treatise on the hôtel-dieu medical clinic in paris. in great britain, chronic nephritis, with its identifying symptoms, was described by richard bright ( - ), paralysis agitans by james parkinson ( - ) and addison's disease, which affects the adrenal glands, by thomas addison ( - ). during the th century, many other famous names signaled the arrival of a medicine that was resolutely anatomoclinical in nature, in line with bernardian doctrine. "experimental medicine is thus a medicine that claims to understand the laws of the organism in sickness and in health, in such a way that it not only predicts phenomena, but also in such a way that it can regulate and modify them, within certain limits." in the introduction to the study of experimental medicine, claude bernard stigmatizes the relics of an empirical medicine that was still being practiced in his day and was forgetful of rationalism. the terms that he uses are without leniency: "i have often heard doctors who, when asked the reason for a diagnosis, reply that they don't know how they recognize such a case, but it is obvious, or who, when asked why they administer certain remedies, reply that they don't really know how to put it exactly, and that anyway they are not required to give a reason, because it is their medical tact and their intuition that guides them. it is easy to understand that doctors who reason in this way are denying science. what is more, it is impossible to be too forceful in rising up against such ideas, which are bad not only because they stifle any scientific seed in the young, but also, above all, because they favor laziness, ignorance and charlatanism." in order to evaluate the meaning of these words, it should be remembered that in claude bernard's time, the medical profession was far from considering the microscope as a useful instrument for the study of cell structures and that the cause and effect relationship between bacterial germs and infections was still to be shown. with the development of increasingly effective instruments for exploration, and of methods for microanalyses concerning a wide range of blood and humoral constants, throughout the th century, medicine, which was once empirical, has now become scientific. claude bernard's dream, experimental medicine, is now operative. this medicine is no longer content simply to determine the cause of an illness and to locate the affected organ, which was the major objective of clinical medicine, but it seeks to detect the mechanisms of pathological processes by means of histological and physicochemical explorations. this medicine is no longer willing to passively monitor the evolution of an infectious disease. after having identified the responsible germ, it tries to target this germ with the chemical weapon that is able to selectively destroy it. this medicine is no longer content simply to find remedies, it aims to understand the mode of action. it sets itself the goal of meeting challenges such as finding the genetic cause of degenerative illnesses or of cancers and developing appropriate therapies. it is supported by statistical data. when a new drug is implemented, the results are now evaluated by the double blind method: neither any of the patients (treated and non-treated) nor any of the investigators are aware of who has been administered with the drug and who has been administered with a placebo. in the surgical domain, audacious techniques have also led to considerable progress, particularly in neurosurgery and in cardiovascular surgery. thanks to robotics and to computer technology, remote surgery or telesurgery has become practicable, although up until not that long ago, it was only to be found in fiction. faced with emerging problems in public health, the task undertaken by experimental medicine is immense. in the middle of the th century, the spectacular recovery from high-incidence infectious diseases such as pneumococcal pneumonia, meningococcal meningitis or acute forms of tuberculosis, which that was brought about by antibiotics, gave rise to the idea that medicine had won a battle against the microbial world and that, from then on, it would be able to control the evolution of infectious diseases and to offer rational treatments. the gradual appearance of a microbial resistance to antibiotics has brought an end to this euphoric era. penicillin, for example, which was put on the market at the end of the s, was active on practically all strains of staphylococcus aureus. sixty years later, more than % of the strains of this same microbe are resistant to penicillin. the incidence of nosocomial infections, which are contracted in health care facilities, never ceases to rise. at present, around % of the hospitalizations that take place are complicated by the patient developing a nosocomial infection. equally worrying are the re-emergence of diseases that were once considered to be under control, such as tuberculosis or poliomyelitis in africa, and the emergence of new diseases such as aids, whose hiv virus (human immunodeficiency virus), which was identified at the beginning of the s, has generated a pandemic that has spread throughout the planet. infectious diseases are currently responsible for more than a quarter of human deaths. the koch bacillus responsible for tuberculosis and the pneumococcus kill three to four million people a year, around the world. in , hiv killed more than three million people, and more than forty million people are infected. one person is infected every seconds. in viral diseases, the role of vectors (insects, various animals) as well as the notions of contagiousness and aggressivity have been emphasized. we have only to remember the dreadful contagiousness and aggressivity of the spanish flu virus (influenzavirus ah n ) which, in - , killed more human beings around the world than the first world war that preceded it. in contrast, the sars (severe acute respiratory syndrome) epidemic of , the vector of which was doubtless the civet, a small carnivore raised in china and desired for its meat, was rapidly contained because of its low contagiousness and also because of the isolation measures that were taken. human behavior is not without its effect on the emergence of viral diseases. the growth in intercontinental travel and human migration, as well as intensive deforestation in africa and south america, which bring virus vectors into contact with man, are factors concerned in the emergence of viral diseases that risk being explosive and devastating. in this context, the history of the ebola virus and of the marburg virus, which cause violent hemorrhaging, is edifying. in , in the german village of marburg, an epidemic of unknown origin broke out, the illness manifesting itself with brutal suddenness by vomiting, diarrhea, a high fever and an increased tendency to bleed. this pathology, which was contained rapidly by means of drastic isolation measures, was found to be of viral origin. the pathogen concerned was a filovirus (filiform virus). a brief enquiry showed that the origin of the epidemic was contact between technicians of a pharmaceutical company and monkeys that had been imported from uganda and that were carrying the virus. in , two other epidemics, characterized by severe and often fatal hemorrhagic fevers, were reported in the sudan and the republic of the congo. here again, the illness was caused by a filovirus, the ebola virus. at the time of writing, only public health organizations, including the nih (national institutes of health) in the usa, have attempted to set up vaccination and therapeutic strategies. research on these dangerous viruses requires high security installations that are particularly costly, so that private companies are reticent about investing in work that is only targeted on poor regions and which concerns epidemics that have so far been contained successfully, although one day the ebola and the marburg virus could quite well escape their african niches. experimental medicine must also understand the colossal challenge of the five thousand hereditary diseases that are currently listed, the most handicapping of which are myopathies and neuropathies. given the means that are available to the contemporary clinician in order to assign each of these diseases to a genetic defect, one can only be amazed by the mass of information about them that has accumulated over a century, since the first diagnosis of a hereditary disease, alcaptonuria, which was made in by archibald garrod ( - ), a doctor at london's st bartholomew's hospital. alcaptonuria is a non-serious genetic flaw that can be detected easily by a blackening of the urine. it is the result of a blockage caused by the mutation of an enzyme involved in the catabolism of an amino acid, tyrosine, this blockage leading to the accumulation of homogentisic acid, the polymerization of which gives rise to a brownish color. the patient examined by garrod was a young boy. investigation of the family history revealed that transmission of the flaw was correlated to cross-cousin marriages and followed mendel's laws for recessive traits. garrod demonstrated other hereditary-type anomalies, cystinuria, porphyria and pentosuria. in , these observations were published in a work that became a classic: inborn errors of metabolism. in , the specific molecular defect of a metabolic anomaly linked to a mutation was identified for the first time by the german-born british biochemist vernon ingram. this was the hemoglobin defect responsible for drepanocytosis or sickle cell anemia: a glutamic acid in the β chain is replaced by a valine. the consequence of this simple change is a modification of the structure of the hemoglobin, leading to a sickle-shaped deformation of the red blood cells, the increased fragility of these cells and also a tendency towards cell lysis. this discovery made use of the electrophoresis and chromatography techniques that had just been introduced in biochemistry (chapter iii- . . ): such a discovery would not have been possible without these techniques. because of the progress made in molecular biology, the nosological framework of hereditary diseases has been greatly enriched over the last twenty years. for example, at present, more than one hundred hereditary-type myopathies have been identified by accurately locating molecular lesions in the genomic dna and characterizing the structural and functional modifications of the mutated proteins. certain health problems present real challenges for experimental research. this is the case for the spongiform encephalopathy caused by a prion (proteinaceous infectious particle) , which has all the more impact on the imagination because its etiology remains a mystery. it is also the case for degenerescence of the central nervous system correlated with aging, alzheimer's disease being a striking example, although, as far as familial forms of this illness are concerned, i.e., those of the hereditary type, it has been possible to link the invasion of the brain by a so-called amyloid peptide, which accumulates in plaques, on the one hand, and, on the other hand, the absence, due to a mutation, of an enzyme, a peptidase, which normally degrades the amyloid peptide. contemporary scientific medicine sometimes acquires a revolutionary aspect. here again, as with other disciplines involved in the study of living beings, it arises from discoveries resulting from the principle of serendipity (chapter iii- . . ). this was the case when, in january , a team in grenoble, france , led by the neurosurgeon alim-louis benabid (b. ) and the neurologist pierre pollack (b. ) discovered by accident that in patients affected by parkinson's disease a beneficial effect was achieved by deep, high-frequency electrical stimulation of the brain. the three major symptoms of parkinson's disease are muscular rigidity, a tremor when at rest and a slowing down of the execution of movements. in the s, the swedish team of arvid carlsson (b. ) , who won the nobel prize for physiology and medicine, demonstrated a relationship between the parkinson syndrome and a deficit in the secretion of a neurotransmitter, dopamine. a group of neurons that is limited to half a million (of the billion contained in the brain) produces this neurotransmitter in a small structure located in the midbrain, called the substantia nigra. the neurons of the substantia nigra have elongations that interact with different nerve formations (called nuclei) including the subthalamic nucleus. in , bergman et al. published an article that describes a curious relationship between a provoked lesion of the subthalamic nucleus and the disappearance of the signs of parkinson's disease in a monkey which had been made parkinsonian by chemical treatment. this publication led the team in grenoble to target their electrical stimulation on the subthalamic nucleus. this was completely successful. this electrical stimulation procedure, which is now well-codified, involves using stereotactic neurosurgical techniques, controlled by magnetic resonance imaging, to implant an electrode into the subthalamic nucleus. the electrode is connected to a generator that is implanted under the patient's clavicle. the generator sends brief electrical impulses of frequencies from to hz. under the effect of this stimulation, the characteristic symptoms of the illness, particularly the static tremor and bradykinesis, regress in a spectacular manner. the mechanism by which this stimulation acts is not yet understood. no doubt this has to do with complex phenomena involving the inhibition of certain neuron relays near to the substantia nigra, which remain to be deciphered. here we have a typical case of a progression from an experimental fact, discovered by accident, towards the analysis of its cause. from the point of view of the experimental method, it is interesting to make parallels between this discovery by serendipitous means of the beneficial role of electrical stimulation of the midbrain in parkinson's disease and cartesian style programmed research that aims to graft into the brain of parkinson's disease sufferers embryonic stem cells differentiated into dopaminergic neurons . civilian society and its armed force, the political authorities, have understood that experimental science has the tools, the method and the thought processes necessary to develop strategies for prevention and healing. immune cells of this person and induces the synthesis of immune proteins. finally, it seems relatively certain that hopes concerning gene therapy for hereditary diseases will be fulfilled within before too long (chapter iv- . . ). one of the traits that is characteristic of the period we live in, and which arises partly from the economic stakes involved, is the shortening of the time that elapses between a discovery being made and the application of that discovery. for example, interfering rnas, which were discovered in the s (chapter iv- . . ) are already the subject of therapeutic investigation. more than a hundred biopharmaceutical companies around the world are using them with a view to producing drugs from them . in mice, a certain number of synthetic interfering rnas have proved their efficacy in silencing genes which, following mutation, have acquired carcinogenic potential. however, the use of interfering rnas as therapeutic agents requires them to be stabilized, because they are fragile molecules. the group headed by achim aigner (b. ), at the school of medicine in marburg, germany, managed to stabilize a synthetic interfering rna by complexing it with polyethyleneimine, and this interfering rna is able to block the expression of a receptor involved in cancerization (c-erbb /neu(her- ) receptor). used in mice, such a drug appears promising. despite the undeniable progress that has been made, experimental medicine is still some way from finding solutions to some of the enigmas that it meets along the way, and which underline the complexity of living beings. some time ago, it was thought that after having invalidated a gene coding for a protein that is indispensable to a function, we would discover the secret of a cause-and-effect relationship. experimental practice has shown that, generally, this is far from being the case. another example of the complex relationships that exist in living beings is the interference of the mental and the organic. one experiment that suggests this interference was carried out on mice who had acquired a form of pathology similar to huntington's chorea, by transgenesis. mice from the same line were separated into two batches, one acting as a control, and the other being subjected to daily mental stimulation, including memorization tests. unexpectedly, the appearance of symptoms was noticeably slowed down in mice who had been subjected to mental gymnastics , as if the brain, by intentionally mobilizing its neuron activity, was able to secrete substances able to alleviate its own defects. in short, by means of possible retroactive mechanisms that are called upon by the mind, the brain appears to act as actor and spectator. at the turn of the st century, experimental medicine was being nourished by techniques inherited from experimental physics, chemistry, and even mathematics and computer technology, in the same way as the other sciences of living beings. the progress made in medical imaging techniques has been particularly impressive since the time, at the end of the th century, when the x-rays discovered by wilhelm rÖntgen made it possible to view the structure of the human skeleton. the saga of x-radiation continued through the th century (chapter iii- . . ). for the last few decades, new imaging techniques have come to the fore. they have spread rapidly, and been refined. ultrasound imaging, which is based on the principle of the reflection of ultrasound waves off of different kinds of surfaces, has become an everyday technique for viewing blood flow in blood vessels and the heart. however, it is mainly in the study of the brain that medical imaging has benefited from technical advances in the domains of physics and computer technology, and it has been innovative in assigning cognitive activities to well-identified anatomical structures. this functional neuroanatomy makes it possible, in a non-trauma-inducing manner, to monitor and locate the operation of neuron networks with great temporal and spatial precision, during various cognitive tasks such as reading and the written or oral expression of thought. the middle of the th century saw the gradual development of two methods for exploring zones of cerebral activity, electroencephalography and magnetoencephalography. at present, these techniques are being taken over by mri (magnetic resonance imaging) ( figure iv. ) . the principle of mri is based on the detection of hydrogen nuclei and their differentiation according to their environment. functional mri leads to the location of the areas of the brain that are active during calculation exercises, the perception of sounds, language and objects, and memorization, with a resolution of just a few millimeters. its power of exploration is such that it has been possible to analyze the brain response, in sleeping or awake babies who are only three months old, to auditory stimuli from language that either makes sense or does not make sense . the response, located in the left hemisphere and the prefrontal cortex, leads to the conclusion that, from the first months of life, there are zones of the brain that are potentially active before the first attempts at language appear. both in france (cea-saclay and the frederic joliot hospital at orsay) and abroad, recent mri performance has encouraged projects concerning the manufacture of instruments able to produce magnetic fields of around ten teslas, which allows an unequaled definition in the identification of areas of the brain assigned to specific cognitive functions and in the highly accurate determination of the location of pathological lesions. a technique that is complementary to mri is positron emission tomography (pet). this generally uses water labeled with oxygen ( o), a radioactive isotope of natural oxygen that has a very short lifetime ( s), produced extemporaneously in a cyclotron by bombardment of an n target with protons. the radiolabeled water is injected into the blood flow of the patient. it is found in greater concentration in the zones that are the most irrigated by blood capillaries. the positrons that it emits collide with the surrounding electrons and give rise to photons that can be detected by the appropriate apparatus. affected by a stimulus (whether this stimulus results from talking, writing or listening), the blood irrigation of the zones of the brain that have been specifically excited increases noticeably. the location of the positron emission provides information about the location of these zones. within a few dozen minutes, it is possible to locate a highly vascularized cerebral tumor. pet can use molecules other than water, such as organic molecules labeled with positron-emitting atoms, ( f) fluorine (half life min) and ( c) carbon (half life min). around twenty years ago, in canada, an analogue of l-dopa, the precursor of dopamine in the brain, f- -l-fluorodopa, was synthesized, and was found to be an excellent probe for determining the capture capability of the endings of the dopaminergic neurons in the striatum. in patients suffering from parkinson's disease, this capture capability is noticeably reduced. at present, pet involving f- -l-fluorodopa is being used to evaluate the survival of dopaminergic cells grafted into the striata of parkinson's disease sufferers , . nowadays, brain imaging techniques can be used to explore the electromagnetic anomalies of neurological or neuropsychiatric illnesses such as huntington's chorea, the different forms of alzheimer's disease or even autism, the genetic origin of which is in the process of being deciphered. a bridge has now been built between the molecular defects identified by genetics and the electromagnetic anomalies that result, analyzed by functional cerebral imaging. it was not so long ago that descartes considered that human thought was unconnected to a material support (chapter ii- . . ). we are not far from the era when broca located the language area in a specific zone of the brain after the autopsy of an aphasic patient (chapter iii- . ), thus opening the door to another scientific domain, neuropsychology, which had previously only been the subject of speculation. the consequences, from the societal point of view, were far from being insignificant. thus autism, which was once suspected of being caused by errors in the mother's behavior with respect to her child, has been shown to be a disturbance in the development of the fetal nervous system, in the temporooccipital region. while the neurosciences occupy a preponderant position in the medicine of the beginning of the st century, because of the development of techniques that aim to analyze even the functions of thought, emerging methodologies of another order, such as gene therapy (chapter iv- . ), are in the process of completely modifying our ways of treating and curing a range of previously incurable human diseases, from incapacitating immune disorders to cardiovascular diseases and cancer. "it is in the domain of thought about the future that man is singled out. we are beings who have an imagination. not content to live in the present, to profit from past experience, we remain haunted by a future that we are conscious of constantly entering. this obsession with the future has been a powerful driving force in cultural evolution. we seek to predict in order to avoid the worst and to better prepare for our tomorrows." by predicting potential dangers in subjects who are in good health, predictive medicine aims to provide the means of avoiding these dangers. these dangers can be intrinsic in nature, being written, for example, into a certain genome dna sequence, or they can be extrinsic in nature, linked to an unsuspectedly deleterious environment. in each generation, mutations occur, certain of which can lead to so-called genetic diseases; between and % of newborns are affected. besides these spontaneous mutations, there are also mutations arising from the genetic inheritance of the parents. the purpose of genetic counseling is to warn parents when the existence of a potentially serious genetic flaw is suspected. the highlighting of genes that give a predisposition for cancer (proto-oncogenes) is a convincing illustration of the power of predictive medicine. this involves genes that control the synthesis of growth factors, the activity of which is essential to embryogenesis and to the repair of damaged tissue. while they are normally subject to strict control by anti-oncogenes, proto-oncogenes are able to become active in an anarchical manner, under different influences, and to transform themselves into cancer-generating oncogenes. recently, mutations have been found in two genes, brca and brca , these mutations giving a predisposition for cancers of the breast and of the ovary. thanks to genetic exploration, it will soon be possible to predict whether a cancer of the breast will have a rapid progression leading to uncontrollable metastases or a slow progression. depending on the case patients will be subject to heavy chemotherapy or to a less aggressive treatment. in this context, targeted therapy with monoclonal antibodies is a source of great hope. while genetic inheritance has a role in cancer, the environment plays a notinsignificant role as well. this is the case, for example, in lung cancer sufferers who smoke tobacco, cancer of oesophagus in those who drink alcohol and job-related cancers in those working in factories producing colorants or materials derived from asbestos or tars. cardiovascular diseases are the primary cause of death in the more developed countries, involving either an infarctus, or a stroke. many risk factors for these diseases are known, i.e., metabolic deviations affecting cholesterol or the blood serum proteins involved in the transport of lipids. these metabolic anomalies result in a syndrome known as atherosclerosis, which is characterized anatomically by the deposit of fats in the form plaques in the arteries. while genetic factors are at the origin of these metabolic problems, the latter are clearly amplified by an inappropriate diet. the role of predictive medicine is to recognize the genes that are responsible, warn individuals of the risks they are running and to advise them about the types of lifestyle and diet that do not increase these risks. being able to predict, predictive medicine should be able to prevent by means of targeted drugs. within this context, it gives rise to reflection upon polymorphism linked to variation in a single nucleotide in the dna of the genome of an individual. known as snp (single nucleotide polymorphism), this polymorphism has proved to be a very useful auxiliary in molecular medicine. hundreds of thousands of snps are present in the human genome and several tens of thousands in genes coding for the proteins. where they are located differs according to ethnic backgrounds. among these snps, some appear to be linked to certain pathologies, such as certain forms of cancer or degenerative illnesses such as alzheimer's disease. in addition, in a small number of patients, the location of certain snps has been connected with previously-inexplicable drug incompatibilities. in line with these observations, pharmacogenomics, a branch of pharmacology that deals directly with genome sequence data, is trying to evaluate the impact of "snp variants" on the efficacy and toxicity of drugs and to understand the genetic bases that explain the differences that are observed in the responses of different individuals to the same medication . rather than using a standard drug that is not very efficacious or causes adverse side effects, it might be possible, depending on the genetic profile of the patient, to use a drug that is more appropriate to his or her genetic map. it is doubtless not just a fantasy to imagine that, in or years' time, a patient visiting the doctor will be offered a genetic map thanks to cells taken from the buccal mucosa. finding snp variants that are known to be responsible for drug incompatibilities in such a map will make a targeted prescription possible. it will allow the detection of genes for susceptibility to an illness, at the same time uncovering targets for new drugs. pharmacogenomics, which is still called new pharmacogenetics, contrasts with old pharmacogenetics in which, having found an adverse clinical response to a certain therapy, an attempt was made to identify the protein target of the incriminated drug, and then to go back to the gene coding for this protein, and to look for the mutation responsible for the aberrant response to the drug. the existence of customized predictive medicine, which would read the destinies of individuals in their genes, would not be without its consequences in the life of a citizen. by registering each citizen with a genetic map, matched with a named identity card, predictive medicine might begin to take on the aspect of a janus, with his beneficent face warning subjects of potential risks of metabolic problems, and guiding them towards the actions to be taken to lower the risks, but also with his evil face delivering each individual's intimate details to the indiscrete inquisitiveness of investigators who are operating towards their own ends (insurance companies, employers…). no less worrying would be the sly but predictable transformation of the individuality of the repaired or even doped human being within a system of imposed, docilely-accepted assistance. in the th and th centuries, the methodology for biological experimentation underwent a revolution caused by the progress made in the domain of chemistry, both analytical chemistry, with the deciphering of increasingly complex molecular structures, and also in synthetic chemistry, with the large-scale production of tens of thousands of new molecules. the effects of these molecules, which might eventually be used as drugs, were tested directly on animals. it was thus that in the german chemist paul ehrlich discovered salvarsan, a derivative of arsenic, which was active against a type of treponeme, the agent of syphilis. this was the result of a systematic analysis of the effect of synthetic products, aromatic derivatives of arsenic acid, on syphilis in rabbits. salvarsan was the th derivative that was tested, and this is why it was called for a long time before it was given the name salvarsan. sometimes, lucky chance shows surprising and unexpected properties in synthetic molecules. this was the case for chlorpromazine, which was initially used as an antihistamine. it was luck that led to its antipsychotic activity being discovered in . a new era opened up in psychiatry with the arrival of synthetic narcoleptics like chlorpromazine. a new chemical science known as combinatory chemistry, which dates from the s, has aroused an increasing amount of interest in pharmacology. this involves making two or more species of organic molecules that carry reactive functional residues react in solution or in the solid phase in such a way as to synthesize, by means of all possible combinations, a number of final and intermediary products that is situated in the hundreds or even the thousands, and which makes up chemical library or drug library. we can directly test all of the products formed on a sample of eukaryotic cells, in order to verify their effects (for example the inhibition of an anarchical proliferation of cancerous cells), or on microorganisms in order to evaluate an antibiotic capability. we can also proceed straight away with the fractioning of the reaction products and the testing of each of the fractions. if the response is positive, fractioning is continued until the molecular species responsible for the desired effect is obtained. other evaluation parameters for this molecule, such as its absorption, its toxicity and its metabolic future (distribution in the organs, chemical modifications and excretion) are then explored, first in cells, and then in animals (rats, mice), thus comprising pre-clinical tests. these screening operations, which are said to be high-throughput, require automation and robotization aided by powerful computer technology. each year, pharmaceutical companies screen tens of thousands of different molecules on hundreds of targets. complementary to combinatory chemistry, in silico chemistry works by molecular modeling and uses computer programs for the rational design of new drugs that are able to fix onto specific protein targets. the purpose of this is to provide a virtual follow up to modifications in the reactivity of a given drug molecule as a function of the modifications imposed on its structure, for example, the addition of residues that differ according to their electrophilic or hydrophilic properties, or according to the length of their side-chain. provided there is a chemical library and we know the three-dimensional structure of a macromolecule, for example an enzyme, as well as the nature of the residues that define its active site, we can hope to select and chemically modify a substance that is able recognize the active site of this enzyme and to make an almost perfect ligand out of it which is able to efficiently block the operation of the target enzyme. this method, which is based on computer-aided chemistry, is called "structure-based drug design", and has had some notable successes. it has made it possible to develop an inhibitor capable of blocking a protease involved in the replication of the aids virus. however, both in combinatory chemistry and in molecular modeling, the many successes that have been achieved remain modest in number compared to the means that have been deployed to achieve them. in terms of statistics, out of ten thousand molecules that are recognized as being efficacious for a given target in vitro, around one hundred are chosen for preclinical trials on animals, around ten are chosen for preclinical trials in man and only one will come out as a drug. the financial and economic effect is far from being negligible. it has even become a preoccupation in a system where merciless competition is the rule. in addition to synthetic chemistry, preparative chemistry, which is based on the isolation of natural molecules, is now the subject of renewed interest, due to the introduction of high-throughput techniques. high-throughput screening, which is an essential tool in combinatory chemistry, is also carried out to ensure the systematic detection and isolation of natural substances having interesting pharmacological activities such as antibiotic activities or anti-cancer activities, based on marine animals, microscopic fungi, prokaryotic organisms and various plants. for example, among the substances that have been isolated recently are cibrostatin, a specific cytostatic of melanoma cells, from a marine sponge, mannopeptimycin, a bacterial antibiotic from an actinobacterium streptomyces hydroscopicus and a whole set of alkaloids with a cytostatic activity with respect to human tumor cells from an exotic plant of the genus daphniphyllum. the molecular diversity of the living world is such that the reserves of natural products having pharmacological activities are far from being exhausted. so far, only a small percentage of the microbial species populating the earth have been listed. the depths of the oceans harbor many unknown species. thousands of insect species remain to be discovered in the canopies of tropical rainforests. exploration of the plant kingdom is far from being complete. the listing of natural molecules having a therapeutic activity has only just begun. the hunt promises to be a fruitful one, all the more so because the highthroughput screening methods that can now be used greatly increase the efficiency of the search. high-throughput screening, applied to natural molecules, has overturned the methodological procedures that were in use until recently, which progress through logical steps, using relatively simple artisanal analytical methods, from observation, often resulting from serendipity, to the isolation of the active substance. thus, in the th century, using inherited traditional knowledge that a decoction of cinchona officinalis bark calms malaria crises, pierre joseph pelletier ( - ) and joseph bienaimé caventou ( - ) decided to isolate the active substance of this bark. from the raw extract, they purified an alkaloid, quinine, which proved to be the anti-malarial substance they were looking for. more recently, the starting point of florey and chain's isolation of penicillin from the microscopic fungus penicillium notatum was the fortuitous observation made by fleming that this penicillium secretes an antibiotic factor (chapter iii- . . ). there are many examples in which serendipity has been the principle factor involved in the discovery of a drug, and this will no doubt continue to be the case. the appearance of a lucky chance, after all, is not incompatible with highthroughput practices. also, it is not impossible that in the future there will be a conjugation of the discovery of new natural substances and the use of combinatory chemistry, with the aim of manufacturing derivatives having a much greater power of action and quality of specificity from these substances . to sum up, the experimental method has caused contemporary medicine to take a giant leap forward, with the discovery of increasingly high-performance functional exploration techniques, the development of therapies using molecules that are already present in nature or are manufactured by synthesis and the more and more advanced understanding of molecular mechanisms that takes into account the basic idea of the pioneers of molecular biology was that the function of a macromolecule depended on its structure. thus, perutz's elucidation of the tetrameric three-dimensional structure of hemoglobin, and of its modifications depending on the degree of oxygenation, shed a considerable amount of light on the cooperative mechanism of the transition from the hemoglobin state to the oxyhemoglobin state (chapter iii- . . ). in the same way, an understanding of the structure of many enzymes, receptors and transporters of metabolites has shed light on their mechanisms. in a parallel manner to the exploration of the structures and functions of proteins, that of genomes has made remarkable progress. the subtle entanglements of genomics and proteomics that have become accessible to the experimental method are the order of the day. one major challenge for post-genomics is to understand how proteins, expressed by genes, interact with one another to generate functions that characterize cellular specificity. even more ambitious are attempts to understand the operation of organs or even of living organisms, based on mechanisms that are implemented at molecular level. these attempts lead straight to an integrated biology, that is, a biology that aims to understand the overall functioning of living beings. taking as its purpose the access to emerging functions, resulting from interactions between macromolecules, integrated biology first tries to invent methods that make it possible to detect these interactions. strengthened by the information obtained, it tries to integrate this information with a mathematized language into modules that attempt to simulate living beings. from the simplistic procedures of the middle of the th century, which were justifiable within the reductionist context of this period, and which involved considering each species of proteins as an autonomous functional entity, we have moved on to the idea that the different species of protein that inhabit a cell have a dialogue with one other, and that they may move from one endocellular organelle to another, depending on post-translational modifications (for example, phosphorylations) that change their conformation and, at the same time, their reactivity and their behavior. thus, an enzyme protein is not only defined by its catalytic performance with respect to a given substrate, but also by its place in a metabolic network where it interacts in a dynamic and transitory manner with a multitude of other protein species (figure iv. a) . the concept of cell signaling has also evolved. instead of considering that a cell membrane receptor, activated by fixation of an extracellular ligand (a hormone, for example), addresses the information received to an endocellular effector protein via a linear cascade of individual proteins, it has come to be postulated that communication between an activated receptor and its effector is mediated by proteins organized into interactive networks ( figure iv. b) . this machinery provides more flexibility in the addressing of messages to effectors. the diagram on the right shows that besides its catalytic function, protein a interacts with other proteins in the cell. b -case of the transduction of a signal that is external to the cell (a hormone, for example). the diagram on the left refers to the classical idea of signaling from a receptor r according to a linear cascade of protein-protein interactions inside the cell, leading to an effector z. the diagram on the right shows that the signal is spread through proteins organized into interactive networks. another subject to be considered is the density of macromolecules of all types, such as proteins, nucleic acids, lipids and polysaccharides, contained by a microorganism or a eukaryotic cell, which reaches values of to g/liter, denoting a semi-solid state or a considerable degree of compacting. however, for technical reasons, kinetic studies carried out in vitro on isolated enzymes have been carried out with solutions that are or times more dilute. conscious of this difference in scale between information obtained from in vitro studies and the in vivo reality, the biology of today is trying to re-evaluate molecular dynamics within the context of a cell. this is why we are seeing the birth of an integrated (or integrative) biology of functions, which, using modeling procedures, aims to achieve an understanding of the temperospatial dynamics of the interactive components inside cells. this holistic conception of biological systems ("systems biology ") has been made possible by progress in technological expertise in domains as varied as biochemistry, molecular biology, physical optics, electronics, nanomechanics, physical and mathematical modeling and computer technology. it is a necessary complement to the classical experimental method based on bernardian determinism which, in order to connect an effect with a cause, explores living beings in a manner that is often monoparametric and is inevitably reductionist. this signals a change in paradigm in the experimental approach to living beings. a particularly effective investigative method used to explore the dialogue between proteins is the double-hybrid by genetic construction, two proteins, p and p , whose interaction is to be tested, are expressed in the form of fusion proteins in yeast. protein p is fused with the binding domain (gal -bd) to the dna of gal , a protein that regulates the transcription of the β-galactosidase gene. protein p is fused with the activation domain of gal (gal -ad). insofar as p interacts with p (b), the gal transcription regulation activity is re-established, which is verified by the transcription of the reporter gene. if the opposite occurs, i.e., in the absence of any interaction between the two domains of gal (a), the reporter gene is not transcribed. the principle of this method is based on the modular nature of numerous transcription factors in eukaryotes. these factors contain both a dna-binding domain that includes a specific dna-binding site and a transcription activation domain that starts up the machinery for transcribing dna into messenger rna. these two domains can be dissociated and then re-associated in a functional manner, by forming hybrids with interacting proteins. a first protein, p , is fused with the dna-binding domain of a transcription factor by genetic manipulation, and a second protein, p , is fused with the activation domain of the same transcription factor. if p is able to interact with p , the transcription factor is reconstituted and the reporter gene upon which it depends can be expressed. the trapping technique, which is complementary to the double-hybrid system, was developed to make it possible to identify a set of interactive proteins within a cell. a protein that is included in this set (protein of interest) is fused by genetic engineering techniques to a short polyhistidine chain (called a tag). using this tag, the protein of interest is fixed to a solid medium containing nickel ions, a material that is reactive with respect to the polyhistidine chain. in the presence of a soluble cell extract, the protein of interest binds the cognate proteins of this extract, making it possible to retrieve a complex whose components, corresponding to interactive proteins, can be resolved after denaturing gel electrophoresis and then characterized ( figure iv . ). the techniques described above are backed up by cell imaging techniques that make use of confocal microscopy, which is more directly in keeping with living reality. the optical performance level of confocal microscopes has improved lately, with the arrival of biphotonic and multiphotonic lasers that illuminate precise points of the cell. as we have seen previously (chapter iii- . . ), it is possible to create a protein chimera made up of a protein of interest fused with a fluorescent protein, in this case gfp (green fluorescent protein), inside a cell. there are currently several variants of gfp that are able to emit fluorescent light at different wavelengths. this has allowed the development of a technique known as fret (fluorescence resonance energy transfer ) which explores the interaction between two fluorescent proteins. in practice, two gfp variants that have neighboring emission spectra are fused, by genetic engineering inside the cell, to two proteins of interest, p and p , that are suspected of being interactive. if this is the case, the fluorochromes that they carry are sufficiently close that the result is a modification in the intensity of the emission fluorescence of the donor fluorochrome (decrease) and of the receiver fluorochrome (increase), which is readily detectable. all of these studies, taken together, have given rise to the idea that endocellular proteins are organized into networks, that these networks are interactive and that their location in defined compartments of the cell is dependent on epigenetic events such as phosphorylations. two attributes can be found in integrated systems: firstly, the presence of modules, i.e., interactive motifs, which, like the pieces of a jigsaw puzzle, fit together to produce a complex, coherent structure, and, secondly, the emergence of functional properties due to the newly created interactions. the protein of interest p is expressed in the form of a protein fused to a protein "tag" t that is able to bind to a solid support with a specific affinity. the assembly is brought into contact with a cell extract. certain proteins of this extract, a, b and c, which are able to interact with the protein p, become fixed to the latter. in a second step, the tag t is freed from its attachment to protein p by means of a specific cleavage enzyme. the pabc complex that is recovered from the solid medium in soluble form is subjected to polyacrylamide gel electrophoresis, in order to separate and identify its components. given an analytical description of the basic building blocks that are used to construct living systems, and an understanding of their modes of association in defined circumstances, it is normal to try to reconstruct, in their entirety, mechanisms that show the functioning of these systems. this idea was first applied to the yeast saccharomyces cerevisiae for different reasons, such as cell homogeneity (in principle, and, in any case, statistically speaking, all yeast cells have the same genome and the same proteome), an in-depth understanding of the genome and the proteome and the presence of a vast directory of well-characterized mutants. the use of techniques for the detection of interactions between proteins has revealed the existence of a potential dialogue of unexpected richness between a multitude of proteins ( figure iv. ) , in the yeast cell. it is necessary to reflect upon this evidence, which leads to the postulate that, for a given protein, there are mechanisms that restrict and select the many partners able to react with it at a precise moment.faced with a situation in which chance has the upper hand, leading to uncontrollable anarchy, it is necessary to have regulation, which is underpinned by darwinian logic. example of an interaction network involving the yeast sup prion protein. the line of dashes refers to experimental data concerning the interaction of sup with another protein, sup . the lines in bold refer to interactions taken from experimental data; while the fine lines refer to predictions, particularly phylogenetic ones. this logic arises from a choice of the most efficient reaction path, which is first of all dictated by the speed constants involved in the association and dissociation of molecular partners, without, however, neglecting any stochastic events that may arise. chemical modifications of proteins participate in this regulation, such as phosphorylation, glycosylation and acylation. the result at cell level is a coherent channeling of the information that is carried from a molecular signal. thus, fixation of a hormone onto a receptor induces a series of modifications to the intracellular proteins that channel information towards an effector terminal, for example an enzyme responsible for the production of a metabolite with a strategic function. how can the sum of the scattered experimental data that we have concerning the catalytic capabilities of a multitude of enzymes of cellular origin be integrated into the operation of a cell? how can we envisage the gene-enzyme relationship according to current evidence concerning the complexity of the genetic message? biocomputing, or bioinformatics, a science that emerged towards the end of the th century, proposes to try to answer these questions. at the turn of the th century, with the development of increasingly powerful computer microprocessors that are able to carry out complex operations with amazing rapidity, the hope arose that it would be possible to simulate processes as varied as the regulation of the cell cycle, molecular flow in metabolic pathways and the reception of molecular signals, for example from hormones by living cells, as well as the transmission of the messages that result. the dream of an in silico virtual biology has become achievable. the first mathematical theory of simple enzyme reaction kinetics was put forward approximately a century ago, by victor henri ( - ). born in marseilles to russian parents, victor henri studied in saint petersburg and then spent time at the universities of göttingen and leipzig before becoming established in paris. having an eclectic mind, studying both psychology and physicochemistry, he had the wonderful intuition that enzyme catalysis arises from a specific mechanism, different from that implemented in a chemical reaction. the study carried out by henri concerned the cleavage of sucrose (table sugar) into fructose and glucose by the action of an enzyme called invertase (sucrase). the term invertase was used because during reaction there was a change in the rotatory power of the sucrose solution, shown by a polarimeter. analysis of the reaction suggested that an enzyme-substrate complex is formed, which breaks down to regenerate the enzyme and liberate the product of the reaction. this analysis gave rise to an equation for the speed of the enzyme reaction as a function of substrate concentration. henri published the results of his experiments both in his thesis, which he presented to the paris faculty of sciences in , and in two articles that appeared in the reports of the academy of sciences , . in , in biochemische zeitschrift (vol. , pp. - ), leonor michaËlis and maud menten ( - ) confirmed the results of victor henri and formulated an equation that became a classic, describing the speed of formation of a product from a substrate in enzyme catalysis. since the period of these first studies, the concepts involved in enzyme kinetics have evolved considerably. the first metabolic pathway be deciphered was that of the degradation of glucose (glycolysis) , either into ethanol in yeast, or into lactic acid in muscle tissue. after this, researchers became aware that the activity of enzymes could be modulated as a function of covalent modifications of amino acid residues of their protein chain (phosphorylation, dephosphorylation, acylation…). metabolic flow analysis led to the idea of the limiting reaction. in the s, the idea that there is a single limiting reaction in a chain or a cycle of reactions gave way to the idea that metabolic control is distributed over several reactions, and that each reaction has its own, more-or-less intense control force. another complexity factor came to light with the discovery of allostery . allosteric enzymes have the particularity that they can fix reversibly onto a site that is different from the active site (allosteric site) molecules that are often the terminal products of a chain of reactions: the consequence of this is a conformational modification of the structure of the enzyme that has repercussions on the geometry of the active site and modifies its reactivity with respect to the substrate. in the s, faced with the complexity of the tangle of listed metabolic and signaling networks, attempts were made to use mathematical modeling to show the progress of the traffic of molecules inside a cell in relatively simple metabolic pathways such as glycolysis. in the modeling procedure, the concentrations of the different molecular species are considered to be variables whose variations over time depend on their speed of production and their speed of disappearance, which leads to a set of paired differential equations. with this procedure, we entered the domain of virtual biology. thanks to the creation of increasingly powerful software, the aim of virtual biology is to simulate signaling and metabolic pathways. in the longer term, the aim is to understand the molecular and cellular processes that direct embryo development, or to test the effects of drugs of known target on the metabolic behavior of the cell. metabolic engineering (which is already well developed) comprises two types of models, stoichiometric models and kinetic models. stoichiometric models describe metabolic networks in the stationary state, based on analytical data. kinetic models combine stoichiometric information and that concerning the catalytic capabilities of the enzymes in a metabolic network. in canada, the cyber-cell project plans to model the overall functioning of the machinery which, in the bacterium, includes its metabolism and its proliferation. the aim of the afcs (alliance for cellular signaling), which was launched in the usa, is to understand how signaling occurs in cells such as the b lymphocyte, the macrophage or the cardiac cell in response to different types of stress. the techniques that are used range from identification of all signaling network proteins to the evaluation of the flow of circulating information and to the integration of the data acquired into theoretical models. the european nerve synapse project makes use of similar procedures, with its long-term hope of linking the functioning of nerve cells with the cognitive and behavioral functions of living beings. this is a sizable challenge. in fact, there is far from being a real consensus concerning the principle of a demarcation between, on the one hand, cognitive functions such as language or memory, which are located in precise zones of the brain, and which could be reduced to physicochemical processes, and, on the other hand, forms reflective thought that are expressed through the creative imagination or by judgements concerning ethics or esthetics, the notion of personal responsibility, or even pictorial, architectural or musical beauty. should we see the human soul as the programmer of a superb computer that never ceases to develop from the embryonic state onwards, like john eccles ( - ) and others, or should we admit, like jean-pierre changeux (b. ), stanislas dehaene (b. ), daniel dennett (b. ) and others, that thought is not transcendent, and that it is intrinsically dependent on the brain, which is considered to be a neurochemical system, and thus look for the secret of the individuation of the human being in brain information storage mechanisms with retrocontrol loops associated with subtle neuron architectures, or, in short, refer to a sort of turing machine? whatever the case, in this domain, as in others, simple animal models are used in order to identify elementary processes that are able to explain easily-tested functions such as the memory in anatomical and physiological terms. this is the case for the sea slug or sea hare (chapter iii- . ) which, despite its rudimentary cognitive capabilities, provides information that can be used to reconstruct higher cognitive functions, present in the brains of mammals. it is clear that the cognitive sciences have reached a stage in which they are emerging from their infancy (chapter iv- . ). now, ingenious computing methods and a basis for reflection that has spread beyond the confines of psychology and philosophy, are available to them. they have set themselves the goal of producing an artificial intelligence, using ultrarapid computers as well as software that is able to model the operation of neural networks and to come close to the performance of human intelligence in terms of the power of their reactivity and their memorization. at present, many other biological systems are being subjected to multiparametric exploration, with the aim of producing models. this is the case, for example, with the program of the differentiation of certain white blood cells, the neutrophils (chapter iii- . . ), from precursors located in the bone marrow, a differentiation that leads to the emergence of functions such as phagocytosis that are implemented in the fight against microbial infections . in a domain that is closer to mechanical science, hydrodynamics, the digital simulation of the cardiovascular system has already made it possible to represent the physical phenomena associated with the propagation of a wave in deformable arteries during a cardiac contraction, in the form of equations, with a good approximation . in short, from a monoparametric approach that often began as being essentially and necessarily reductionist, the experimental method, applied to living beings, has become a "globalized", or synthetic, multiparametric approach, the aim of which is to understand the dynamics of molecular interactions in defined biological systems. making use of data obtained, the hope is to use mathematical processing to simulate the overall functioning of a cell, organ or organism. this new paradigm of the experimental method ("systems biology" ) is not limited to a simple accumulation of observations concerning a given biological system and their abstraction in mathematical form. the originality of this approach is that it formulates predictions of changes in the behavior of a system as a function of the manipulation of parameters such as substrate concentration, the presence of inhibitors, and so on. the mathematical processing of experimental data, with a view to learning about the functioning of complex systems by modeling, is supported by the technosciences, particularly biocomputing. it is linked not only to the enormous sum of accumulated knowledge concerning the structures and functions of living beings in the post-genomic era, and to the notion that the life of a cell depends on multiple networks of molecular interactions and thousands of enzyme reactions located in its different organelles, but also to the information that comes to it from its environment. a first type of modeling is based on observations made or experiments carried out on an easy-to-study model system. laws are drawn up from this. this so-called "bottom-up" (or synthetic) procedure, which proceeds from the simple to the complex, makes it necessary to have a set of very precise biochemical data. this great precision is all the more imperative in that any deviation, even a minimal one, in the integration of an experimental result can generate a mathematical model that is apparently plausible but which is unconnected to the living reality. the reverse, "top-down" (analytic) procedure proceeds from the overall operation of an organ and its theoretical analysis towards the specific mechanisms of its components. it takes into account the functioning of complex integrated systems such as the nerve and endocrine systems, immune and reproduction systems and the system controlling homeostasis, descending in stages towards the cellular, molecular and genetic levels. in the end, an understanding of living beings involves the management of an amazing capital of experimental data. this makes it necessary to consider all of the genes (genome), all of the transcripts coding for the proteins (transcriptome), all non-coding rnas (rnaome), all proteins expressed in a particular cell type (proteome) and all metabolites (metabolome) as a function of the enzyme catalyzers expressed and the energetics that underlie the catalyzed reactions, and, finally, to connect upstream events (mutations of genes and interference of messenger rnas, chemical modifications to amino acid residues in the proteins) to phenotypical modifications on the scale of the whole organism (phenome) (figure iv. ) . the goal of integrated or integrative biology ("systems biology") is therefore to put living beings into equations, that is, to represent them in virtual systems for which the behavior, accessible by means of calculation, can be predicted as a function of modifying parameters. in addition to the possibilities that are opened up in terms of a deeper understanding of physiological mechanisms, such virtual systems could be used for the design of new drugs or for the manufacture of economically valuable biomolecules. the diagram illustrates the different levels of complexity in the pathway that goes from all the genes together (genome) to all of the expressed characteristics (phenome) in the living being, passing via coding rnas (transcriptome) and non-coding rnas (non-coding rnaome), all the proteins (proteome), all addressing systems in the cell compartments (localisome) and all of the metabolic pathways (metabolome). at a scientific meeting held in sheffield, england, in january of , with the theme systems biology: will it work?, an argumentative discussion of the advantages as well as the disadvantages of an integrated, mathematized biology was useful in that it included a reminder that most of the parameters used in "systems biology" come from studies that are carried out in vitro on purified enzymes, and that it is not sufficient to know the value of the michaelian parameters (v max and k m ) in order to reach biological reality. in fact, in vivo, many enzymes record variations in activity that are hard to control due to allosteric type regulation or interenzyme contact; several enzymes of a metabolic pathway being able to interact to form a metabolon. however, by compacting several enzymes that catalyze contiguous reactions in a metabolic pathway, a metabolon considerably increases the catalytic efficiency of this pathway. another element of uncertainty arises from the protein density of the cell medium, and also from the fact that covalent modifications of enzymes can introduce a change in endocellular location (nucleus, organelles of the cytoplasm…). nevertheless, an approximative approach could limit itself to dealing with biological systems in modular terms, i.e., to considering them as being made up of a number of black boxes, each black box containing a series of reactions being processed mathematically together, with an input and an output. there is still a long way to go if we place ourselves on the cellular scale, but the end of the pathway seems even further away if we envisage the organism as a whole, taking into account the remote interactions between organs involving the interplay of chemical mediators. the brain plays a critical role in the dialogue between different organs, and in the regulation of the energy equilibrium in higher animals. this equilibrium can be disturbed by fasting or intense, prolonged muscular activity, or by an overabundant diet. the corrective response comes from a deep region of the brain, the hypothalamus, via the secretion of different types of peptides, some of which stimulate the appetite and others of which suppress it . while taking into account the multitude of parameters that affect the complexity of living beings on an individual level, the theoretical approach to the study of cell function by modeling has the advantage that it produces predictions and provides information about the validity of conclusions and of theories based on experiments that are old and accepted in the absence of contradictory elements. this was the case for the theory that stated that the state of activation of a gene is determined only by the presence in its environment of transcription factors. recent studies concerning the level of gene transcription in isolated cells have shown that there are probabilistic-type factors which mean that a given gene in a given cell can be activated at any moment. a review which came out in sums up this subject. in this review, the authors use modeling to analyze the behavior of cells in the process of differentiation during embryogenesis. their darwinian model, which associates contingency and selectivity, competes advantageously with the determinist (or instructive) model, based on an all-or-nothing logic, that has been implicitly accepted up until now. the darwinian model takes into account the occurrence of stochastic events at gene expression level, events that are partially linked to the structural modifications to the chromatin that depend on covalent modifications of an epigenetic nature (phosphorylation, methylation…). by basing itself on the existence of mutational fluctuations that arise by chance, associated with a selfregulation of gene expression, the model that is obtained shows that during embryogenesis a cell has a choice either to differentiate into another cell type or to remain in its initial state. differentiated cells stabilize their own phenotype and, in their surroundings, stimulate the proliferation of foreign cell phenotypes. a harmonious equilibrium between these two processes is the necessary condition for the setting up of the steps that lead to the arrangement of different cell types during organogenesis, which take place in an apparently inescapable order, in the absence of disturbances. a break in this equilibrium leads to an anarchical cell proliferation. generally speaking, from the point of view of experimental science, the lesson that can be drawn from current modeling experiments is that the bernardian determinism that has prevailed as the essential foundation stone of the methodology applied to the study of living beings may find itself being requalified by the taking into account of stochastic phenomena. this is the case when the number of reacting molecules is low and the probability of stochastic events is non negligible. the modeling of such systems necessitates having recourse to a complex mathematical formalism. it remains true that determinist models for simulation of the dynamics of living beings, represented by classical differential equations, are more-or-less valid when the number of reacting molecules involved is high and the reactions supposedly take place in a homogeneous medium. should "systems biology" be regarded as a resurgence of a physiology that has been somewhat neglected over the last few decades, but has been reinvigorated by a salutary hybridization of biologists and model-makers? in any case, this is the intention of the "physiome" project which has recently been launched on an international scale. it is also doubtless due to this state of mind that a trend which had gone out of fashion, involving the simulation of the performance of living beings by very elaborate concrete models, robots, is being reborn. an immense distance has been covered in just over two centuries, since the time when vaucanson presented automata in the forms of human figures, moved by ingenious springs and cogs, and giving the illusion that their movements were controlled by an intelligence, to a marveling public (chapter ii- . ). in the last decades of the th century, considerable progress was made in the understanding of the operation of the nervous system and in the development of technologies in which miniaturized electronics have come to the aid of already high-performance micromechanics. the brain being considered as an information processing machine, the aim is to understand the logic of this information machine by means of simulations on computers and, based on the results obtained, to construct robots whose electrical circuits take their inspiration from the operation of animal neurons. these robots are called biorobots or animats. insects have been chosen as a reference for the construction of such creatures because of the relative simplicity of their nervous systems: several hundred thousand neurons, in comparison with the billions of neurons present in mammals ( billion in man). the fly's system of vision has been favored as a subject of study because of the possibility it offers of being able to record the electrical response of neurons that can be identified one by one. in the middle of the s, in france, this inspired the pioneering work in biorobotics carried out by nicolas franceschini (b. ) and his team , (figure iv. ) . their objective was to study how an animal can avoid obstacles by means of its ocular perception and its movement-detecting neurons, the operation of which the team just analyzed using microelectrodes and a microscope-telescope specially built for the purpose. the fly's composite eye has elementary units or ommatidia, each carrying eight light receptor neurons. the electrical signals emitted by these neurons in response to captured light (at most a few dozen millivolts) are sent to subjacent neurons that are organized into three levels that correspond to the optical ganglions called the "lamina", "medulla" and "lobula". the lobula is a strategic decoding center which, because of the small number of neurons contained in it (sixty), has been the subject of in-depth electrophysiological investigation. each of the sixty neurons of the lobula operates as a signal integrator. the neurons of the lobula send their messages to motor neurons involved in the contraction of small muscles that control the guidance and stabilization of the fly's flight. based on an exhaustive study of the neuron wiring of the fly's eye, franceschini and his colleagues were able to reconstruct a facetted artificial eye that can retranscribe the light signals received optoelectronically. this artificial eye, the electronic components of which correspond to around one hundred movement detectors in the fly, was incorporated into the head of a robot. the recorded light signals were transmitted to the moving components of the robot. a -head of the blowfly, calliphora, seen from the front, showing the two compound eyes with their multifacetted array. each eye hides , photoreceptors that drive various image processors based on a few hundred thousand neurons. b -"elementary motion detector" (emd) neuron and its evolution over fifteen years: on the left, first generation ( ), using surface mounted device (smd) technology, compared to a one franc coin from that period; on the right, the version of the highly-miniaturized hybrid (analog + digital) emd circuit (mass . grams), compared with a one euro coin. c -autonomous vehicle ( kg) able to move around in a field of obstacles that it does not know about in advance. its vision is based on a genuine compound eye, whose circuits are inspired by those of the fly. it includes a network of "motion detecting neurons", transcribed electronically according to the principle analyzed in the fly's eye by means of microelectrodes and a specially-constructed microscope-telescope. this network is arranged around a ring that is about thirty centimeters in diameter. the recently-constructed roboflies, oscar and octave, only weigh around one hundred grams. d -routing of the electronic components (resistances, condensers, diodes and amplifiers that operate in their thousands) soldered onto the six-layer printed circuit-board that provides the connection between the sensors and the steering motor on board the autonomous mobile robot shown in (c). figure iv . illustrates the neuromimetic biorobofly constructed according to this principle. completely autonomous because of its on-board power supply, this robot was able to move around at high speed ( cm/s) in a cluttered area, avoiding the obstacles. this first "terrestrial" robofly, which was completed in , was followed by several much lighter brothers and sisters: fania, oscar and octave are aerial roboflys . constructed in , oscar is a captive robot that weighs around one hundred grams. it is equipped with an eye that reproduces the retinal microscanning of the fly's eye discovered by franceschini, oscar is able to rotate around a vertical axis because of its two diametrically opposed helices and can thus orient its view towards an object. if this object moves, oscar follows it with its eye, up to an angular speed comparable to the tracking speed of the human eye. produced in , octave is another aerial robofly that is able not only to take off and distinguish a relief, but also to land automatically and to react sensibly to a contrary wind in a turbulent atmosphere. on board, it has an electronic visuomotive self-regulation system, the operation of which is based on the signal processing operations that, in the insect, carry out the automatic pilot functions . the age of biorobotics, in which robots take their inspiration from animals, has only just begun . if specimens are still so rare, this is because behaviors for which we have a good understanding of the underlying neuronal bases are also rare. at the time of writing, a robot rat named psikharpax, with artificial muscles and a vision system that enables it to perceive objects in three-dimensional space, is being developed at the university of paris vi. almost in the realm of science fiction, we find hybrid robots obtained by hybridization of the living and the non-living. this is the case for the hybrid robot produced by japanese researchers, based on the silkworm moth. control of the nervous system of this insect is spread throughout its body. if its head is cut off, it continues to fly, which gave rise to the idea of replacing the head with an electronic transistor system . using a remote measurement device, it was possible to explore certain behavioral aspects of the insect. although the construction of hybrid robots may raise ethical objections, such technology is capable of giving rise to spectacular applications in the domain of prostheses. the neurological prostheses of the future will nevertheless require that a contact be made between living neurons and the electronic chips that are able to improve the inadequate processing of the physio- logical signal. such a contact was produced recently in a german laboratory directed by peter fromherz (b. ) . a small network of snail neurons, chosen because of their large size, was cultured on the surface of a silicon chip. a signal emitted at one location of the chip was able to be transmitted to another location via the synapse connection between two neurons ( figure iv. ) . on the molecular scale, mitochondrial atpase or atp synthase, with a size of around ten nanometers (chapter iii- . . ) was used recently for the manufacture of a biorobot that made its mark in the media as the smallest known rotating molecular motor. the membrane-type enzyme catalyzes the reversible reaction atp + h o adp + pi. this enzyme therefore has a double function; hydrolysis and synthesis. for this reason it is called atpase or atp synthase depending on the physiological context in which it is involved. in the mitochondria that oxidize metabolites, the enzyme operates like atp synthase. it catalyzes the synthesis of atp coupled to oxidation reactions. in the absence of respiration or of oxidizable substrates, the enzyme operates like atpase; it catalyzes the hydrolysis of atp. for ease of language, the enzyme will be designated here by the term atpase. it should be remembered that mitochondrial atpase includes two sectors, a hydrophobic sector, fo, characterized as a proton channel located inside the mitochondrial membrane, and a hydrophilic sector, f , carrying catalytic subunits that are arranged as if they were on a turret (see figure iii . c). fo contains two master parts of the atpase motor, i.e., a rotor comprising an assembly of around ten so-called "c" subunits and a stator that corresponds to the "a" subunit. the "c" subunit assembly is attached to the "γ" subunit of the catalytic sector f , which thus functions as a rotor. in , the british biochemist peter mitchell ( - ) showed that phosphorylative oxidation in the mitochondria is associated with a transmembrane transfer of protons. the mechanism involved is said to be chemiosmotic. the most important experiment involved an almost serendipitous observation, carried out with a simple ph meter. when a current of oxygen was passed through a suspension of mitochondria in an unbuffered saline medium, in the absence of adp and phosphate, an instantaneous acidification of the extramitochondrial medium occurred, shown by means of the ph meter electrode immersed in this medium. it was concluded that the sudden switch from anaerobiosis to aerobiosis, i.e., the start up of respiration, is correlated with an ejection of protons from the mitochondrial matrix to the extramitochondrial medium. afterwards, this fact was linked with several others, the whole leading to the formulation of the chemiosmotic theory. briefly, mitochondrial respiration generates a vectorial movement of protons from the interior to the exterior of the mitochondrion. because of this, a proton concentration difference is established on either side of the mitochondrial membrane. the electrical potential that is created in this way is used by the mitochondrial atpase in order to synthesize atp from adp and mineral phosphate. this process involves two correlated events: return movement of protons towards the inside of the mitochondrion across the fo sector of the atpase; rotation of the assembly of c subunits and the γ subunit that is interdependent with it. we have therefore moved from electrical to mechanical energy. during its rotational movement, the γ subunit establishes contacts with the three catalytic subunits of the f sector, in succession. one after the other, each of the three catalytic subunits in contact with the γ subunit undergoes a change in the conformation of its active site, which is at the origin of the synthesis of atp. in the absence of mitochondrial respiration, the reverse process occurs. the atp is hydrolyzed into adp and mineral phosphate, and the energy released at each of the three catalytic subunits is used to rotate the γ subunit in the reverse direction to that which accompanies the synthesis of atp. the existence of a rotational movement of mitochondrial atpase, which had been suggested on the basis of biochemical arguments and of structural data was authenticated by masasuke yoshida and his co-workers in japan in , thanks to an imaging technique . in a first step, the molecular system was simplified by being limited to the catalytic f sector of the enzyme. a methodological trick was employed: genetic engineering was used to modify the α and β subunits of this sector by fixing polyhistidine chains to them. because of the strong affinity between polyhistidine and nickel ions, the f sector α and β subunits were immobilized on a medium covered with nickel ions (carried by an organic molecule). an actin filament labeled with a fluorescent ligand was attached to the end of the f sector γ subunit. this assembly made it possible, under a fluorescence microscope, to display a rotational movement of the actin arm carried by the g subunit affected by the addition of atp and by its hydrolysis into adp and mineral phosphate. a similar rotational movement of the γ subunit carrying a metal microbar was observed by an american research group . remarkably, in , after having fixed a metal microbead onto the γ subunit, the japanese researchers demonstrated synthesis of atp from adp and mineral phosphate by rotating the γ subunit by means of rotation of the magnetic bead, induced by magnets. thus, the experimental coupling of a mechanical force and a chemical synthesis was demonstrated. in , the japanese research team succeeded in photographing the rotational movement of the enzyme powered by atp under the microscope, this time looking at the entire atpase complex, f fo. after having attached a gold microbead onto the "c" subunits of the fo sector, to act as a probe, the researchers were able to confirm that the rotational movement of these subunits depended on the hydrolysis of atp into adp and phosphate ( figure iv. ) . the whole of the mitochondrial atpase (atp synthase) does, in fact, function as a molecular rotational motor powered by a proton flow, rather like an industrial rotational motor powered by a fossil fuel or electricity. the analogy is a striking one; the γ subunit of the enzyme corresponds to the motor driveshaft and the "c" subunits correspond to the motor itself. because of its association with non-living structures, for example metal bars or gold beads, which are carried along in the rotational movement of the enzyme, it is possible to speak of molecular biorobots. this domain, in which nanomachines use macromolecules from the living world, has only just opened up, but its future is full of promise. the story of scientific progress made with respect to the mechanisms of phosphorylative oxidation via the functioning of mitochondrial atpase, from the time of mitchell's experiment with the ph meter until the time of the manufacture of yoshida's biorobots, is an exemplary one. it is typical of the way in which a mode of thought evolves over time, from a primary discovery resulting from serendipity or an experiment "to see what happens", leading to the proposal of the existence of a mechanism, to a carefully programmed project which, because of its inventive technicity, shows the validity of the proposed mechanism, and, in addition, demonstrates its future utilitarian value. nowadays, certain biotechnologists dream of being able to "synthesize life" in terms of cells that are able to imitate the performance of living cells. the concept of the "lab-in-a-cell " is coming to the fore , . nevertheless, it would be necessary to design an artificial cell that is an authentic replica of a living cell, and which benefits from all the attributes of a living cell. this is not achievable at the moment. thus, the current aim of nanobiotechnology is limited to scheduling the construction of artificial cells that are relatively simple both in composition and in function, for example, a microvesicle edged with a lipid membrane, containing a system of protein synthesis expressed from a short sequence of dna, as well as a system of atp synthesis able to supply the energy necessary for this protein synthesis. demonstration of a rotational movement of f fo mitochondrial atpase (atp synthase) , induced by atp. atpase or atp synthase (reversible catalysis enzyme that hydrolyzes or synthesizes atp) has two sectors (see figure iii . ). the membrane sector, fo, comprises an assembly of a dozen so-called c (rotor) subunits and an a (stator) subunit. the other, extra-membrane sector, f , is catalytic. it comprises three β catalytic subunits and three α non-catalytic subunits arranged in a ring, in alternating order. at the center of the ring is the γ subunit which is attached to the c subunits of the fo sector. subunits δ, ε and b stabilize the whole of the molecular complex. in the experiment illustrated in this figure, subunits α and β of the f sector of the enzyme have been genetically modified to include polyhistidine chains (his-tag, artificial ligand). due to the interaction of these chains with nickel ions (linked to an organic molecule) covering a solid support medium, the α and β subunits are immobilized. in addition, a gold microbead is fixed onto the ring of the fo sector c subunits by means of a chemical device (streptavidin molecule, artificial ligand). following the addition of atp, rotation of the microbead attached to the ring of fo sector c subunits is observed by microscopy on a black background. this rotation is dependent on (and at the same time an indicator of) the rotation of the c subunits, itself led by the rotation of the γ subunit in contact with the catalytic β subunits. note the ejection of protons. when the enzyme functions as atp synthase, the proton movement takes place in the opposite direction. "progress in biology is possibly mainly tributary to the drawing up of concepts or principles […] . in the process of elaborating concepts, which marks scientific progress in biology, there is sometimes a crucial step, when we realise that a more-or-less technical term that we had previously considered to cover a given concept, in fact covers a mixture of two (or more) concepts." ernst mayr translated from a french translation entitled history of biology. diversity, evolution and heredity - on the margins of the modeling and the difference in mathematized systems that comprise theoretical biology, particularly in silico biology, concepts are mental representations, often image-filled and idealized ones, of fundamental mechanisms that are deduced on the basis of experimental results. from the imaginary domain of the probable, they extrapolate constructions of the mind that are in phase with the facts and experimental data, within a reflective projection that gives them their meaning and makes it possible to make certain predictions. there are premonitory concepts. this was the case for the concept of the reflex arc that associates movement with sensation. this concept was already present in the ideas of descartes (chapter ii- . ), but it took more than a century before the theory of the existence of a reflex arc was supported by bell and magendie's demonstration of the existence of relay centers for sensory and motor nerves in the spinal chord (chapter iii- ). there have been premonitory concepts that, while they were demolished at the time they were first proposed, were shown to be completely accurate a few decades later. in the middle of the th century, the german pathologist jacob henle ( - ) needed a healthy dose of imagination and audacity in order to oppose the theory of the "miasma", a theory that was taught as a dogma, with a new theory that not only explained the spread of contagious diseases by microscopic beings, but also formulated the criteria for validating this theory, i.e., isolation of the pathogenic agent and its development in culture away from the diseased organism, then reproduction of the original pathology after injection of the pathogenic agent, which has been isolated, characterized and multiplied in a culture, into a model animal. thirty years would go by before the formulation of koch's postulates, based on experimental evidence (chapter iii- ). we may ask ourselves whether or not history is currently repeating itself in the case of spongiform encephalopathies that affect humans and animals, for which, according to the thesis of stanley prusiner (b. ) , the prion, as an infectious protein, is responsible. other evocative concepts hold the keys that open doors to domains that are unknown, but are potentially rich in information. it is thus that the double helix dna structure proposed by crick and watson, based on the complementarity of adenine-thymine and cytosine-guanine bases (chapter iv- . . ), led to the concept of dna replication with reconstruction of a double strand that is identical to the original double strand. the concept of dna replication spurred on matthew meselson (b. ) and franklin stahl (b. ) to develop an experimental protocol based on the labeling of the dna nucleotide bases of the enterobacterium e. coli with a heavy isotope of nitrogen, n, and on the differentiation of monocatenary dna strands in the process of synthesis by measurement of their density, as analyzed by centrifugation in cesium chloride gradients. in the same vein, jacob and monod's discovery of regulatory genes (chapter iv- . . ) gave rise to the concept of the operon which, in the bacterium, defines a genetic unit comprising structural genes and regulatory genes. the concept of the regulation of gene expression, extended to higher eukaryotes, makes it possible to explain the phenomenon of differentiation in cells with specific activities (muscle cells, nerve cells, epithelial cells…) by the silencing of certain genes and the activation of others. within the framework of bioenergetics, the chemiosmotic theory put forward by mitchell, in order to explain the coupling of mitochondrial respiration with atp synthesis (chapter iv- . ), gave rise to consideration of the concepts of transmembrane transport of metabolites and of vectorial metabolism. some generalizing concepts that carry a unifying virtue within them are known. one such is the concept of compartmentation. the cell is no longer considered to be a bag of enzymes, as used to be the case. it is now considered to be a compartmented structure in which each type of compartment corresponds to a type of organelle delimited by a membrane and characterized by specific functions. thus, because of the genetic material that is present in it, the nucleus of the cell holds the information necessary for the manufacture of proteins. the mitochondria, which are called cell power plants, are in charge of oxidizing the products of cell catabolism and using the resulting energy for the synthesis of atp from adp and mineral phosphate. the lysosomes are the garbage collectors of the cell. among the functions carried out by peroxisomes is the partial breakdown of very long chain fatty acids. the endoplasmic reticulum and the golgi apparatus are involved in the maturation and the secretion of proteins. the ribosomes represent the machinery upon which messenger rnas are displayed in order to be decoded into proteins. a sign of the extreme sophistication of this setup is that the membranes of the endocellular compartments are not sealed common walls. they contain proteins that act as selective transporters of metabolites or highly specific ion channels, allowing the exchange of messages throughout the cell. thus, each organelle, informed of the condition of the others, is able to adjust its own activity to ensure the greatest harmony of the whole. this conditioned compartmentation at cell level may be compared to the socialization of human communities. while endocellular organelles are compartments delimited by membranes, there are non-membrane-bound compartments in the cell, such as protein complexes in which two, three or even more proteins are closely linked. often, these are enzymes that catalyze reactions that are contiguous in a metabolic pathway. being compacted into a complex known as a metabolon results in an increased efficiency of the flow of metabolites by facilitating the channeling of this flow. concepts evolve, often adjusting their representations according to accumulated knowledge. a good example of this is the evolution of the concept of the gene since its formulation at the beginning of the th century. the term "genetics" was created in by the english naturalist william bateson ( - ) . the term "gene" was introduced three years later by the dane wilhelm johannsen. this term designated a principle which, in the chromosomes of fertilized egg, and in an intentionally vague manner, was supposed to have an influence on the phenotype of the progeniture. during the same period, the term "locus" appeared out of the experiments carried out by the american thomas hunt morgan on the drosophila, a locus being defined as a region of a chromosome which, when altered by a mutation, leads to a modification of the phenotype of the living organism. based on cross-breeding experiments carried out on hundreds of drosophila mutants, morgan and his co-workers drew up the first genetic maps. by chance, the salivary glands of the drosophila have a particular characteristic; the nuclei of their cells contain giant chromosomes called polytenes, which result from the association of a hundred replicate copies of chromosomes that, after staining, are visible under the optical microscope. on these chromosomes, it is possible to distinguish colored bands separated by clear bands. it was observed that specific mutations had specific effects on the arrangement and number of these bands. the material contained in the bands was therefore the site of mutations. in the middle of the s, the listing of more than bands made it possible to construct a cytological map that was already highly detailed. the concept of the gene, the material basis of inheritance, took root. the sporadic mutagenic effect of x-radiation in the drosophila, which was shown by the geneticist and biophysicist hermann mÜller ( - ), led the austrian physicist erwin schrÖdinger to question the sporadic event which, at the level of a target of a few dozen atoms, determines a mutation. he postulated that the target is located in the chromatin of the chromosomes, organized as an aperiodic crystal. the chemical nature of this target was identified with dna, following bacterial transformation experiments (avery, macleod and mccarthy, ) and experiments concerning bacterial infection by the bacteriophage (hershey and chase, ) (chapter iv- . . ). this is how the idea that gene = dna was born. the simple and reassuring idea that one gene → one enzyme, which was deduced from mutation experiments carried out by beadle and tatum on the mold neurospora crassa (chapter iii- . ), had only a limited lifetime. a first stumbling block appeared when it was shown that the activity of a gene, and in consequence its contribution to the phenotype, depends on nucleic elements outside the gene. the definition of the term "gene" was then extended to include promoting and regulatory sequences. in the case of the lac operon of escherichia coli, these sequences are located just upstream of the site where transcription begins. however, in eukaryotes, a regulatory sequence may be distant from the gene that must be transcribed and sometimes it may be involved in the regulation of several genes (chapter iv- . . ). in the s, the idea of the existence of the mosaic gene in eukaryotes appeared. a gene was now thought of as an assembly of several exons that originally in the chromosome are separated by introns. the alternative splicing of these pieces of genes gives rise to numerous possibilities for reconstitution, i.e., many messages coding for many different proteins. thus, however useful the concept of the gene has been with respect to its ability to generate discussion and to provoke experimentation concerning the molecular machinery responsible for the transmission of the hereditary characteristics, we can see that the term itself has not ceased to be the subject of readjustments, since the time it was first formulated. certain concepts are matched with metaphors. while some metaphorical concepts, particularly those that make use of images designed to grab the imagination, and to be easy to understand, tend to take liberties with the realities of living beings, they can also shed light on unsuspected mechanisms in sectors that have been neglected. metaphorical concepts are not a current fashion. it should be remembered that in his passions of the soul ( ), descartes, when asked "how limbs can be moved by objects of the senses and by the mind without the help of the soul," responds that this takes place "in the same way as the movement of a watch is produced only by the force of its spring and the arrangement of its cogs." later on, with lavoisier's clear vision of the vital role of oxygen, and his comparison of respiration with combustion, the concept of the chemistry of life, combined with that of bioenergetics, came to the fore, and was at the heart of studies on the metabolism. chemical reactions that liberate and absorb heat were substituted for the cogs of cartesian mechanics. the second half of the th century saw the birth and development of the concept of the program, a concept with computer technology connotations, which was destined to explain the phenomena of inheritance. this concept began to fill out from the moment when it became certain that, in its nucleotide sequence, dna contains the necessary information for the construction of the protein material of cells. for a certain period of time, the passion for molecular genetics eclipsed the interest that had previously been given to metabolic chemistry. the powerfulness of the metaphorical concept may be measured according to the effect it has in pushing scientific research in particular directions, with the results this has on society. thus, during the th and th centuries, the study of human pathology was impregnated with a strong iatromechanical current. physiological chemistry and its corollary, pathological chemistry, which emerged as disciplines in their own right in the th century and achieved full expansion in the th century, are our inheritance from lavoisier and the concept of discussion about concepts necessarily leads to a brief discussion of scientific semantics, as shown by the few examples given in the previous pages. as we have just seen, the word gene that was put forward by johannsen around one century ago did not have the same meaning at that time as it has now, a meaning that still remains fluid. the gmo, an acronym meaning genetically modified organism, which has been the subject of vehement diatribes over the last few years, becomes much less of an object of passion if it is considered within the context of evolution. after all, for the last two to three billion years, living organisms have been genetically modified constantly by spontaneous mutations, which is why the human beings that we are today are able to discuss them! the term cloning is another example of a semantic misunderstanding that leads to inaccurate interpretation and arouses the passions. the primary meaning of the term cloning is the multiplication and the identical reproduction of a living cell. the simplest and most unambiguous example is that of bacterial cloning, a bacterial cell producing millions of cells that are identical to the original cell by its multiplication in a nutritive medium. the term animal reproductive cloning does not carry exactly the same semantic weight. it should be remembered that, in eukaryotes, the preliminary act of the cloning procedure involves the injection of the nucleus (with n chromosomes) from a somatic cell into an enucleated oocyte (chapter iv- . . ; see also chapter iv- . ). the somatic cell nucleus, by providing its genetic equipment, gives the being that will develop in the uterus a phenotype that is practically identical to that of the somatic cell donor, but nevertheless not completely identical, as the cytoplasm of the enucleated ovum, with its mitochondria, provides a small but non-negligible fraction of genes, the mitochondrial genes. as for therapeutic cloning (in the absence of uterine implantation), this is used for the manufacture of differentiated cells that may be grafted into the individual who has donated the original somatic cell, with no immune-related rejection occurring. this is non-reproductive cloning. the passionate argument that has arisen because the term cloning is bandied about in an ill-considered fashion illustrates the confusion that can result from a lack of precision in the use of certain terms with a high level of media impact. "all the major problems of the relations between society and science lie in the same area. when the scientist is told that he must be more responsible for his effects on society, it is the applications of science that are referred to […] . no government has the right to decide on the truth of scientific principles, nor to prescribe in any way the character of the questions investigated." the progress of science is linked to that of civilization. it is in keeping with the state of mind, the beliefs, the lifestyle and the thought patterns of societies. in ancient greece, where manual work was considered to be servile, science remained essentially theoretical, confined to logic and dialectics, and strongly attached to questions of philosophy. the birth of experimental science in the th and th centuries went hand-in-hand with the rehabilitation of manual work. the technical side dominates in modern biology, which seeks to solve problems concerning the "how", rather than to address philosophical problems concerning the "why". as ian hacking (b. ) says in representing and intervening ( ), nowadays engineering, and not theorizing, is the greatest proof of scientific realism, which leads to the minimization of philosophical thought. in a skeptical biochemist ( ) , the polish-born american biochemist joseph fruton ( fruton ( - emphasizes the contrast between the th century and the first half of the th century, when eminent scientists were still interested in the ideas of the professional philosophers of the history of the sciences concerning the progress of experimental research and, in contrast, the end of the th century, when philosophy and the experimental sciences pretended to ignore one another. this is doubtless partly because the history of biology has become the history of biotechnologies to such an extent that, according to some, the objects being explored are so familiar that they are now part of the life of society. in pandora's hope ( ), bruno latour (b. ) considers that the current confrontation between subject and object, in which the researcher-subject explores the structure and function of the object, is being transformed into a human-nonhuman dialogue, in which the nonhuman-object becomes "socialized". taking yeast as an example, latour writes that it has been "working for millenia in the brewing industry, but now it works in a network of thirty laboratories where its genome is mapped, humanized and socialized like a code, a book, or a program of action that is compatible with our ways of coding, counting and reading […] . non-humans have become automatons, admittedly without rights, but much more complex than material entities." latour visualizes the human-nonhuman associations in the form of collectives that are organized into strata that implement the technical, the political, the social, the ethical, the ecological… the technosciences correspond to one of these strata, the sociotechnical stratum that is directly linked to the stratum of political ecology. in the same spirit, the belgian philosopher gilbert hottois (b. ), in his philosophies of the sciences, philosophies of techniques ( ) remarks that "laboratories produce things that go off to live their lives in society and in nature." thus, bacteria, yeasts or genetically modified plants are able to produce drugs such as insulin, growth hormone and vaccines for human medicine. these drugs become part of and indispensable to life in society. they are evaluated according to their market value by the companies that patent, manufacture and sell them, and according to the comfort they bring to the patients to whom they are administered. the financial management that results from their consumption becomes a worry for those responsible for public health, while their manufacture by specialized companies generates industrial activity and economic growth which may be measured according to how fashionable they are and how they sell. for a long time, society, while benefiting from scientific progress, remained indifferent to the experimental method, that is to say, the way in which knowledge progresses. in the last decades of the th century, society became aware, via information concerning the occasionally demonized exploits of genetic engineering, that science can "take liberties" with the human being. populations were well informed about the effects that genetic engineering could have on the mortality rates of pathologies such as cancer and diabetes or on degenerative illnesses of the nervous system, and about the closeness of possible solutions. however, they were also warned about the risks to which science was exposing humankind. remembering certain tragic episodes concerning hiv-contaminated blood transfusions, growth hormone and mad cow disease, and certain cassandra-like predictions, such as a catastrophic epidemic of spongiform encephalopathy that has happily yet to appear, society shows reservations when the media inform its members of new feats of modern technology. political authorities, for their part, afraid of potential problems, tend to follow the principle of precaution, which in fact hides a fear of risk. however, evaluating risk involves not being afraid of it but understanding it in a lucid and courageous fashion. informed by the media, which often use sensationalism, the citizen is increasingly calling into question whether certain practices involving the biosciences, such as cloning, or certain mercantile transactions such as the taking out of patents concerning gene sequences, or even experimentation on live animals, are well-founded. "the problem of experimentation on man is no longer a simple problem of technique. it is a problem of value. from the moment that biology concerns man no longer simply as a problem, but as instrumental to the search for solutions concerning him, the question arises of deciding whether the price of knowledge is such that the subject of the knowledge is able to consent to become the object of his or her own knowledge. we have no difficulty here in recognizing the still open debate concerning man as a means or an end; an object or a person. this is to say that human biology does not contain in and of itself the answer to questions concerning its nature and its significance." knowledge of life - written at a time when people were far from imagining how molecular biology was going to expand, the prophetic words of georges canguilhem ( - ) have maintained their philosophical validity. manipulation of the human embryo, whether this involves its creation by cloning or the modification of its genetic inheritance, obviously leads to the need to consider the societal, religious and political points that arise from the domain of bioethics and are a reflection of the period in which we are living. until recently, advances made in biology left moralists indifferent. this ceased to be the case when scientific experimentation began to look at the human embryo with a view to utilitarian ends in the health domain. the specter of cloning was brandished without any clear distinction being made between reproductive cloning and therapeutic cloning. biology became demonized. however, as biologist pierre chambon (b. ) said in an interview in the french journal biofutur: "in absolute terms, biology is unable to tell us whether the cloning of a human being is moral or immoral, it simply tells us whether it is biologically possible." the birth dolly the sheep in (chapter iv- . . ) triggered a virulent debate because now that the cloning of an animal had become possible, that of a human being became envisageable. the media sensationalized this debate all the more in that it was exacerbated by debate concerning gmos (chapter iv- . ). the dolly affair became a problem of society. up until then, the biosciences had been happy just to try and understand the mechanisms that explained the functions of living beings, but now, with the advent of gmos and cloning, it became obvious that a forbidden barrier had been crossed and that man had the power not only to transform but also invent himself. faced with this desacralisation of nature, the need arose for some philosophical reflection. this was given the name of bioethics, which is the title of the book, bioethics, a bridge to the future, which was written by the american biologist van rensselaer potter ( - ) in . the term bioethics covers philosophical considerations that range from the biosphere to the human person. bioethics tries to give a wider meaning to the moral codes which, in human societies, depend on ancestral traditions. it aims to prescribe that which is desirable according to the kantian maxim of the categorical imperative. in his what is bioethics? ( ), the belgian historian gilbert hottois reminds us that bioethics are above traditional morals, the latter being a set of norms that are most often spontaneously respected as good habits, without any critical reflection being involved, while bioethics, on the other hand, arises out of critical thought, analysis, discussion and the evaluation of established mores. over the last few years, the problems that are targeted by bioethics have moved towards today's burning issues. human cloning is an example. while allegations of the transcendence of man in nature may lead to human reproductive cloning being considered as a crime, strictly scientific considerations lead to an emphasis on the lack of responsibility shown by a few zealots, given the hazards involved in cloning in animals, such as the need to use a large number of oocytes in order to achieve success in cloning, the very low viability of the cloned embryos and the development of serious functional anomalies in the clones that survive. even supposing that scientific progress will one day overcome these difficulties, human reproductive cloning will come up against an insurmountable obstacle, the cloned subject's fear of finding that he or she is identical to the relative from whom his or her genetic inheritance comes. after all, the notion of manipulation of the human ovule with the aim of serial reproduction has often haunted science fiction stories. in brave new world ( ), aldous huxley ( - ) gives an apocalyptic vision of the budding of human eggs that produce hundreds of identical twins which are conditioned into classes and subclasses while being raised in jars, depending on the quality of the nutritive substances they are given. in the artificial uterus ( ), henri atlan (b. ) predicts that the raising of human fetuses in jars could well become an alternative to uterine gestation in a distant future. let it be understood that human reproductive cloning, which is no longer part of the domain of science fiction, as it has become feasible, must be considered as being reprehensible because it goes beyond the limits of reason, and is a denial of human transcendence. man as subject cannot be considered as an object. the problem of therapeutic cloning is quite different, although it leads to reticence and prohibition because the demarcation between therapeutic and reproductive cloning depends mainly on whether a cloned embryo is implanted in a uterus. while, at the time of writing, therapeutic cloning has been prohibited in france, germany and other countries, it is tolerated in great britain. in the usa, the prohibition only applies to publicly-financed researched, while each state has its own legislation, which is relatively flexible. the objective of therapeutic cloning is to provide patients with tissues that arise from their own selves, and are therefore immunocompatible and able to be grafted without there being any risk of rejection (chapter iv- . . ). it is based on the removal of somatic cells from the subject to receive the graft and the transfer of the nuclei of these cells into enucleated oocytes. the stem cells that are obtained after the first divisions are stimulated using appropriate growth factors. depending on the factor used, the stem cells differentiate to form a type of tissue (hepatic, muscular, nerve…) that can be used as a graft. such a procedure may be envisaged for patients who have suffered a serious, invalidating trauma, for example section of the spinal chord. a graft of immunocompatible nerve cells might make it possible to re-establish nerve continuity. a similar type of therapy has been considered for parkinson's disease, the cause of which is a degenerescence of certain cells of the encephalon (chapter iv- . . ). given the hopes that are raised by the possibility of such therapies, and the fact that, after all, such therapeutic cloning is the equivalent to an autograft, even if the ways in which the graft is obtained are slightly tortuous, the demonization and rejection of such practices should be reconsidered, calmly and coolly. another option for therapeutic cloning is the correction of mutations identified in the mitochondrial genome of a woman wishing to have children. it is, in fact, the mother's ovum that provides the fertilized egg with its complement of mitochondria that are indispensable for its viability. the manipulation involves inserting the nucleus of a fertilized ovum from the mother, obtained by artificial insemination, into an enucleated oocyte taken from a woman who is not suffering from the mitochondrial defect. the cytoplasm of the enucleated oocyte provides the stock of functional mitochondria that are indispensable to normal cell function in the future embryo. in a domain of the bioethics, in which rational objectivity comes up against deliberately technophobic religious and cultural considerations, it is useful to remember certain legal and legislative paradoxes. thus, in france, after having been considered to be a criminal act that was subjected to severe repression by the law up until , the right to have an abortion before the end of the third month of pregnancy became not only authorized but also protected by law. it is interesting to note that in the th century, thomas aquinas, the father of the church, had acknowledged that a fetus only becomes "animated" by the implantation of the soul by holy will in the third month after fertilization. another subject to be considered is pre-implantation genetic diagnosis (pgd), in which human embryos that have been fertilized in vitro are sorted in order to find those that are without defects, a practice which is on the verge of being a deviation in the direction of eugenics. nevertheless, pgd is the basis of a practice that is either already legalized or is in the process of being so in several european countries, the creation of so-called designer babies. a typical example is that of a designer baby arising from an embryo whose immune profile to that of an older sibling who is suffering from leukemia. in this case, there is good reason to hope that a graft of immunocompatible blood cells from the designer baby into the sibling who is suffering from leukemia will save the latter from death. out of the disharmony of opinions that arising from cultural tradition, religious conviction or simply scientific pragmatism, the american biologist and philosopher h. tristram engelhardt (b. ) , in the foundations of bioethics ( ) proposes a lay bioethics that is based upon the principle of permission. lay bioethics advocates tolerance while admitting that this tolerance in no way prevents anyone from taking up a personal position; it means that each human being has a moral sensitivity as well as the ability to reason and to choose within a defined limit of non-harmfulness and of justice. the individual is free to modify his or her destiny, or to manipulate his or her nature by genetic interventions because, adds engelhardt, "there is no lay moral foundation to prohibit such an intervention." when a researcher or the research organization that the researcher belongs to files for a patent for an invention with a patent office, it is necessary to demonstrate the novel and utilitarian nature of this invention. if a patent is accepted, this gives the person or body that filed it the exclusive right to make use of the invention over a pre-determined period of time, generally years, which is a means of protection, or, if desired, to allow others to make use of the invention by issuing a license to do so. in the domain of living beings, there has sometimes been confusion between invention and discovery. in , craig venter, known for his participation in the sequencing of the human genome, filed a demand for a patent covering the sequences of fragments of recombinant dna (cdna) called est (expressed sequence tags) that are obtained by reverse transcription from human brain messenger rnas, in the name of the nih (national institutes of health) at bethesda (usa). the patent specified that ests could be used as probes to characterize genes that are potentially involved in neurological ailments. the resulting outcry led the nih to withdraw its patent demand. in fact, the patenting of living beings has a long history that goes back to the patent that was filed in in france by louis pasteur, and then in in the usa, for the use, in brewing, of a yeast culture that was free from pathogenic bacteria. from this historical perspective, the case of ananda chakrabarty (b. ) set a legal precedent. in , chakrabarty filed a demand with the us patent office for a patent relating to a pseudomonas type bacterium which, by genetic modification, had acquired the ability to digest crude oil. his demand was refused. after an appeal and many legal battles, the united states supreme court overturned the patent demand refusal, the basis of the judgement being that any modified microorganism is a product of human ingenuity and has a specific name, characteristics and use. thus, from onwards, the arrival of an era of patents derived from genetic engineering was indicative of how this discipline was growing. in december of that year, stanley cohen and herbert boyer, acting on behalf of the university of stanford, patented a nucleic chimera comprising a recombinant dna carried by a vector. in , a patent concerning the growth hormone gene was awarded to the university of san francisco. in , the university of california at berkeley obtained a patent for the human insulin gene. in , the american company pioneer hi-bred succeeded in patenting a variety of corn in which genetic modification has led to an increased synthesis of tryptophan, an amino acid that is indispensable for animal feed. in , the genentech company acquired a patent for the gene coding for human gamma interferon. this was followed in japan by a patent for the gene coding for beta interferon. in the same year harvard university patented the oncomouse, a transgenic mouse whose susceptibility to cancer is greatly increased. after this, several species of transgenic animals were patented for utilitarian purposes, such as the production of human alpha- -antitrypsin taken from the milk of transgenic goats and used for the treatment of cystic fibrosis. the frenetic patenting of living beings has reached the domain of natural products arising from the plant world in tropical regions, the immensely varied essences arising from these plants being full of pharmacological potential. the potential for producing drugs of a considerable commercial value from such plants is very high. here we return to the problem of the patenting of genetically modified, cultivatable plants (gmps) (chapter iv- . ). thus, the experimental method, the principle of which is to acquire pure knowledge, finds itself led astray in its applications. whatever the motives that are given, particularly for manipulations that give rise to the manufacture of marketable products, the patenting of genomes for mercantile ends shows the regrettable, but unfortunately inevitable, direction in which the very spirit of a science, molecular biology, which half a century ago wished to be at the heart of an understanding of living beings, has drifted. the suffering of animals that are being experimented upon gives rise to a moral problem. the end of the th century saw large-scale demonstrations against vivisection and repeated demands for it to be abolished. today, there is renewed vigor in the call for the abolition of vivisection, without any real coherent basis. this desire to stop experimentation on animals ignores the imperatives of contemporary medicine, which must meet the challenge of pathologies whose increasing incidence is worrying, such as cardiovascular diseases, diabetes, cancer, and the degenerative illnesses that are linked with aging or are of genetic origin. it is true that animal experimentation inevitably leads to questions. are the stakes involved in a particular experiment, in terms of the acquisition of new knowledge, worth the suffering of an animal used in that experiment? is it not necessary to ensure that the experimental protocol is well-documented, that it is not redundant, or even that it has been the subject of previous studies carried out on cells in culture? it is easy to see the size of the methodological chasm that separates contemporary physiology from that of the time of claude bernard, when cell culture techniques were not yet being used, when the main instrument used was the scalpel and the researcher, using his or her imagination and creativity, had to develop specific protocols that were able to validate or refute a working hypothesis. each period in history operates in its own way according to its moral laws and its technical capabilities. the bloody operations carried out by magendie and by claude bernard in the th century, which were tolerated at this time despite criticisms from antivivisectionists, would not be permitted today. nevertheless, it is true that the physiologists of the th century, by means of the results of their experiments, wove a tapestry of new knowledge on which contemporary biologists were going to work and without which the level of understanding the modern science would be much lower than it is. animal experimentation remains indispensable in many areas of physiological investigation, in genomics, in toxicology and in pharmacology. it is a precondition for clinical trials of any new drug, being used to test for the drug's efficacy, its metabolism and any toxicity. however, not all data arising from animal experi-mentation can be extrapolated to man. the margin of uncertainty can be reduced by means of comparative trials on several animal species. because of their phylogenetic proximity to man, primates may seem to be the solution for experimentation prior to the application of a drug in man. this was the case for the development of a vaccine against hepatitis b. it has been proposed the grafting of stem cells in man should be preceded by experimentation in apes, in order to ensure the absence of tumorization over the long term. however, the researcher is confronted with a dilemma: should he or she ensure the safety of man with respect to possible deleterious effects or respond to ethical demands that recognize the very great genomic similarities between man and the chimpanzee. a consideration of cloning, patenting and animal experimentation practices illustrates the excesses of the experimental method in domains where political authorities consider themselves able to legislate. administrative decisions, often made in the absence of any dialogue with scientific authorities, can have serious consequences. thus, given the pretext of strict obedience to the principles of bioethics, which are a matter of tradition, and while certainly respectable, are nevertheless arguable, and also given the pretext of a sickly and unconsidered fear of the risk involved in certain experimental practices, and the absence of an intelligent evaluation of this risk, research, which until recently took place in a motivating atmosphere of liberty, may, over the long term, be weighed down with a highly prejudicial handicap and a limitless sense of discouragement. in the th and th centuries, experimental research, which was still in an emergent phase, was mainly artisanal, and in the hands of rare scholars. it took form during the th century in the west, particularly actively in germany, and became operational in the th century, under the aegis of governmental authorities, with the creation of institutes, the programmed recruitment of researchers and the allocation of renewable budgets. modern science, based on the principles of the experimental method, came to the fore much later in the east than in the west. the globalization of knowledge has meant that at present experimental science, in all domains, including that of the life sciences, has spread throughout the world, with even those countries that had become relatively backward in these domains because of their isolation catching up rapidly. nevertheless, it is true that the progress of the experimental sciences in the usa and in the united kingdom has been distinguished by pragmatic management of these countries' science policies, based on the excellence and the high degree of autonomy of their universities and research institutes with respect to recruitment and choice of subjects of study. the efficacy of this policy in the life sciences may be judged by the number of researchers who have won nobel prizes since the second world war (at the time of writing, more than in the usa and twenty or so in great britain as opposed to only in france). in france, research on living beings is carried out in the laboratories of universities, in institutes connected with higher education and in laboratories that are run by large organizations such as the national scientific research center (cnrs), the national institute of health and medical research (inserm), the national institute of agronomic research (inra), the atomic energy commission (cea), the national institute of research in computer processing and automation (inria), the national center for space studies (cnes) and the french institute of research on the seas and oceans (ifremer). equivalent bodies exist in countries other than france, some of them being institutes that are dedicated solely to research, and some being university laboratories that associate research and teaching. at the beginning of the th century, the function of researcher was most often associated with that of a professor occupying a chair at a university, surrounded by a few assistants, the professor directing the research work in his area of specialization. now, within a period of a few decades, the status of researcher has been modified greatly. today we talk of research careers classified according to level of expertise and technicality. management, or the supervision of career paths and the control of financing, is carried out by an administration that is itself highly hierarchical. the scientific process has undergone a metamorphosis, shown by changes in the behavior of researchers not only within the institutions in which they work but also in their relationships with the media, the political sphere and society. the teaching of the life sciences needs to take this into account. "long ago, there was a time when scientists recounted the exact circumstances of their discoveries, without shame, even when their recital showed up the fragility of their forecasts or an indecent collaboration on the part of every bit of luck. such times are past, and the researchers of today often like to make us believe that they only find what they are looking for. the thousands of pages pasteur's lab books provide an opportune reminder to us (and to program-makers or impatient users) that it is just as difficult to ask a question as to answer it, that a scientific discovery often occurs after a long, winding path, that rather than following the fashion, it is preferable to follow one's ideas, particularly if they are good ones, and are in advance of the fashion." jean jacques molecular dissymmetry, in "pasteur, workbooks of a scholar" - current technological progress, the accumulation of the scientific knowledge, the institutionalization of the public research and many other factors are disrupting a ritual of the experimental process that had survived until the middle of the th century, and even beyond. the experimental life sciences of the st century will necessarily see themselves remodeled with respect to their objectives and procedures. faced as it is by an increasingly tough international competition, the scientific community is also subject to restrictions in terms of operation and prospectives. an organization into small teams of a few researchers gathered around a boss, working in friendly interaction, is increasingly giving way to large groupings that sometimes seem like consortiums. focused on research subjects that are deemed to be "cost-effective", these superstructures are encouraged, or even imposed, in the sadly illusive hope that the will lead to greater efficacy. the person in charge of such large groups is taken up with everyday management tasks and by maintaining good relations with the administrative bodies on which his or her organization's survival depends. he or she may become distanced from the experimentation and forget the intellectual motivations that in the past caused his or her competence to be recognized. it should be emphasized that the secret of future successes lies in situations where young researchers are in direct contact with their bosses, and where friendly interaction with a known master teaches the apprentice researcher how to learn, how to think and how to experiment in a critical fashion. preoccupied by the rapid expansion of the scientific population, accompanied by the creation of laboratories whose operation necessarily requires financing, often on a large scale, political authorities, giving way to the requirements of media-fed public opinion, are interfering more and more, via administrative relays, in the control of the objectives of experimental research. short-term objectives, considered to be "visible", are favored. a priori, the viability of a project is judged according to the scientific context of the period and its impact on society, insofar as the project looks at health problems with a high degree of media coverage (cancer, degenerative illnesses, viral infections…) and often in agreement with a consensus that avoids going against the orthodoxy of the moment. this leads to a rigid management of projects that are financed and controlled according to objectives that have been fixed in advance, and that are all the more easily accepted by state authorities when they are somewhat fantastic in character. however, fundamental research proceeds from a playful activity, and for this reason, its efficacy is dependent on the passion of the researcher for the problem that he or she is studying. in contrast to what is believed by the narrow-minded, the effectiveness of a researcher in terms of discoveries depends upon the liberty that is given to this researcher, assuming, of course, that this liberty is underpinned by criteria of confidence such as the researcher's scientific past, his or her motivation, and judgements made concerning the researcher by impartial peers. it should not be forgotten that the determination of the three-dimensional structure of hemoglobin by max perutz (chapter iii- . . ) took around twenty years of solitary, uninterrupted and untiring labor. the theoretical and technical tricks that led to this success helped to open up the domain of the structures of giant macromolecules, several dozen kilodaltons in size, which no-one had dared study before. anyone who uses the experimental method realizes that while fundamental research must be organized, it cannot be scheduled. such a person knows that the pathways to discovery are convoluted, and that an inexplicable observation that appears unexpectedly during an experiment can sometimes, if the researcher is sufficiently perspicacious, be the beginning of an adventure that leads to a discovery. it was to just such a convoluted path that the belgian biologist christian de duve (b. ) alluded in his speech when he received the nobel prize for medicine and physiology in . after working at the university of saint louis in the usa, de duve, who had taken up a post at the university of louvain, belgium, decided to look at a research theme that had received a great deal of media coverage, diabetes and insulin. it was while operating on one of the subcellular fractions obtained from ground rat's liver, and analyzing certain of the enzyme activities of these fractions, that he was surprised to find, in one of them, enriched with mitochondria, a phosphatase activity that, paradoxically, increased with time, while the enzyme activities specific to the mitochondria declined. this was an activity belonging to organelles that were contaminating the mitochondria. dropping all research on diabetes, de duve set out to identify and characterize these unknown organelles. he discovered that they were involved in the breakdown (lysis) of molecules that are undesired by the cell and, for this reason, he called them lysosomes. the discovery of lysosomes helped to open a new chapter in cell biology and to attribute a molecular cause to diseases with serious prognoses whose etiology had remained a mystery up until then. these diseases were given the label lysosomal diseases. these diseases result from the absence of a lysosomal enzyme that is responsible for the breakdown of a given metabolite. the accumulation of this non-broken-down metabolite in the lysosomes leads to cell malfunction, which causes the lysosomal disease. as de duve said jokingly, if he had carefully followed the experimental process laid down in his diabetes research project, and if he had not given way to the temptation of "playing hooky" or "playing truant" he would never have mounted the podium in stockholm. in the same way, henri-gèry hers ( - ), a cell pathologist at the internationally renowned louvain school, remarked in an article published in the review médecine/sciences: "i believe we would obtain maximum value for the money devoted to research if we were willing to distribute it to those who have been shown to be productive, according to their needs, and without asking them for a program." hers concluded, in a tone that was deliberately playful, but thought-provoking, "such a simple system would lead to unemployment for a large number of administrators, which is why i suspect that it will never be adopted." research has its own set of ethics, driven by anticonformity and the creative imagination, capable of shaking up firmly-anchored ways of thinking and established hierarchies, and of leaving the researcher the freedom to express him or herself and to experiment off the beaten paths. as eccles says in evolution of the brain and creation of the conscience, it is important to distinguish between intelligence and imagination. intelligence is measured according to the rapidity and depth of understanding and clearness of expression. it may be measured and even given a numerical value. the same is not true for the imagination, a more subtle, unmeasurable phenomenon that cannot be learned. the imagination is one of the levers that is able to lift the boulder that hides scientific truth. the imagination is the ultimate weapon of research, which shakes up the knowledge acquired by the intelligence. nevertheless, the imagination must be tempered by a good critical sense that is able to perceive potential sources of artifacts, both in sophisticated instruments that act as so many black boxes from which already manufactured information emerges and in genetic or chemical cell exploration methods whose specificity must be carefully checked. the benefits that can sometimes be gained from prospective research that is far from dogma that is rooted in sterilizing tradition, the way in which knowledge progresses, most often by moving away from any orthodoxy, the way discoveries appear unexpectedly on the fringes of carefully put together projects, all of these points are matters for reflection for those in power in the worlds of politics, economics and industry. publication is an essential tool for communicating scientific knowledge, and is the judgement criterion for committees in charge of evaluating the creativity of a researcher. in order to have meaning, a publication must provide information that is sufficiently innovative with respect to parallel work carried out in other laboratories. here again, media coverage has quietly infiltrated the scene. its role is all the more perverse in that the rating of a publication is estimated according to its impact index, or, roughly speaking, the renown of the scientific journal in which it is published. curiously, it has happened that articles that would later be considered to be of primary importance have been rejected by highly prestigious journals, simply because the facts mentioned in the article and the conclusions made have not coincided with the orthodox opinions of the period and the traditionalist spirit of the journal's editorial committee. this was the case for an article which the biochemist hans krebs ( krebs ( - submitted to the british journal nature in . in this article krebs described a series of experiments showing that an endocellular metabolite, pyruvate, product of glycolysis, is completely degraded during a cycle of enzyme reactions. this degradation cycle would later be recognized as the central pivot of the intermediate metabolism. called upon to judge revolutionary scientific considerations, and unable to perceive their importance, nature's editorial committee rejected the article. krebs then sent his article to a journal with a relatively restricted audience, enzymologia. it was accepted and published in the two months that followed. the importance of the concept that was put forward in the article ensured that its author gained international recognition, leading to his winning the nobel prize for physiology and medicine in . for the researcher, publication is a way of making his or her work known. it is also the way in which the researcher learns about the work of others. while the rhythm at which publications in the life sciences appeared increased slightly in the first half of the th century, the second half of that century saw a great acceleration in this rhythm, leading to a difficult-to-manage proliferation of reviews and books. it has been estimated that in the last thirty years the volume of publications in the biological domain has increased five-fold; in the preceding twenty years it had already doubled. this accumulation of publications makes it harder for the researcher to judge the quality of the huge mass of published articles, even in the highly targeted domains that are within his or her area of expertise. the researcher, therefore, will deliberately choose a particular article according to the prestige of the journal in which it is published, which is not an inviolable criterion of quality. in addition, any judgement concerning the pertinence of a scientific article necessitates a dissection of the subtleties of the methodology, the well-groundedness of the experimental protocol and the validity of the results, by means of a careful examination of tables of results and graphs, and, finally, the logic of the discussion. this restrictive yet absolutely necessary requirement limits the number of articles that are likely to be screened. however, this is not the worse fault of publication today. there is another problem that is much more worrying. many documentation centers have reacted to this inflation in the scientific press by equipping themselves with computing facilities that are able to find, in data banks, articles that have been selected on the basis of a key word index, and to display them on screens. while acknowledging that this constitutes an inescapable change in the transmission of scientific know-how, it should be recognized that in browsing through the pages of a highquality scientific review, it is possible to come across an article containing an innovative idea or a useful technique, an advantage that is less available when using the on-line system of scientific publication that is most prevalent nowadays. mention should also be made of the requirement to publish frequently and within short time frames, for reasons of competitivity, when aspiring to obtain jobs or promotions, or even just to obtain recognition, this requirement being another factor that is prejudicial to fundamental research. it is the cause of worrying excesses, such as experiments that are hastily published and non-reproducible, or even the falsification of experimental results, occasionally within a context of considerable media coverage. although such practices, which are the exception rather than the rule, are rapidly detected and condemned in a scientific culture where information circulates freely, the publicity that they incite, which reaches society at large via the media, leads to an overall discrediting of experimental research. at present, one of the most noticeable trends in scientific publication is that of collectivism. while, in the th century, scientific articles were usually published in the name of a single author, occasionally two authors, and very rarely more than two, nowadays publications are often co-authored by several people, and when the work involves the analysis of structures, or the sequencing of genomes, several dozen researchers may be co-authors. from being the work of individuals, research has become collective. in domains whose complexity requires a wide selection of techniques that may range from physics to genetics, the hybridization of specific areas of expertise is certainly indispensable, and this requires the collaboration on a particular project of researchers who are sometimes physically remote from one another. the downside for the researcher, particularly one who is young, is that this requires him or her to abandon individuality and creativity. both collectivism and inflation in scientific publication are facts that are an integral part of contemporary science, facts which reflect an irreversible trend that it would be difficult to obviate. over the last few years, scientific publication has been subject to a type of restraint, in that certain "sensitive" data in the domain of molecular biology might be used for the manufacture of biological weapons in a form of terrorism known as bioterrorism. thus, the means of synthesizing de novo viruses (influenza virus, poliomyelitis) and the possibility of modifying their tropism by "directed molecular evolution" (change from a sexual tropism to a respiratory tropism for the aids virus) have been the subject of publications in prestigious journals. given sufficient means, terrorist pharmacists could well make use of such data in order to carry out malicious actions with catastrophic consequences . in order to please a public that is eager for progress and the sensational, politicians favor, by means of targeted financing, the types of organization that appeal to their sensibilities, such as the technological platforms. while recognizing that such platforms are now an integral part of the landscape of research on living beings, and that they must therefore be taken into account, and while acknowledging that projects which implement the latest technologies in different domains need to be federated, it is nonetheless vital not to underestimate the potential creativity of small groups of researchers, a point that was expressed by one of the greatest of contemporary biologists, arthur kornberg ( kornberg ( - , winner of the nobel prize for physiology and of medicine, in a speech given in : "as i view the steady growth of collective science and big science, the greatest danger i see is a dampen-ing of individual creativity and reversion to the old politics -the inevitable local politics that infects every group and institution." however, conscious of the metamorphosis that is occurring in the experimental method, and faced with a particularly inventive and all-conquering technology, fundamental research in the life sciences must come to terms. a century ago, fundamental research and technological research interacted all the more directly because they were both in their infancy. this is no longer the case. management of the ever-increasing amount of knowledge in the life sciences, and the degree of sophistication achieved by bioengineering techniques and instruments, is widening a gap that makes dialogue increasingly laborious. however, dialogue appears to be a guarantee of future progress. the solution can only come from an increase in cross-disciplinarity, which should begin with university teaching and the establishment of a recruitment policy that advocates the cohabitation of talents from different educational backgrounds in the same laboratory. fortified by such hybrid expertise, while maintaining its share of originality and liberty in the choice of problems to be studied, fundamental research on living beings can only be enriched by a marriage of reason with biotechnology. convinced of the necessity for such a marriage, stanley fields, the inventor of the double hybrid method (chapter iv- . ), in an article entitled "the interplay of biology and technology" (proceedings of the national academy of sciences, usa, , vol. , pp. - ), concludes,: "it is at the interfaces of biology and other sciences that many of the future discoveries will be made, at the interfaces of biology and engineering that these discoveries will come to be exploited, and at the interfaces of biology and ethics and law that their consequences for society will be decided." the desired dialogue between biology and technology also implies the breaking down of barriers that too often isolate fundamental research and so-called applied research, and the facilitating of consistent interaction between the discoveries made in the academic institutions and their application for utilitarian ends in private companies. this is where the twin demons of money and power raise their heads. already, at the turn of the s, a. bartlett giamatti ( - ) , who was then president of yale university in the usa, commenting on american university policies, spoke of a "ballet of antagonisms" between, on the one hand, commercial companies that are interested in the rapid cost-effectiveness of any new therapeutic advance and, on the other hand, non-profit university laboratories. recently, james j. duderstadt (b. ), emeritus president of the university of the michigan, argued that the university is a "counter-hierarchical" organism. in fact, its members are free to carry out the research that pleases them and to think in the ways that they wish to think, in any case within an academic norm that considers itself as being free from the constraints dictated by private interest groups. until recently, such behavior was considered as a sort of ethic which arose out of the university conscience and dignity. the crumbling away of this ethic in the final decades of the th century coincided with the rise of biotechnologies and the large-scale filing of patents relating to molecular genetics techniques that could be applied to the manipulation of living beings, by researchers in the public sector. the intrusion of the american private sector into public research laboratories, in the form of collaborations with transfer of "sensitive" information from the public to the private, has become such a worrying problem that drastic control measures have had to be taken. within this context, the american federal government, in february , issued a certain number of prohibitions targeting the national institutes of health (nih) of bethesda, particularly with respect to the retribution of researchers for services rendered to industry . these stands call for thought concerning the place that is currently held in universities with respect to fundamental research. without arguing against the efficacy of major research institutes, it is nevertheless necessary to remember the part played by the university in this domain. the university is not only the place where knowledge, both as it is now, in its current state of advancement, and as it has been, it is also the place where knowledge must be created by fundamental research. for the last few decades, under pressure from state policies, and also as a function of an improvement in social status, the world of the university has opened up to a wider public, leading to an influx of students that is sometimes so enormous that the task of teaching them has become overwhelming. because of this, the share of their time that university researchers can, in practice, devote to their research tasks has shrunk. this situation is highly prejudicial to the mission to innovate, which should be a priority. it is, in fact, during their university studies that the thought patterns of young students are forged by contact with teachers who not only instruct them, but also educate them by inspiring in them a motivation and an enthusiasm that gives rise to hope. how could this be true if the teaching faculty did not itself participate in scientific creation? "what can teaching, ex cathedra, do to guide the researcher? nothing, obviously. the researcher is trained in the laboratory. and the first stroke of genius on the part of a future researcher is to find a good boss. such a find will open up the royal road to success. the road will be opened -but the researcher must travel along it. a researcher may be taught many things. he or she can become familiar with techniques and with equipment. she or he can be assigned a problem to resolve. however, what is essential for the researcher is to know how to understand relationships between phenomena that seem unrelated, and to be able to progress from the particular to the general. a boss may develop such qualities in a gifted young researcher, but intuition is a gift; it cannot be taught." while the bernardian style experimental method, based on a working hypothesis aroused by an observation, followed by implementation of an experimental protocol, is still extant in the life sciences, and while "serendipity" is still the origin of great discoveries, "big science" , underpinned by sophisticated biocomputing or bioinformatics procedures, is intruding more and more, while genomics and proteomics are not far behind. the methods and instruments developed by the biotechnosciences have led to profound modifications in the ways that the structures and functions of living beings are investigated. for example, by varying multiple parameters in dna chips or protein chips, at the same time, the experimenter is able to ask questions that lead to grouped all-or-nothing answers (chapter iv- . . ). in combinatory chemistry, screening makes it possible to detect a molecule that is active for a given pathology from among a multitude of molecules (chapter iv- . ). the mathematical simulation of metabolic networks or of signaling chains is already well under way (chapter iv- . ). given this new technological outlook and the hope that it can provide rapid solutions to health problems subject to considerable media coverage, the teaching of biology in universities must not be limited to a description of current advances, no matter how brilliant and promising they may be. this teaching should return to its origins, be a reminder of history, and should not hesitate to use examples to illustrate how a major discovery can arise from a long period of wandering in the wilderness. in practical terms, while being conscious of the extraordinary complexity of living nature, and carefully avoiding the dangers of simplification, it is important to remember that the reductionist method was a necessary path to an understanding of the integrated, modelized biology that is emerging nowadays. at present, certain people call reductionism naive, but this is only the case insofar as we have faith in recent advances in integrated biology . with this in mind, it should be noted that the deciphering of the protein synthesis mechanism in prokaryotic microorganisms (chapter iv- . . ) was, along with the discovery of the genetic code, a jumping-off point for an inventory of similar, but noticeably more sophisticated, mechanisms in eukaryotic organisms. the reductionist "one gene, one enzyme" dogma, formulated on the basis of beadle and tatum's experiments on the mold neurospora crassa (chapter iii- . ) was a necessary prerequisite to a considerably more elaborate understanding of the relationship between the genotype and the phenotype. the way in which the nucleic acid and protein units in the tobacco mosaic virus spontaneously organize themselves (chapter iii- . ) acted as a basis for thought concerning the self-organization of macromolecular complexes in the cell. these few examples underline the fact that it is difficult to comprehend the scientific research process if we only refer to experiments carried out in the present, and if we do not have a clear idea not only of the way in which hypotheses, even false ones, were once formulated, but also of the way in which experimental work, which may have led to failures, was once carried out, or, in brief, if we do not look back at the past. let us add that it is occasionally good for us to show some humility when we take the trouble to examine the past. thus, the processes involved in the phagocytosis of bacteria by innate immune cells (neutrophils, macrophages), which are today studied in the greatest detail with particularly refined technical facilities, had already been perceived more than a century ago by metchnikoff, and even analyzed, admittedly with the clumsy means at his disposal, but with such accuracy that none of the conclusions formulated at that time have yet been disproved (chapters iii- . . and iii- . . ). the experimental method applied to the life sciences, the history of its birth and of its development, the way in which it is regarded by political and societal authorities, and, finally, the dependencies that are developing at present between the technosciences, human medicine and the different branches of the economic sector, all of these aspects should be covered by university teaching that includes not only the pure sciences, but also the human, political and economic sciences, as well as philosophy. the student should not be saturated with book-learning, but he or she should be taught to reason, to imagine and to criticize, not to accumulate knowledge in an indigestible catalogue, but to ask questions about the way in which certain, carefully chosen, items of knowledge have been acquired, and not to deliberately accept science in its current state without knowing what it was like in the past. he or she should understand what pathways of thought led to dogmas that were established and taught as truths being refuted, and favor experimentation, with its risks and questions, rather than well-smoothed, abstract theoretical presentations without rough edges. these should be the principles of teaching that is designed to open up young minds to creativity. in anglo-saxon countries, the worlds of industry and research that welcome the graduate manage to communicate with one another, but these worlds ignore one another in france, or at least remain reserved, a situation which is prejudicial from the economic point of view. if we look at the pharmaceutical industry in particular, we see that only half a century ago the pharmacopeia was limited to plant extracts or active agents isolated from these plants, with antibiotics quietly beginning to make their appearance. in the last decades of the th century, a great technological leap forward was made, with completely new methods in bioengineering, combinatory chemistry, and the finding of therapeutic targets in macromolecules, and this created a hiatus that severely handicapped countries that were unprepared for it. france, with its biological fundamental research training that is out of phase with that of the anglo-saxon countries, fell behind, and continues to be behind, a situation that is prejudicial for its economy. the remedy for this does not lie in incantatory speeches. it requires a volontarist policy for the management of experimental research. generally speaking, the fact that the major engineering schools in france, which recruit the scientific intellectual elite, students being chosen by competitive exams that select for intelligence rather than imagination, are unable to impose upon their students an end-of-course thesis that would authenticate their engineering degree, should not be tolerated. in contrast to other countries, in france only a small percentage of engineers have received doctoral training or had to present a thesis before entering their careers. the french dual system of major engineering schools and universities, which, a century ago, made sense for the economy of that period, has become completely obsolete, and deserves a courageous revision. "there is a question, much older than modern science, which has never ceased haunting certain men of science: that of the conclusions that the existence of science and the contents of scientific theories can lead to concerning the relationships that humankind has with the natural world. such conclusions cannot be imposed by science as is, but they are an integral part of the metamorphosis of this science." the new alliance. metamorphose of science - ( nd edition) in the s - s, the hybridization of the techniques of genetics, biochemistry and biophysics gave birth to molecular biology. with the resolution of the double helix structure of dna, the demonstration of its replication, the elucidation of the mode of expression of its nucleotide sequence as a sequence of amino acids in proteins and finally the deciphering of the genetic code, biology underwent a revolution of an amplitude similar to that which, at the end of the th century, saw a blossoming of the seeds of cell biology. the last decades of the th century represented the utilitarian era of molecular biology. the introduction of genetic engineering into biological experimentation dates to the beginning of the s. it was at this time that techniques were developed that made it possible to transfer a fragment of genomic dna from one species into the genome of another species. genetic engineering now fills a predominant position in the life sciences, supported by increasingly effective biocomputing or bioinformatics techniques. it is easy to understand that expertise and a high degree of knowledge about fundamental research is necessary in order to be able to master or even invent the genetic engineering techniques that are indispensable if we are going to produce biomolecules with a therapeutic impact, such as those that are currently being used in the pharmaceutical domain: insulin, growth hormone, blood coagulation factors, vaccines, etc. the engineering sciences that make up the greater part of contemporary biotechnology have now come to the fore in many domains of the life sciences. it is thus that a modernistic and original way of investigating nature has come into being. a multiparametric model, in which biocomputing or bioinformatics and high-throughput screening reign, is added to, or even substituted for, the bernardian model for the experimental method, based on observation, an a priori hypothesis, and experimentation to verify this hypothesis by varying a single parameter at a time. the aim of this globalized approach is to integrate the multiple reactions that take place almost simultaneously in different locations of a cell into a coherent whole, to rationalize the interpretation of the dialogue that operates between the different endocellular organelles, and finally to discover how the exchanges of information between cells in an organ and between organs in multicellular organisms are set up. we are therefore witness to the emergence of an integrated biology that has been labeled "systems biology". its long-term objective is to model the functioning of living beings and to theorize them. its development is encouraged by the perspective of consequences that could revolutionize certain sectors of the human economy and of public health. today, concrete, mechanical models, in the form of biorobots and hybrid robots, and, very recently, molecular motors are added to abstract models that are based on the logic of mathematics and algorithms, ushering in the era of nanobiomachines. becoming more utilitarian, the life sciences are imperceptibly detaching themselves from traditional philosophical concepts that try to explain the modes of reasoning of the researcher, or even to impose a framework for thought that is likely to orient his or her way of doing research. looking at genetic inheritance, contemporary experimentation has shown that at all levels of the tree of nature, including man, this inheritance can be modified. aware of his or her ability to influence the functioning and the destiny of living beings, the researcher is confronted with the dilemma of a desire for knowledge versus a questioning of the use to which discoveries may be put. there has never been such a real divorce between the world of phenomena that are understood by the experimenter and the world of noumena whose intelligibility is foreign to our senses. there has never been such a wide gap between the biotechnosciences, whose possibilities are coming to be seen as limitless, and a reflective analysis of thought, which wanders between freedom of action and prohibition. as society becomes aware of the potential applications of discoveries made concerning living beings, problems of bioethics, particularly those involving reproduction, have become problems of public interest. cloning and the production of stem cells are subjects that give rise to diatribes and passions. in the near future, genotyping, which is the result of progress in pharmacogenetics, could usher in a new form of customized medicine. elsewhere, the cognitive sciences that are bringing together philosophy and psychology in the domains of computer technology and artificial intelligence, and which are tackling the processes of thought, the creative imagination and memory, will no doubt be the subject of the considerable questioning concerning research on living beings with which the experimental method will be confronted in the st century. when faced with the way in which biotechnologies have erupted into the life of society, the mind travels back to the allegorical illustration that embellishes francis bacon's novum organum (see figure ii. ) , showing vessels returning from unknown lands, loaded with precious cargoes and returning to port having sailed past the pillars of hercules. at present, the challenge has been partially met, but a great deal remains to be done. innumerable cargoes have already reached port, but what will be the destiny of this precious merchandise? after all, the seeds of the idea of technoscience were already in place in the th century, in the philosophy of francis bacon and robert boyle (chapter ii- ). bacon recommended that the governments of the time promote experimental science by the creation of laboratories equipped with high-performance instruments and libraries, by the organization of researchers into teams and by appropriate financing. the utilitarian ends of scientific research were underlined. boyle imagined a situation in which laboratories were open to society and researchers were able to accept criticism. given innovations that upset tradition, protestations arose. the pneumatic machine or vacuum pump was the subject of the fameuse diatribe between boyle and the philosopher hobbes (chapter ii- . ). hobbes criticized the validity of boyle's conclusions, drawn from experiments that he qualified as doubtful. following his words, he came to see in the discoveries of experimental science a possible threat to the power of governments and the hierarchical layout of society. such overcautious opposition to the pursuit of knowledge is in no way anecdotal, it is still a reality, with the uprooting of genetically modified plants and the veto that has been placed in certain areas on stem cell research. this type of opposition is also shown when pressures or even vetoes are in operation that take into account more the opportunism of the moment than an in-depth understanding of science and of its history and that forget that freedom of the mind is a guarantee of its creativity, because, just as in the world of arts and letters, the world of scientific research is situated outside those norms that can be modulated by state decrees. the creativity of the researcher cannot be manufactured on demand. where it exists, it still needs to be detected and encouraged. the atp-synthase -a splendid molecular machine structure at . Å of f -atpase from beef heart mitochondria direct observation of the rotation of f -atpase powering an inorganic nanodevice with a biomolecular motor mechanically driven atp synthesis by f -atpase atp-driven stepwise rotation of fo-f atp synthase key: cord- -f j fsc authors: chamboredon, p.; roman, c.; colson, s. title: covid‐ pandemic in france: health emergency experiences from the field date: - - journal: int nurs rev doi: . /inr. sha: doc_id: cord_uid: f j fsc aim: this paper describes the situation regarding covid‐ emergency in france as of early may , the main policies to fight this virus, and the roles and responsibilities of nurses regarding their work at this time, as well as the challenges facing the profession. background: europe continues to be affected by the covid‐ pandemic. at the time of writing france was the fourth country with the highest number of detected cases and cumulative deaths. sources of evidence: websites of the world health organization, french government, french agency of public health, french national council of nurses and clinicaltrials.gov database, as well as the experiences of the authors. discussion: the history of the development of the pandemic in france helps explain the establishment of the state of health emergency and containment of the population. many decisions made had undesirable repercussions, particularly in terms of intra‐family violence, mental health disorders and the renunciation of care. hospitals and primary care services, with significant investment by nurses, played a key role in the care of persons with and without covid‐ . conclusion: france has suffered a very high toll in terms of covid‐ morbidity and mortality, and effects on its people, health systems and health professionals, including nurses. implications for nursing practice: nurses are recognized for their social usefulness in france. however, it is important to consider the collateral effects of this crisis on nurses and nursing and to integrate the health emergency nursing skills established during the pandemic into the standard field of nursing competence. implications for nursing policy: the nursing profession has expectations of a reflection on and revision of nursing skills as well as of its valorization in the french healthcare system, notably carried out by the french national council order of nurses. • present a brief history of the development of the pandemic in the country, including the political decisions that have been taken to combat it; • explain the repercussions of containment measures on the health of the population; • describe the roles and responsibilities of nurses regarding their work during the pandemic, as well as the challenges facing the profession; and • summarize the current french research studies in progress about covid- . the covid- pandemic is undoubtedly the most serious global health crisis in decades, causing more than deaths worldwide as of may (world health organization [who] a). this is a devastating new virus. first reported in wuhan, china, on december , the virus gradually spread to europe and the rest of the world (who b). the emergency situation was declared by who on january . within days of the outbreak of the virus, the director-general of who found that more than . million people were confirmed as infected, of whom nearly died (who c) . at the time of writing on may , the situation in europe remains catastrophic: more than reported cases and more than cumulative deaths (who d) . the most affected countries are spain ( detected cases, cumulative deaths), the united kingdom ( detected cases, cumulative deaths), italy ( detected cases, cumulative deaths), germany ( detected cases, cumulative deaths) and france ( detected cases, cumulative deaths). the situation has necessitated the reorganization of healthcare systems and changes in population lifestyles and has led to particularly difficult economic consequences. to date, the primary strategy has been to utilize cross-contamination measures to prevent the spread of the virus such as good hand hygiene, avoiding close contact with others or social distancing and respecting respiratory hygiene rules. population containment measures have been implemented in many countries, and particularly in france, from march . france is the fourth most affected country in europe. the number of deaths is important, but just as important are the more than people who underwent hospitalization for covid- (french public health ) . the data are updated daily. the most reliable indicator to date remains the incidence of covid- cases entering resuscitation/critical care every day, which is beginning to plateau. france must manage the first wave of the pandemic while deploying all means to avoid a second wave. health policies must then adapt to a virus whose spread is not fully known and whose treatments are currently being evaluated. these many unknowns in the equation lead to the need to adjust policy measures in france on an almost daily basis. in preparing this report from the field, relevant information was taken from the websites of who, french government, french agency of public health and french national council of nurses. the clinicaltrials.gov database was also examined. we have also drawn on our experiences as french nurses. the identification of the first three cases of covid- positive patients was announced by the ministry of solidarity & health ( a) on january . the virus began to circulate in france, considered to have been transmitted by people who had stayed in china or singapore and had been in contact with infected people. the first death in france was announced on february (ministry of solidarity & health b). despite the isolation of the cases identified and the reminder to the public to practice barrier actions, covid- spread. subsequently, the minister of solidarity and health, olivier v eran, initiated the plan d'organisation de la r eponse du syst eme de sant e en situations sanitaires exceptionnelles (orsan) (organizational plan for health system response in exceptional health situations) under the epidemic and biological risk section on february , enacting the various protocols to be implemented in the context of a health crisis (ministry of social affairs, health and women's rights ). stage of this plan consisted of isolating the identified cases and the people they had been in contact with, at the time numbering about people, to slow down the spread of the virus in the country. a few days later, on february , france moved to stage , which consisted of slowing down the viral spread, following the identification of several epidemic outbreaks and the first deaths linked to covid- . barrier measures were widely disseminated to the population, and containment measures were implemented locally in areas with identified infectious outbreaks. on march , when who declared the status of a pandemic concerning the novel coronavirus (who e), crisis measures were taken by the president of the french republic ( a) and his government, to control the epidemic and manage the health situation, namely, the closure of the nurseries, schools and universities for users as of march ; the introduction of short-time work hours for employees whose companies cannot carry out their activities and of teleworking for all employees who have this possibility of adjusting the exercise of their profession (ministry of solidarity & health c). however, a few days later, the number of cases and deaths increased. stage was declared to reduce the circulation of the virus in the population and mitigate its effects. all nonessential public places were closed, and several measures put in place by the french government to manage what was becoming the country's biggest health crisis in several decades. on march , the president of the french republic spoke live on television, declaring that 'we are at war' against covid- ( b) . the white plan corresponded to the provisions of orsan to organize health facilities in response to a major health crisis (ministry of social affairs, health and women's rights ). it consisted of four points: mobilizing health establishments to respond to a crisis situation, mobilizing health professionals, mobilizing the material and logistical resources of establishments and adapting their medical activity. initiated in health establishments close to identified epidemic outbreaks, the white plan was generalized throughout france when the epidemic reached stage . a new gradation of care began to be implemented: university and public hospitals as the first line to receive patients with covid- , private hospitals with at least an emergency department and critical care service as the second line and private hospitals with critical care service as the third line. all other care facilities were placed in the fourth line. covid- units were set up in more than public hospitals, and new resuscitation places were being created, increasing the capacity from to beds (prime minister of the french government ). healthcare professionals were mobilized as well as health students on internships or volunteers, and retired people were also called upon to strengthen healthcare teams. the french system of mobilization by the state of volunteer health professionals in exceptional health circumstances, known as the health reserve, was activated to provide support in the areas most affected by the epidemic (ministry of solidarity & health d). non-urgent medical activities were deprogrammed, and the monitoring of chronic pathologies was reorganized. primary care teams, especially home care nurses, were also referred to as backup, to manage not only the usual care of the population but also the aftercare of covid- patients discharged from hospital or those who did not require hospitalization, only simple monitoring at home. however, as the existing legislative and regulatory measures were not sufficient to deal with the crisis, the french state introduced the state of health emergency (president of the french republic c). this new state of health emergency covered parts or all of the territory (including overseas territories) in the event of a health disaster that, by its nature and severity, endangered the health of the population. within this framework, the prime minister, as head of the french government, could decree measures listed by the law: order home confinement, requisition personnel and equipment, and prohibit gatherings. the prime minister could also take temporary measures to control the prices of certain products, allow patients to have access to medicines and decide on any regulatory limits to entrepreneurial freedom. the minister responsible for health could, by ministerial order, determine other general and individual measures. the military operation 'resilience' was launched on march (ministry of the army ). the french army served as a reinforcement to provide assistance and support to the population and public services in terms of health, logistics and protection of the entire territory. mistral and dixmude helicopter carriers were deployed in the southern indian ocean (reunion, mayotte) and in the antilles-guyana regions. implementation of containment throughout france up to may to decelerate the circulation of the virus, the government implemented a containment of the french population (prime minister of the french government b). travel was severely restricted. a certificate justifying individual movements was required to leave the home, and checks were carried out by the police and the army to ensure that these restrictions were respected by the population. those not respecting the confinement were fined or even sentenced to imprisonment according to the severity of the situation. economic measures were put in place urgently by the french state (president of the french republic c). to safeguard jobs and reduce the risks of job insecurity, a shorttime working scheme was launched for the duration of the confinement, enabling more than million people to receive at least three-quarters of their wages. an adapted sick leave scheme was set up for parents of children under years old who could not telework, pregnant women in the third trimester, and vulnerable or fragile persons. unemployment benefit entitlements were extended for persons reaching the end of their entitlement. several types of aid were likewise offered to companies affected by the crisis, to safeguard them and secure jobs in france. concerning children's schooling, pedagogical continuity was achieved at a distance, in virtual classes, or through homework assignments to be carried out with the help of parents. this system had major limitations, including the absence of computer equipment in low-income families, saturation of the bandwidth of internet connections and saturation of educational platforms, which are not accustomed to such a large number of simultaneous connections. containment measures were applied in medical establishment for dependent older adults for dependent older adults (ehpads), where the circulation of the virus was particularly harmful. older adults were initially confined to their rooms, without visiting rights, and these measures were recently relaxed, with permission for visits without physical contact. the french government conferred a broadening of competences and recognition of the role of home care nurses. the health context made it possible to create the first telecare procedure related to the management of patients with covid- by home nurses during the period of the state of health emergency (high authority of health ; prime minister of the french government c). for the duration of the epidemic, a patient diagnosed with covid- could benefit from telecare on prescription, as long as the patient guarantees their availability and mastery of the tele-monitoring tools (smartphone, computer with wi-fi connection, or, failing that, telephone). telecare would be fully covered by the french health insurance. before any care was provided to the patient with covid- , a nurse collected general information and the care plan prescribed by the doctor for the patient (e.g. points of vigilance, monitoring rhythm). during the first contact, the nurse assessed the patient to confirm the criteria for inclusion in the telecare system, supplemented by measures related to the current situation and, in particular, the implementation of hygiene and prevention measures for the family caregiver. then, as part of the follow-up set-up according to the severity of the patient's condition as indicated by the doctor, the nurse carried out the following: determining the patient's general condition, looking for signs of worsening symptoms, collecting clinical observations at a distance (e.g. temperature, weight), looking for signs of altered consciousness, looking for signs of dehydration, reminding the family and friends of the hygiene and prevention instructions, coordinating with the doctor regarding an alert without delay if the patient's condition required it, or call for emergency medical assistance in case of distress, in parallel with the information from the doctor. if the nurses considered that the conditions would no longer enable them to carry out the follow-up, they would then go to the patient's home to carry out face-to-face monitoring and inform the attending physician, who will adjust the prescription for nursing follow-up as necessary. this new system, requested by the order of nurses, made it possible to monitor patients while drastically reducing exposure to the risk of contamination for caregivers. if telecare was not possible for patient follow-up, and to avoid the risk of spreading the coronavirus within home nursing structures, nurses could opt to follow-up their patients at home, even without specific instruction from the medical prescription. the related procedures were subject to specific coverage and price re-evaluation by the health insurance. prolonged containment can have several implications for the health of the population. the first concern to be feared was the impact on mental health, brought by social isolation, fear of illness and uncertainties in relation to the illness. a survey was conducted by sant e publique france with a sample of internet users to characterize the impact of covid- on the general population and to influence the political measures to be implemented to care for the population (french public health b). because the abovementioned repercussions may be more severe for people with disabilities, particularly psychiatric disorders, specific measures were recommended by the high council of public health ( a) to adapt containment measures to the problems of each person concerned. these containment measures were applied in ehpads, where the circulation of the virus was particularly harmful. the second concern was that a large, difficult-to-measure proportion of the french population seemed to have given upon their usual, acute or chronic care, mainly because of covid- containment measures and the fear of being contaminated. according to a recent survey by a telemedicine platform, the number of consultations with general practitioners decreased by % since the beginning of containment, and by % for specialist physicians (doctolib ) . to date, the effects of this situation remain difficult to assess, especially for people with particular health vulnerabilities. meanwhile, paediatricians alerted the authorities to the decrease in the number of families requesting paediatric consultations, particularly for consultations in connection with the programming of children's vaccinations (french association of outpatient pediatricians ). the risk of a resurgence of infectious diseases in children is becoming significant because it is not possible to identify the proportion of children who are not vaccinated according to the vaccination schedule issued by the high council of public health. third, confinement unfortunately endangers a certain number of women and children who are victims of domestic violence (usher et al. ) . the french government ( ) widely publicized the possibility of contacting a telephone hotline to report situations of violence. recently, these reports have doubled; however, it is difficult to obtain reliable data to date to estimate the number of collateral victims in confinement. for these reasons, the government has wished to introduce deconfinement for children, who seem less sensitive to the virus, so that a certain number of them can return to school, eat at least one balanced meal a day and escape intrafamily contexts that are harmful to them. finally, several french nurses faced threats or were subjected to malicious acts, often anonymous, by neighbours in particular: posters or anonymous letters asking the nurse to move to avoid contaminating an entire residence, vandalism on personal vehicles or in professional premises, theft of equipment and assault. the french national council order of nurses ( a) assisted nurses who were victims of these malicious acts in legal proceedings. gradual deconfinement was being implemented as of may (prime minister of the french government d). the national deconfinement strategy was based on three main principles: protecting the population through barrier gestures and the wearing of masks in certain situations, testing the population on a large scale and isolating sick people and contact cases. departmental (territorial division in france) maps were established to report on situations that may or may not be conducive to deconfinement, according to three main indicators: the rate of new cases in the population over seven days, hospital resuscitation capacity, and organization of the local testing and contact case detection system. the deconfinement plan announced the opening of some public places, including schools, but advised the maintenance of teleworking as much as possible. new rules for social life were also introduced. if the indicators were unfavourable, then a department would not be deconfined. two phases were planned: a first period of deconfinement from may to june , followed by a second period before the summer holidays. despite the exceptional purchasing and requisitioning measures by the french government of personal protective equipment (ppe) and other urgent health supplies, caregivers were left with a real lack of protection, as was the case elsewhere in the world. france was counting on its main supplier, china, without foreseeing that if china itself was exposed to a health crisis such as covid- , stocks of chinese products would then be used primarily by china. to obtain more precise information on the situation, the french national council order of nurses ( b) carried out an online consultation from april to april , in which more than nurses participated (a sample of % of the french nursing population). the main results were as follows: • nearly three-quarters of the nurses consulted stated that they did not have enough ppe. • of the nurses consulted, % said they did not have enough gowns, and % said they did not have enough masks. • of the nurses consulted, more than two-thirds ( %) stated that they did not have enough protective goggles. • more than half ( %) said they did not have enough overshoes. • more than half ( %) stated that they did not have enough mob caps. • nearly half ( %) stated that they did not have a sufficient quantity of hand sanitizers. the french state set up an emergency system for the purchase of ppe. it has been able to count on the solidarity of the french population and companies, which, on a voluntary basis, have developed the production of masks, gowns and hand sanitizers, although this was not their primary function. to date, french studies on covid- have been referenced in clinical trials, of which are in the process of gathering participants. these studies cover the epidemiology of covid- , clinical trials of drug treatments and their side effects, and the effects of containment. different drug strategies are being investigated, and the results of these studies are expected to be published soon. the results of these studies are eagerly awaited by the french government, by the scientific community, as well as the population. france has suffered a very high toll in terms of covid- morbidity and mortality, and adverse effects on its people, economy, health systems and health professionals, including nurses. the context of the health crisis caused by covid- in france is leading to strategic and political changes on a daily basis. health professionals in hospitals and primary care facilities are in the front line of the health management of the crisis. however, the population, through political decisions, has a duty to support healthcare workers to reduce the circulation of the virus. after a confinement of almost two months, france is preparing to live a new life, partly deconfined, but with new habits to implement, and above all, a deep reflection on the aftermath of the pandemic. nurses play a key role in the context of the covid- health crisis, in hospitals, medical and social care institutions and primary care. the public is largely grateful for nurses' involvement and dedication in this context. although public gratitude may bring satisfaction and value to the profession, the collateral effects of this crisis on the nurses themselves need to be studied. the authorities likewise need to ensure that nurses remain in their profession. derogating measures that would extend the scope of nursing activities during crises also need to be considered to develop and establish them on a permanent basis in nursing practice. it would be inappropriate to withdraw recognized skills acquired during the crisis once the crisis is over. the french concerns are completely in line with the global concerns raised by the international council of nurses (icn), which calls for the recognition, respect and protection of nurses (international council of nurses a). the context of this health crisis places the nursing profession in a social mandate recognized by the french population. it is imperative that nursing practice be adapted and evolved so that france can win the fight against this virus. the french national council order of nurses ( c) has asked the french government to deploy several means to help nurses accomplish their daily mission: an intensification of efforts to equip nurses working in residential institutions for dependent older people, medico-social establishments or at home with ppe and systematic screening of health personnel; additional efforts to promote tele-nursing; the introduction of differentiated spaces and rounds of home visits (covid- /non-covid- ); a more efficient system to ensure the quality and continuity of care for all, particularly for at-risk populations and those suffering from chronic pathologies; a strong fight against any malicious act or discrimination towards healthcare workers with regard to their employment and the covid- risk; the possibility for nurses to carry out the entire procedure relating to releasing death certificates instead of a doctor; and the prescription of covid- tests. the french national council order of nurses has also called for an accurate count of nurses infected with and died from covid- , the recognition of occupational disease for infected caregivers, and the granting of the status of ward of the nation for the children of deceased nurses. these latter concerns appear to be global, as the icn also notes that the number of nurses who died from covid- appears to be underestimated (international council of nurses b). these requests were made during the time of the covid- crisis, but the french national council order of nurses asked the french government to rethink completely its vision of the nursing profession. today, the nurse is an essential link in the patient's care journey. the nurse is a clinician, and this must be reflected in a progressive evolution of nursing skills to include skills regarding medical prescription. the international council of nurses has positioned itself to ensure that the critical role of nurses in the management of covid- , as well as in day-to-day operations, is fully recognized by governments around the world (international council of nurses c). the state of the world's nursing report provides a basis for reflection on the evolution of the nursing practice and better recognition of nurses in all countries (who f). covid- : doctolib alerts on drop in practice attendance and commits to allowing patients to return for consultation press release april. available at protecting children, continuing to care for them in the midst of the pandemic (in french) the government fully mobilized against domestic and intra-family violence the order assists nurses who are victims of pressure or aggression in their legal proceedings (in french) covid- : the national order of nurses alerts on the situation of the profession and announces new emergency measures (in french) the national order of nurses makes recommendations for priority measures for deconfinement (in french) covid- france covid- : a survey to monitor behavioural and mental health changes during confinement rapid responses under covid- teleconsultation and telecare epidemic at covid- : support for people with disabilities (in french). notice international nurses day: nurses deserve praise, thanks, protection amid covid- . press release may icn says worldwide death toll from covid- among nurses estimated at may be far higher international council of nurses: nursing the world to health prime minister of the french government . introduction of the emergency law to deal with the covid- epidemic address transcript march assistance in organising the provision of care in exceptional health situations (in french) three cases of coronavirus ( -ncov) infection in france (in french) covid- : a twelfth case confirmed in france (in french) order of march laying down various measures to combat the spread of the covid- (in french). regulatory text march ministry of solidarity and health d. order of march on the mobilization of the health reserve (in french) ministry of the army . operation resilience (in french) address to the french population address to french population as an emergency measure to deal with the covid- epidemic decree no. - of march prescribing the general measures necessary to deal with the covid- epidemic within the framework of the state of health emergency (in french). regulatory text march decree no. - of adopting adapted conditions for the receipt of cash benefits for persons exposed to coronavirus (in french). regulatory text march presentation of the national deconfinement strategy (in french) family violence and covid- : increased vulnerability and reduced options for support who health emergency dashboard who (covid- ) homepage world health organization b. coronavirus disease (covid- ) pandemic who director-general's opening remarks at the mission briefing on covid- rector-general-s-opening-remarks-at-the-mission-briefing-on-covid covid- situation in the who european region who director-general's opening remarks at the media briefing on covid- state of the world's nursing report - firstly, we thank all nurses in france, from all sectors of activity, for their involvement in this covid- crisis. secondly, we thank the french national council order of nurses for the financial support for the linguistic revision of this article. manuscript design: sc data collection: pc, cr, sc manuscript writing: pc, cr, sc critical intellectual revisions of manuscript: pc, cr, sc key: cord- -vxhta a authors: véran, emilie; gallay-lepoutre, julie; gory, guillaume; guillaumot, pierre; duboy, julie title: chyloabdomen in a cat with pancreatic carcinoma date: - - journal: open vet j doi: . /ovj.v i . sha: doc_id: cord_uid: vxhta a a -year-old spayed female domestic shorthair cat was evaluated for a -week history of abdominal distension. chyloabdomen secondary to pancreatic carcinoma was diagnosed. the cat was palliatively managed using rutin and a low-fat diet. the etiology, diagnosis and management of chyloabdomen are discussed. in human and veterinary medicine, chyloabdomen is a rare condition. it results from leakage of lipid-rich lymph into the peritoneal cavity, secondarily to obstruction or increased permeability of lymphatic vessels. in cats, it is believed to be mostly of neoplastic origin. medical investigation relies on a systematic approach and definitive diagnosis often requires histopathological samples. this report describes the exploration and management of a chyloabdomen secondary to pancreatic carcinoma in a -year-old cat. case details a -year-old neutered female domestic shorthair cat was referred for evaluation of an abdominal effusion. the owners reported an abdominal distension of three weeks duration, with conservation of general demeanor and appetite. the referring veterinarian detected an abdominal effusion. a chyloabdomen was suspected, according to the gross appearance of the liquid. the cat was housed mainly outside. her vaccination status was not up-to-date. on physical examination, the cat was bright, alert and normothermic. the body condition score was of out of and she had an unkempt haircoat. a marked abdominal distension was noted. no pain was elicited on abdominal palpation. cardiorespiratory parameters were within normal limits. abdominocentesis was performed under sedation. approximately ml of milky fluid were removed. triglycerides content in the effusion was highly increased ( . g/l; reference range . - . ). the cytologic analysis of the fluid showed erythrocytes/µl and leukocytes/µl, including neutrophils ( %), monocytes ( %) and small, mature lymphocytes ( %). no bacteria nor neoplastic cells were visualized. based on these findings, chyloabdomen was confirmed. complete blood count and biochemistry profile were within normal limits. in-house tests for feline immunodeficiency virus antibody and feline leukemia virus antigen were negative. a feline coronavirus research by polymerase chain reaction on effusion was negative. echocardiography was unremarkable. abdominal ultrasound showed remaining abdominal effusion and an ill-defined heterogeneous mesenteric aggregate in the cranial abdomen, assumed to be mesenteric fat reaction due to chronic effusion. no mass was identified and the pancreas showed no significant ultrasonographic abnormalities. computed tomodensitometry (ct) with lymphangiography was planned. thoracic ct scan was within normal limits. on abdominal ct scan, the amorphous mesenteric aggregate, previously identified on ultrasound, was seen, surrounding the portal vein ( fig. ) . the caudal extremity of the right lobe of the pancreas was slightly thickened and it was in contact with the mesenteric aggregate. it had ill-defined heterogenous contrast enhancement (fig. ) . ct scan lymphangiography was performed by injection of contrast media in perianal subcutaneous tissue, as previously described in dogs by ando et al. ( ) . the perianal area was clipped and surgically prepared. using a -gauge needle, a warmed water-soluble contrast media (iodixanol, visipaque tm , ge healthcare sas, vélizy-villacoublay, france) was injected in the subcutaneous tissue surrounding the anus at . ml/kg. the administration site was subsequently massaged for minutes. images were obtained with a multi-detector helical ct scan at , , , , and minutes after injection. only two lymph nodes in the sacral region and one hypogastric were marked with iodixanol. the remaining lymphatic system was not correctly visualized. http://www.openveterinaryjournal.com e. véran et al. open veterinary journal, ( ) , vol. ( ): - ________________________________________________________________________________________________________ http://www.openveterinaryjournal.com e. véran et al. open veterinary journal, ( ) , vol. ( ): - ________________________________________________________________________________________________________ as no definitive diagnosis had been made, and given the mesenteric lesion of undetermined origin, an exploratory laparotomy was performed. diazepam (valium® roche mg/ ml, roche, boulogne-billancourt, france), . mg/kg body weight (bw), iv, was given as premedication. anesthesia was induced using alfaxalone titrated to effect (alfaxan® mg/ml, dechra veterinary products sas, montigny-le-bretonneux, france), mg/kg bw, iv. oro-tracheal intubation was readily performed with a -mm cuffed tube. anesthesia was maintained with isoflurane (vetflurane® mg/g, virbac, carros, france) vaporized in oxygen. analgesia was provided by morphine (morphine lavoisier mg/ml, c.d.m. lavoisier, paris, france), . mg/kg bw, iv, q h. a midline laparotomy was performed. a large amount of chylous effusion was removed. the mesentery was folded upon itself by necrotic adhesions. those were released and biopsies of the abnormal mesentery close to the pancreas and of mesenteric and pancreaticoduodenal lymph nodes were obtained. no other abnormalities were seen on thorough examination of abdominal cavity. abdomen wall was closed routinely. the cat recovered well from the anesthesia. she was discharged days after surgery with amoxicillin/clavulanic acid, mg/kg bw, po, q h for days (késium® . mg, ceva santé animale, libourne, france), rutin, . mg/kg bw, po, q h, and a low-fat diet (royal canin gastro-intestinal low fat, royal canin sas, aimargues, france). biopsies revealed exocrine pancreatic tissue infiltrated by solid sheets of large polygonal cells with oval nuclei, prominent nucleoli and coarse chromatin (fig. ) . anisokaryosis was moderate and mitotic rate was low. mesenteric fat tissue showed necrotic areas surrounded by vacuolated macrophages (fig. ) . lymph node biopsies were free from metastatic lesions. pancreatic carcinoma with mesenteric necrotic and granulomatous remodeling was diagnosed. standard chemotherapy and targeted therapy (tyrosine kinase inhibitor) were declined. the cat was palliatively maintained under rutin and low-fat diet (royal canin gastro-intestinal low fat, royal canin sas, aimargues, france). three days after discharge, the cat was presented to the referring veterinarian for inappetence and depression. non-steroidal anti-inflammatory drugs (metacam® , mg/ml suspension orale pour chats, boehringer ingelheim france division santé animale, reims, france) were prescribed for a few days and the cat improved. on telephonic follow-up, three weeks after discharge, the patient was in good general condition. no relapse of the abdominal effusion was reported. however, one month after surgery, the cat was presented to her regular veterinarian for decreased appetite. ml of abdominal effusion were removed. despite administration of prednisolone (dermipred® mg, ceva santé animale, libourne, france), maropitant (cérénia® mg, zoetis, paris, france), and mirtazapine (norset® mg, msd france, courbevoie, france), the cat's condition worsened with rapid recurrence of effusion. she was euthanized days after the surgery. discussion chyloabdomen is an uncommon condition in veterinary medicine, with sparse data available in the literature. it results from leakage of triglycerides-rich lymph formed in intestinal lacteals into the peritoneal cavity, due to damage or obstruction of the lymphatic system or one of its tributaries (al-busafi et al., ) . unlike chylothorax, which is frequently diagnosed as idiopathic, no idiopathic chyloabdomen has been http://www.openveterinaryjournal.com e. véran et al. open veterinary journal, ( ) , vol. ( ): - ________________________________________________________________________________________________________ described. the differential diagnosis includes tumor, congestive heart failure, infectious diseases (feline infectious peritonitis, feline immunodeficiency), ruptured cisterna chyli, mesenteric root strangulation or lymphatic vessels malformation. cases in dogs were reported in association with intestinal lymphangiectasia (peterson, ) , mediastinal lymphangiosarcoma (myers et al., ) , acute pancreatitis (lott et al., ) , abdominal lymphatic rupture (fossum et al., ) , complication of mesenteric lymphangiography for chylothorax (fossum et al., ) , and lymphatic obstruction secondary to thrombus formation (fossum et al., ) . in contrast, chylous ascites in cats have mainly been reported in association with neoplastic disease. in the unique published case series of feline chyloabdomen, seven of cats had intra-abdominal malignancy: were diagnosed with a nonresectable solid tumor (hemangiosarcoma, paraganglioma), with lymphoma of the small intestine infiltrating the mesenteric lymph nodes, and one with lymphangiosarcoma of the abdominal wall (gores et al., ) . the remaining cats had nonneoplastic diseases: biliary cirrhosis, and steatitis caused by vitamin e deficiency (gores et al., ) . chyloabdomen has also been associated with feline immunodeficiency virus (börkü et al., ) , feline infectious peritonitis (savary et al., ) and hypertrophic cardiomyopathy (nelson, ) . a case of chylous pleural and peritoneal effusion with no underlying cause was described in a cat, with no postmortem examination performed (thompson and carr, ) . in this case report, pancreatic carcinoma was identified as the underlying cause of the chyloabdomen. linderman et al. ( ) previously reported feline cases of pancreatic carcinoma; chylous ascites was present in one cat in this series. in human medicine, direct malignant cells invasion into lymphatic vessels and obstruction of lymphatic flow by lymph node metastatic infiltration are thought to explain chyloabdomen formation during neoplastic disease (al-busafi et al., ) . in this particular case, the mesenteric adipose tissue showed necrotic and granulomatous remodeling on histopathology. this might have led to an obstruction of lymphatic flow and subsequent leakage of chyle. ct lymphangiography is an imaging technique used to assess lymphatic networks through injection of contrast agents. it is frequently used for exploration of chylous effusion when first-line diagnostic procedures have failed to identify the underlying cause of the effusion. ultrasound-guided percutaneous lymphography by mesenteric or popliteal lymph node injection has been recently described in cats (kim et al., ; lee et al., ) . however, the mobility of the lymph nodes and the volume of contrast agent to administer ( . ml) can make the procedure tedious (kim et al., ; lee et al., ) . laparotomy or more recently laparoscopy may be needed to perform the mesenteric injection (brisson et al., ) . in this case report, an approach described by ando et al. ( ) was applied. ando et al. ( ) reported an appropriate visualization of the thoracic duct minutes after contrast media injection in a healthy beagle dog. iwanaga et al. ( ) successfully used this protocol in a shiba inu suffering from thoracic duct rupture, with visualization of the duct minutes after injection. in this cat, however, no interpretable visualization of lymphatic system was obtained, with only a few caudal lymph nodes detected. failure of the lymphangiography in this case might be explained by species differences in absorption of subcutaneously injected contrast media, inappropriate dose of iodixanol, a different contrast media (iopamidol in the previous descriptions) or differences in the underlying disease responsible for chylous effusion. in human medicine, the precision of magnetic resonance lymphangiography images is improved if subjects have ingested a high fat meal - h prior to examination rather than fasting (chen et al., ) . it could have been a way to enhance lymphatic networks visualization in our case. management of chyloabdomen first relies on treatment of the underlying cause when possible, as in this case pancreatic carcinoma. pancreatic carcinoma is an uncommon tumor in cats with a high metastatic rate and a poor prognosis (linderman et al., ) . abdominal effusion is a negative prognostic indicator, with a median survival time of only days (linderman et al., ) . gemcitabine is a nucleotide analogue used as a firstline agent in human pancreatic carcinoma (teague et al., ) . it has been evaluated in cats alone or in combination with other drugs, like carboplatin or tyrosine kinase inhibitors (martinez-ruzafa et al., ; linderman et al., ) . chemotherapy provided an improvement of quality of life, but survival time remained poor, with a median of days (linderman et al., ) . only four cats were reported to live longer than a year after diagnosis; all of them received gemcitabine-based chemotherapy (martinez-ruzafa et al., ; linderman et al., ) . in our case, chemotherapy was declined, due to poor long-term prognosis. a palliative treatment with rutin and a low-fat diet was instaured. a low-fat diet may decrease the amount of fat in the effusion, which may improve the animal's ability to resorb fluid from the cavity (hawkins and fossum, ). rutin is a benzopyrone flavonoid extracted from plants. the exact mechanism of action is unknown; it might reduce leakage from blood vessels, increase proteolysis and removal of protein from tissues, and enhance http://www.openveterinaryjournal.com e. véran et al. open veterinary journal, ( ) , vol. ( ): - ________________________________________________________________________________________________________ macrophage phagocytosis of chyle (meadows et al., ; gould, ; kopko, ) . in cats, it is recommended for management of idiopathic chylothorax (gould, ; kopko, ) and it was successfully used in a case of chylothorax due to cryptococcal mediastinal granuloma (meadows et al., ) . by analogy, rutin has been unsuccessfully used in a case of chyloabdomen secondary to hypertrophic cardiomyopathy and a chylous pleural and peritoneal effusion with no underlying condition (nelson, ; thompson and carr, ) . in this present case, owners have reported a great improvement of the cat general condition after initiation of rutin therapy. the hair coat was shinny and smooth, and the cat was bright and alert. however, effusion rapidly relapsed despite rutin. in conclusion, chylous ascites is an uncommon condition in dogs and cats. neoplastic disease is a leading cause of chylous abdominal effusion in cats. the identification of the underlying cause relies on a systematic and often fastidious approach with analysis of the effusion as a first step. regarding the lymphangiography method, the results obtained in our case were disappointing, even if the injection into the perianal tissue is easier and less invasive than into popliteal or mesenteric lymph nodes. a prospective study would be needed to validate this technique in small animal imaging and establish a standardized protocol for cats. management of chyloabdomen relies on treatment of the underlying cause. by analogy with chylothorax, management with rutin and low-fat diet may be attempted, but to this date, no studies have demonstrated its efficacy. chylous ascites: evaluation and management. isrn hepatology computed tomography and radiographic lymphography of the thoracic duct by subcutaneous or submucosal injection chylous pleural and peritoneal effusion in a cat with feline immunodeficiency virus; diagnosis by lipoprotein electrophoresis comparison of mesenteric lymphadenography performed via surgical and laparoscopic approaches in dogs non-enhanced mr lymphography of the thoracic duct: improved visualization following ingestion of a high fat mealinitial experience chylous ascites in three dogs chylous ascites in cats: nine cases ( - ) the medical management of idiopathic chylothorax in a domestic long-haired cat kirk's current veterinary therapy xiv thoracic duct lymphography by subcutaneous contrast agent injection in a dog with chylothorax ultrasound-guided mesenteric lymph node iohexol injection for thoracic duct computed tomographic lymphography in cats the use of rutin in a cat with idiopathic chylothorax ct thoracic duct lymphography in cats by popliteal lymph node iohexol injection feline exocrine pancreatic carcinoma: a retrospective study of cases acute chylous peritonitis associated with acute pancreatitis in a staffordshire bull terrier tolerability of gemcitabine and carboplatin doublet therapy in cats with carcinomas chylothorax associated with cryptococcal mediastinal granuloma in a cat chylothorax and chylous ascites in a dog with mediastinal lymphangiosarcoma chyloabdomen in a mature cat postcaval thrombosis and delayed shunt migration after pleuro-peritoneal venous shunting for concurrent chylothorax and chylous ascites in a dog chylous abdominal effusion in a cat with feline infectious peritonitis advanced pancreatic adenocarcinoma: a review of current treatment strategies and developing therapies hyponatremia and hyperkalemia associated with chylous pleural and peritoneal effusion in a cat the authors declare that there is no conflict of interest. ___________________________________________ key: cord- -jrl fowa authors: abry, patrice; pustelnik, nelly; roux, stéphane; jensen, pablo; flandrin, patrick; gribonval, rémi; lucas, charles-gérard; guichard, Éric; borgnat, pierre; garnier, nicolas title: spatial and temporal regularization to estimate covid- reproduction number r(t): promoting piecewise smoothness via convex optimization date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: jrl fowa among the different indicators that quantify the spread of an epidemic such as the on-going covid- , stands first the reproduction number which measures how many people can be contaminated by an infected person. in order to permit the monitoring of the evolution of this number, a new estimation procedure is proposed here, assuming a well-accepted model for current incidence data, based on past observations. the novelty of the proposed approach is twofold: ) the estimation of the reproduction number is achieved by convex optimization within a proximal-based inverse problem formulation, with constraints aimed at promoting piecewise smoothness; ) the approach is developed in a multivariate setting, allowing for the simultaneous handling of multiple time series attached to different geographical regions, together with a spatial (graph-based) regularization of their evolutions in time. the effectiveness of the approach is first supported by simulations, and two main applications to real covid- data are then discussed. the first one refers to the comparative evolution of the reproduction number for a number of countries, while the second one focuses on french departments and their joint analysis, leading to dynamic maps revealing the temporal co-evolution of their reproduction numbers. the ongoing covid- pandemic has produced an unprecedented health and economic crisis, urging for the development of adapted actions aimed at monitoring the spread of the new coronavirus. no country remained untouched, thus emphasizing the need for models and tools to perform quantitative predictions, enabling effective managements of patients or an optimized allocations of medical ressources. for instance, the outbreak of this unprecedented pandemic was characterized by a critical lack of tools able to perform predictions related to the pressure on hospital ressources (number of patients, masks, gloves, intensive care unit needs,. . .) [ , ] . as a first step toward such an ambition goal, the present work focuses on the pandemic time evolution assessment. indeed, all countries experienced a propagation mechanism that is basically universal in the onset phase: each infected person happened to infect in average more than one other person, leading to an initial exponential growth. the strength of the spread is quantified by the so-called reproduction number which measures how many people can be contaminated by an infected person. in the early phase where the growth is exponential, this is referred to as r (for covid- , r * [ , ] ). as the pandemic develops and because more people get infected, the effective reproduction number evolves, hence becoming a function of time hereafter labeled r(t). this can indeed end up with the extinction of the pandemic, r(t)! , at the expense though of the contamination of a very large percentage of the total population, and of potentially dramatic consequences. rather than letting the pandemic develop until the reproduction number would eventually decrease below unity (in which case the spread would cease by itself), an active strategy amounts to take actions so as to limit contacts between individuals. this path has been followed by several countries which adopted effective lockdown policies, with the consequence that the reproduction number decreased significantly and rapidly, further remaining below unity as long as social distancing measures were enforced (see for example [ , ] ). however, when lifting the lockdown is at stake, the situation may change with an expected increase in the number of inter-individual contacts, and monitoring in real time the evolution of the instantaneous reproduction number r(t) becomes of the utmost importance: this is the core of the present work. monitoring and estimating r(t) raises however a series of issues related to pandemic data modeling, to parameter estimation techniques and to data availability. concerning the mathematical modeling of infectious diseases, the most celebrated approaches refer to compartmental models such as sir ("susceptible-infectious-recovered"), with variants such as seir ("susceptible-exposed-infectious-recovered"). because such global models do not account well for spatial heterogeneity, clustering of human contact patterns, variability in typical number of contacts (cf. [ ] ), further refinements were proposed [ ] . in such frameworks, the effective reproduction number at time t can be inferred from a fit of the model to the data that leads to an estimated knowledge of the average of infecting contacts per unit time, of the mean infectious period, and of the fraction of the population that is still susceptible. these are powerful approaches that are descriptive and potentially predictive, yet at the expense of being fully parametric and thus requiring the use of dedicated and robust estimation procedures. parameter estimation become all the more involved when the number of parameters grows and/or when the amount and quality of available data are low, as is the case for the covid- pandemic real-time and in emergency monitoring. rather than resorting to fully parametric models and seeing r(t) as the by-product of their identification, a more phenomenological, semi-parametric approach can be followed [ ] [ ] [ ] . this approach has been reported as robust and potentially leading to relevant estimates of r(t), even for epidemic spreading on realistic contact networks, where it is not possible to define a steady exponential growth phase and a basic reproduction number [ ] . the underlying idea is to model incidence data z(t) at time t as resulting from a poisson distribution with a time evolving parameter adjusted to account for the data evolution, which depends on a function f(s) standing for the distribution of the serial interval. this function models the time between the onset of symptoms in a primary case and the onset of symptoms in secondary cases, or equivalently the probability that a person confirmed infected today was actually infected s days earlier by another infected person. the serial interval function is thus an important ingredient of the model, accounting for the biological mechanisms in the epidemic evolution. assuming the distribution f to be known, the whole challenge in the actual use of the semi-parametric poisson-based model thus consists in devising estimatesrðtÞ of r(t) with satisfactory statistical performance. this has been classically addressed by approaches aimed at maximizing the likelihood attached to the model. this can be achieved, e.g., within several variants of bayesian frameworks [ , , , ] , with even dedicated software packages (cf. e.g., https://shiny.dide.imperial. ac.uk/epiestim/). instead, we promote here an alternative approach based on inverse problem formulations and proximal-operator based nonsmooth convex optimisation [ ] [ ] [ ] [ ] [ ] . the questions of modeling and estimation, be they fully parametric or semi-parametric, are intimately intertwined with that of data availability. this will be further discussed but one can however remark at this point that many options are open, with a conditioning of the results to the choices that are made. there is first the nature of the incidence data used in the analysis (reported infected cases, hospitalizations, deaths) and the database they are extracted from. next, there is the granularity of the data (whole country, regions, smaller units) and the specificities that can be attached to a specific choice as well as the comparisons that can be envisioned. in this respect, it is worth remarking that most analyses reported in the literature are based on (possibly multiple) univariate time series, whereas genuinely multivariate analyses (e.g., a joint analysis of the same type of data in different countries in order to compare health policies) might prove more informative. for that category of research work motivated by contributing in emergency to the societal stake of monitoring the pandemic evolution in real-time, or at least, on a daily basis, there are two classes of challenges: ensuring a robust and regular access to relevant data; rapidly developing analysis/estimation tools that are theoretically sound, practically usable on data actually available, and that may contribute to improving current monitoring strategies. in that spirit, the overarching goal of the present work is twofold: ( ) proposing a new, more versatile framework for the estimation of r(t) within the semi-parametric model of [ , ] , reformulating its estimation as an inverse problem whose functional is minimized by using non smooth proximal-based convex optimization; ( ) inserting this approach in an extended multivariate framework, with applications to various complementary datasets corresponding to different geographical regions. the paper is organized as follows. it first discusses data, as collected from different databases, with heterogeneity and uneven quality calling for some preprocessing that is detailed. in the present work, incidence data (thereafter labelled z(t)) refers to the number of daily new infections, either as reported in databases, or as recomputed from other available data such as hospitalization counts. based on a semi-parametric model for r(t), it is then discussed how its estimation can be phrased within a non smooth proximal-based convex optimization framework, intentionally designed to enforce piecewise linearity in the estimation of r(t) via temporal regularization, as well as piecewise constancy in spatial variations of r(t) by graph-based regularization. the effectiveness of these estimation tools is first illustrated on synthetic data, constructed from different models and simulating several scenarii, before being applied to several real pandemic datasets. first, the number of daily new infections for many different countries across the world are analyzed independently. second, focusing on france only, the number of daily new infections per continental france départements (départements constitute usual entities organizing the administrative life in france) are analyzed both independently and in a multivariate setting, illustrating the benefit of this latter formulation. discussions, perpectives and potential improvements are finally discussed. datasets. in the present study, three sources of data were systematically used: • source (jhu) johns hopkins university provides access to the cumulated daily reports of the number of infected, deceased and recovered persons, on a per country basis, for a large number of countries worldwide, essentially since inception of the covid- crisis (january st, time series. the data available on the different data repositories used here are strongly affected by outliers, which may stem from inaccuracy or misreporting in per country reporting procedures, or from changes in the way counts are collected, aggregated, and reported. in the present work, it has been chosen to preprocess data for outlier removal by applying to the raw time series a nonlinear filtering, consisting of a sliding-median over a -day window: outliers defined as ± . standard deviation are replaced by window median to yield the pre-processed time series z(t), from which the reproduction number r(t) is estimated. an example of raw and pre-processed time series is illustrated in fig . when countries are studied independently, the estimation procedure is applied separately to each time series z(t) of size t, the number of days available for analysis. when considering continental france départements, we are given d time series z d (t) of size t each, where � d � d = indexes the départements. these time series are collected and stacked in a matrix of size d × t, and they analyzed both independently and jointly. model. although they can be used for envisioning the impact of possible scenarii in the future development of an on-going epidemic [ ] , sir models, because they require the full estimation of numerous parameters, are often used a posteriori (e.g., long after the epidemic) with consolidated and accurate datasets. during the spread phase and in order to account for the on-line/on-the-fly need to monitor the pandemic and to offer some robustness to partial/ incomplete/noisy data, less detailed semi-parametric models focusing on the only estimation of the time-dependent reproduction number can be preferred [ , , ] . let r(t) denote the instantaneous reproduction number to be estimated and z(t) be the number of daily new infections. it has been proposed in [ , ] that {z(t), t = , . . ., t} can be modeled as a nonstationary time series consisting of a collection of random variables, each drawn from a poisson distribution p p t whose parameter p t depends on the past observations of z(t), on the current value of r(t), and on the serial interval function f(�): the serial interval function f(�) constitutes a key ingredient of the model, whose importance and role in pandemic evolution has been mentioned in introduction. it is assumed to be independent of calendar time (i.e., constant across the epidemic outbreak), and, importantly, independent of r(t), whose role is to account for the time dependencies in pandemic propagation mechanisms. for the covid- pandemic, several studies have empirically estimated the serial interval function f(�) [ , ] . for convenience, f(�) has been modeled as a gamma distribution, with shape and rate parameters . and . , respectively (corresponding to mean and standard deviations of . and . days, see [ ] and references therein). these choices and assumptions have been followed and used here, and the corresponding function is illustrated in fig . in essence, the model in eq ( ) is univariate (only one time series is modeled at a time), and based on a poisson marginal distribution. it is also nonstationary, as the poisson rate evolves along time. the key ingredient of this model consists of the poisson rate evolving as a weighted moving average of past observations, which is qualitatively based on the following rationale: whenr is above , the epidemic is growing and, conversely, when this ratio is below , it decreases and eventually vanishes. non-smooth convex optimisation. the whole challenge in the actual use of the semiparametric poisson-based model described above thus consists in devising estimatesrðtÞ of r (t) that have better statistical performance (more robust, reliable, and hence usable) than the direct brute-force and naive form defined in eq . to estimate r(t), and instead of using bayesian frameworks that are considered state-of-the-art tools for epidemic evolution analysis, we propose and promote here an alternative approach based on an inverse problem formulation. its main principle is to assume some form of temporal regularity in the evolution of r(t) (we use a piecewise linear model in the following). in the case of a joint estimation of r(t) across several continental france départements, we further assume some form of spatial regularity, i.e., that the values of r(t) for neighboring départements are similar. univariate setting. for a single country, or a single département, the observed (possibly preprocessed) data {z(t), � t � t} is represented by a t-dimensional vector z r t . recalling that the poisson law is pðz ¼ njpÞ ¼ p n n! e À p for each integer n � , the negative log-likelihood of observing z given a vector p r t of poisson parameters p t is where r r t is the (unknown) vector of values of r(t). up to an additive term independent of p, this is equal to the kl-divergence (cf. section . . in [ ] ): given the vector of observed values z, the serial interval function f(�), and the number of days t, the vector p given by ( ) reads p = r � fz, with � the entrywise product and f r t�t the matrix with entries f ij = f(i − j). maximum likelihood estimation of r (i.e., minimization of the negative log-likelihood) leads to an optimization problem min r d kl (zjr � fz) which does not ensure any regularity of r(t). to ensure temporal regularity, we propose a penalized approach usinĝ r ¼ argmin r d kl ðz j r � fzÞ þ oðrÞ where o denotes a penalty function. here we wish to promote a piecewise affine and continuous behavior, which may be accomplished [ , ] using o(r) = λ time kd rk , where d is the matrix associated with a laplacian filter (second order discrete temporal derivatives), k�k denotes the ℓ -norm (i.e., the sum of the absolute values of all entries), and λ time is a penalty factor to be tuned. this leads to the following optimization problem: spatially regularized setting. in the case of multiple départements, we consider multiple vectors (z d r t , � d � d) associated to the d time series, and multiple vectors of unknown (r d r t , � d � d), which can be gathered into matrices: a data matrix z r t�d whose columns are z d and a matrix of unknown r r t�d whose columns are the quantities to be estimated r d . a first possibility is to proceed to independent estimations of the (r d r t , � d � d) by addressing the separate optimization problemŝ which can be equivalently rewritten into a matrix form: is the entrywise ℓ norm of d r, i.e., the sum of the absolute values of all its entries. an alternative is to estimate jointly the (r d r t , � d � d) using a penalty function promoting spatial regularity. to account for spatial regularity, we use a spatial analogue of d promoting spatially piecewise constant solutions. the d continental france départements can be considered as the vertices of a graph, where edges are present between adjacent départements. from the adjacency matrix a r d�d of this graph (a ij = if there is an edge e = (i, j) in the graph, a ij = otherwise), the global variation of the function on the graphs can be computed as ∑ ij a ij (r ti − r tj ) and it is known that this can be accessed through the so-called (combinatorial) laplacian of the graph: [ ] . however, in order to promote smoothness over the graph while keeping some sparse discontinuities on some edges, it is preferable to regularize using a total variation on the graph, which amounts to take the ℓ -norm of these gradients (r ti − r tj ) on all existing edges. for that, let us introduce the incidence matrix b r e�d such that l = b > b where e is the number of edges and, on each line representing an existing edge e = (i, j), we set b e,i = and b e,j = − . then, the ℓ -norm krb > k = kbr > k is equal to p t t¼ p ði;jÞ:a ij ¼ jr ti À r tj j. alternatively, it can be computed as krb > k ¼ p t t¼ kbrðtÞk where rðtÞ r d is the t-th row of r, which gathers the values across all départements at a given time t. from that, we can define the regularized optimization problem: optimization problems ( ) and ( ) involve convex, lower semi-continuous, proper and non-negative functions, hence their set of minimizers is non-empty and convex [ ] . we will discuss right after how to compute these using proximal algorithms. by the known sparsity-promoting properties of ℓ regularizers and their variants, the corresponding solutions are such that d r and/or rb > are sparse matrices, in the sense that these matrices of (second order temporal or first order spatial) derivatives have many zero entries. the higher the penalty factors λ time and λ space , the more zeroes in these matrices. in particular, when λ space = , no spatial regularization is performed and ( ) is equivalent to ( ) . when λ space is large enough, rb > is exactly zero, which implies that r(t) is constant at each time since the graph of départements is connected. optimization using a proximal algorithm. the considered optimization problems are of the form where f and g m are proper lower semi-continuous convex, and k m are bounded linear operators. a classical case for m = is typically addressed with the chambolle-pock algorithm [ ] , which has been recently adapted for multiple regularization terms as in eq. of [ ] . to handle the lack of smoothness of lipschitz differentiability for the considered functions f and g m , these approaches rely on their proximity operators. we recall that the proximity operator of a convex, lower semi-continuous function φ is defined as [ ] prox φ ðyÞ ¼ arg min in our case, we consider a separable data fidelity term: as this is a separable function of the entries of its input, its associated proximity operator can be computed component by component [ ] : where τ > . we further consider g m (�) = k.k , m = , , and k (r) ≔ λ time d r, k (r) ≔ λ space rb > . the proximity operators associated to g m read: where (.) + = max( ,.). in algorithm , we express explicitly algorithm of [ ] for our setting, considering the moreau identity that provides the relation between the proximity operator of a function and the proximity operator of its conjugate (cf. eq. ( ) of [ ] ). the choice of the parameters τ and σ m impacts the convergence guarantees. in this work, we adapt a standard choice provided by [ ] to this extended framework. the adjoint of k m , denoted k � m , is given by the sequence ðr ðkþ Þ Þ k n converges to a minimizer of ( ) (cf. thm . of [ ] ). input: data z, tolerance � > ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi p m¼ ; kk m k to assess the relevance and performance of the proposed estimation procedure detailed above, it is first applied to two different synthetic time series z(t). the first one is synthesized using directly the model in eq ( ), with the same serial interval function f(t) as that used for the estimation, and using an a priori prescribed function r(t). the second one is produced from solving a compartmental (sir type) model. for such models, r(t) can be theoretically related to the time scale parameters entering their definition, as the ratio between the infection time scale and the quitting infection (be it by death or recovery) time scale [ , ] . the theoretical serial function f associated to that model and to its parameters is computed analytically (cf., e.g., [ ] ) and used in the estimation procedure. for both cases, the same a priori prescribed function r(t), to be estimated, is chosen as constant (r = . ) over the first days to model the epidemic outbreak, followed by a linear decrease (till below ) over the next days to model lockdown benefits, with finally an abrupt linear increase for the last days, modeling a possible outbreak at when lockdown is lifted. additive gaussian noise is superimposed to the data produced by the models to account for outliers and misreporting. for both cases, the proposed estimation procedure (obtained with λ time set to the same values as those used to analyze real data in the next section) outperforms the naive estimates ( ), which turn out to be very irregular (cf. fig ) . the proposed estimates notably capture well the three different phases of r(t) (stable, decreasing and increasing), with notably a rapid and accurate reaction to the increasing change in the last days. the present section aims to apply the model and estimation tools proposed above to actual covid- data. first, specific methodological issues are addressed, related to tuning the hyperparameter(s) λ time or (λ time , λ space ) in univariate and multivariate settings, and to comparing the consistency between different estimates of r(t) obtained from the same incidence data, yet downloaded from different repositories. then, the estimation tools are applied to the estimation of r(t), both independently for numerous countries and jointly for the continental france départements. estimation of r(t) is performed daily, with t thus increasing every day, and updated results are uploaded on a regular basis on a dedicated webpage (cf. http://perso.ens-lyon.fr/patrice. abry. regularization hyperparameter tuning. a critical issue associated with the practical use of the estimates based on the optimization problems ( ) and ( ) lies in the tuning of the hyperparameters balancing data fidelity terms and penalization terms. while automated and data-driven procedures can be devised, following works such as [ ] and references therein, let us analyze the forms of the functional to be minimized, so as to compute relevant orders of magnitude for these hyperparameters. let us start with the univariate estimation ( ). using λ time = implies no regularization and the achieved estimate turns out to be as noisy as the one obtained with a naive estimator (cf. eq ( )). conversely, for large enough λ time , the proposed estimate becomes exactly a constant, missing any time evolution. tuning λ time is thus critical but can become tedious, especially because differences across countries (or across départements in france) are likely to require different choices for λ time . however, a careful analysis of the functional to minimize shows that the data fidelity term ( ), based on a kullback-leibler divergence, scales proportionally to the input incidence data z while the penalization term, based on the regularization of r(t), is independent of the actual values of z. therefore, the same estimate for r(t) is obtained if we replace z with α × z and λ with α × λ. because orders of magnitude of z are different amongst countries (either because of differences in population size, or of pandemic impact), this critical observation leads us to apply the estimate not to the raw data z but to a normalized version z/std(z), alleviating the burden of selecting one λ time per country, instead enabling to select one same λ time for all countries and further permitting to compare the estimated r(t)'s across countries for equivalent levels of regularization. considering now the graph-based spatially-regularized estimates ( ) while keeping fixed λ time , the different r(t) are analyzed independently for each département when λ space = . conversely, choosing a large enough λ space yields exactly identical estimates across départments that are, satisfactorily, very close to what is obtained from data aggregated over france prior to estimation. further, the connectivity graph amongst the continental france départements leads to an adjacency matrix with non-zero off-diagonal entries (set to the value ), associated to as many edges as existing in the graph. therefore, a careful examination of ( ) shows that the spatial and temporal regularizations have equivalent weights when λ time and λ time are chosen such that the use of z/std(z) and of ( ) above gives a relevant first-order guess to the tuning of λ time and of (λ time , λ space ). estimate consistency using different repository sources. when undertaking such work dedicated to on-going events, to daily evolutions, and to a real stake in forecasting future trends, a solid access to reliable data is critical. as previously mentioned, three sources of data are used, each including data for france, which are thus now used to assess the impact of data sources on estimated r(t). source (jhu) and source (ecdpc) provide cumulated numbers of confirmed cases counted at national levels and (in principle) including all reported cases from any source (hospital, death at home or in care homes. . .). source (spf) does not report that same number, but a collection of other figures related to hospital counts only, from which a daily number of new hospitalizations can be reconstructed and used as a proxy for daily new infections. the corresponding raw and (sliding-median) preprocessed data, illustrated in fig , show overall comparable shapes and evolutions, yet with clearly visible discrepancies of two kinds. first, source (jhu) and source (ecdpc), consisting of crude reports of number of confirmed cases are prone to outliers. those can result from miscounts, from pointwise incorporations of new figures, such as the progressive inclusion of cases from ehpad (care homes) in france, or from corrections of previous erroneous reports. conversely, data from source (spf), based on hospital reports, suffer from far less outliers, yet at the cost of providing only partial figures. second, in france, as in numerous other countries worldwide, the procedure on which confirmed case counts are based, changed several times during the pandemic period, yielding possibly some artificial increase in the local average number of daily new confirmed cases. this has notably been the case for france, prior to the end of the lockdown period (mid-may), when the number of tests performed has regularly increased for about two weeks, or more recently early june when the count procedures has been changed again, likely because of the massive use of serology tests. because the estimate of r(t) essentially relies on comparing a daily number against a past moving average, these changes lead to significant biases that cannot be easily accounted for, but vanishes after some duration controlled by the typical width of the serial distribution f (of the order of ten days). confirmed infection cases across the world. to report estimated r(t)'s for different countries, data from source (ecdpc) are used as they are of better quality than data from source (jhu), and because hospital-based data (as in source (spf)) are not easily available for numerous different countries. visual inspection led us to choose, uniformly for all countries, two values of the temporal regularization parameter: λ time = to produce a strongly-regularized, hence slowly varying estimate, and λ time = . for a milder regularization, and hence a more reactive estimate. these estimates being by construction designed to favor piecewise linear behaviors, local trends can be estimated by computing (robust) estimates of the derivativeŝ bðtÞ ofrðtÞ. the slow and less slow estimates ofrðtÞ thus provide a slow and less slow estimate of the local trends. intuitively, these local trends can be seen as predictors for the forthcoming value of r:rðt þ nÞ ¼rðtÞ þ nbðtÞ. let us start by inspecting again data for france, further comparing estimates stemming from data in source (ecdpc) or in source (spf) (cf. fig ) . as discussed earlier, data from source (ecdpc) show far more outliers that data from source (spf), thus impacting estimation of r and β. as expected, the strongly regularized estimates (λ time = ) are less sensitive than the less regularized ones (λ time = . ), yet discrepancies in estimates are significant, as data from source (ecdpc) yields, for june th, estimates of r slightly above , while that from source (spf) remain steadily around . , with no or mild local trends. again, this might be because late may, france has started massive serology testing, mostly performed outside hospitals. this yielded an abrupt increase in the number of new confirmed cases, biasing upward the estimates of r(t). however, the short-term local trend for june th goes also downward, suggesting that the model is incorporating these irregularities and that estimates will return to unbiased after an estimation time controlled by the typical width of the serial distribution f (of the order of ten days). this recent increase is not seen in source (spf)based estimates that remain very stable, potentially suggesting that hospital-based data are much less affected by changes in testing policies. this local analysis at the current date can be complemented by a more global view on what happened since the lifting of the lockdown. considering the whole period starting from may th we end up with triplets [ th percentile; median; th percentile] that read as given in table : source (ecdpc) provides data for several tens of countries. figs to reportrðtÞ and bðtÞ for several selected countries. more figures are available at perso.ens-lyon.fr/patrice.abry. as of june th (time of writing), fig shows that, for most european countries, the pandemic seems to remain under control despite lifting of the lockdown, with (slowly varying) estimates of r remaining stable below , ranging from . to . depending on countries, and (slowly varying) trends around . sweden and portugal (not shown here) display less favorable patterns, as well as, to a lesser extent, the netherlands, raising the question of whether this might be a potential consequence of less stringent lockdown rules compared to neighboring european countries. fig shows that whiler for canada is clearly below since early may, with a negative local trend, the usa are still bouncing back and forth around . south america is in the above phase but starts to show negative local trends. fig indicates that iran, india or indonesia are in the critical phase withrðtÞ > . fig shows that data for african countries are uneasy to analyze, and that several countries such as egypt or south africa are in pandemic growing phases. phase-space representation. to complement figs to , fig displays a phase-space representation of the time evolution of the pandemic, constructed by plotting one against the other the local average (over a week) of the slowly varying estimated reproduction numberrðtÞ and local trend, ð � rðtÞ; � bðtÞÞ, for a period ranging from mid-april to june th. country names are written at the end (last day) of the trajectories. interestingly, european countries display a c-shape trajectory, starting with r > with negative trends (lockdown effects), thus reaching the safe zone (r < ) but eventually performing a u-turn with a slow increase of local trends till positive. this results in a mild but clear reincrease of r, yet with most values below today, except for france (see comments above) and sweden. the usa display a similar c-shape though almost concentrated on the edge point r(t) = , β = , while canada does return to the safe zone with a specific pattern. south-american countries, obviously at an earlier stage of the pandemic, show an inverted c-shape pattern, with trajectory evolving from the bad top right corner, to the controlling phase (negative local trend, with decreasing r still above though). phase-spaces of asian and african countries essentially confirm these c-shaped trajectories. envisioning these phase-space plots as pertaining to different stages of the pandemic (rather than to different countries), this suggests that covid- pandemic trajectory resembles a clockwise circle, starting from the bad top right corner (r above and positive trends), evolving, likely by lockdown impact, towards the bottom right corner (r still above but negative trends) and finally to the safe bottom left corner (r below and negative then null trend). the lifting of the lockdown may explain the continuation of the trajectory in the still safe but. . . corner (r below and again positive trend). as of june th, it can be only expected that trajectories will not close the loop and reach back the bad top right corner and the r = limit. continental france départements: regularized joint estimates. there is further interest in focusing the analysis on the potential heterogeneity in the epidemic propagation across a given territory, governed by the same sanitary rules and health care system. this can be achieved by estimating a set of localrðtÞ's for different provinces and regions [ ] . such a study is made possible by the data from source (spf), that provides hospital-based data for each of the continental france départements . fig (right) already reported the slow and fast varying estimates of r and local trends computed from data aggregated over the whole france. to further study the variability across the continental france territory, the graphbased, joint spatial and temporal regularization described in eq is applied to the number of confirmed cases consisting of a matrix of size k × t, with d = continental france départements, and t the number of available daily data (e.g., t = on june th, data being available only after march th). the choice λ time = . leading to fast estimates was used for this joint study. using ( ) as a guideline, empirical analyses led to set λ space = . , thus selecting spatial regularization to weight one-fourth of the temporal regularization. first, fig ( top row) maps and compares for june th (chosen arbitrarily as the day of writing) per-département estimates, obtained when départements are analyzed either independently (r indep using eq , left plot) or jointly (r joint using eq , right plot). while the means of r indep andr joint are of the same order (' . and ' . respectively) the standard deviations drop down from ' . to ' . , thus indicating a significant decrease in the variability across departments. this is further complemented by the visual inspection of the maps which reveals reduced discrepancies across neighboring departments, as induced by the estimation procedure. in a second step, short and long-term trends are automatically extracted fromr indep and r joint and short-term trends are displayed in the bottom row of fig (left and right, respectively) . this evidences again a reduced variability across neighboring departments, though much less than that observed forr indep andr joint , likely suggesting that trends on r per se are more robust quantities to estimate than single r's. for june th, fig also indicates reproduction numbers that are essentially stable everywhere across france, thus confirming the trend estimated on data aggregated over all france (cf. fig , right plot) . video animations, available at perso.ens-lyon.fr/patrice.abry/deptregul.mp , and at barthes.enssib.fr/coronavirus/ixxi-sisyphe/., updated on a daily basis, report further comparisons betweenr indep andr joint and their evolution along time for the whole period of data availability. maps for selected days are displayed in fig ( with identical colormaps and colorbars across time). fig shows that until late march (lockdown took place in france on march th),r joint was uniformly above . (chosen as the upper limit of the colorbar to permit to see variations during the lockdown and post-lockdown periods), indicating a rapid evolution of the epidemic across entire france. a slowdown of the epidemic evolution is visible as early as the first days of april (with overall decreases ofr joint , and a clear north vs. south gradient). during april, this gradient rotates slightly and aligns on a north-east vs. south-west direction and globally decreases in amplitude. interestingly, in may, this gradient has reversed direction from south-west to north-east, though with very mild amplitude. as of today (june th), the pandemic, viewed hospital-based data from source (spf), seems under control under the whole continental france. estimation of the reproduction number constitutes a classical task in assessing the status of a pandemic. classically, this is done a posteriori (after the pandemic) and from consolidated data, often relying on detailed and accurate sir-based models and relying on bayesian frameworks for estimation. however, on-the-fly monitoring of the reproduction number time evolution constitutes a critical societal stake in situations such as that of covid- , when decisions need to be taken and action need to be made under emergency. this calls for a triplet of constraints: i) robust access to fast-collected data; ii) semi-parametric models for such data that focus on a subset of critical parameters; iii) estimation procedures that are both elaborated enough to yield robust estimates, and versatile enough to be used on a daily basis and applied to (often-limited in quality and quantity) available data. in that spirit, making use of a robust nonstationary poisson-distribution based semiparametric model proven robust in the literature for epidemic analysis, we developed an original estimation procedure to favor piecewise regular estimation of the evolution of the reproduction number, both along time and across space. this was based on an inverse problem formulation balancing fidelity to time and space regularization, and used proximal operators and nonsmooth convex optimization. this tool can be applied to time series of incidence data, reported, e.g., for a given country. whenever made possible from data, estimation can benefit from a graph of spatial proximity between subdivisions of a given territory. the tool also provides local trends that permit to forecast short-term future values of r. the proposed tools were applied to pandemic incidence data consisting of daily counts of new infections, from several databases providing data either worldwide on an aggregated percountry basis or, for france only, based on the sole hospital counts, spread across the french territory. they permitted to reveal interesting patterns on the state of the pandemic across the world as well as to assess variability across one single territory governed by the same (health care and politics) rules. more importantly, these tools can be used everyday easily as an onthe-fly monitoring procedure for assessing the current state of the pandemic and predict its short-term future evolution. updated estimations are published on-line every day at perso.ens-lyon.fr/patrice.abry and at barthes.enssib.fr/coronavirus/ixxi-sisyphe/. data were (and still are) automatically downloaded on a daily basis using routines written by ourselves. all tools have been developed in matlab™ and can be made available from the corresponding author upon motivated request. at the methodological level, the tool can be further improved in several ways. instead of using o(r) ≔ λ time kd rk + λ space krb > k , for the joint time and space regularization, another possible choice is to directly consider the matrix d rb > of joint spatio-temporal derivatives, and to promote sparsity with an ℓ -norm, or structured sparsity with a mixed norm ℓ , , e.g., kd rb > k , = ∑ t k(d rb > )(t)k . as previously discussed, data collected in the process of a pandemic are prone to several causes for outliers. here, outlier preprocessing and reproduction number estimation were conducted in two independent steps, which can turn suboptimal. they can be combined into a single step at the cost of increasing the representation space permitting to split observation in true data and outliers, by adding to the functional to minimize an extra regularization term and devising the corresponding optimization procedure, which becomes nonconvex, and hence far more complicated to address. finally, when an epidemic model suggests a way to make use of several time series (such as, e.g., infected and deceased) for one same territory, the tool can straightforwardly be extended into a multivariate setting by a mild adaptation of optimization problems ( ) and ( ), replacing the kullback-leibler divergence d kl (zjr � fz) by p i i¼ d kl ðz i j r � fz i Þ. finally, automating a data-driven tuning of the regularization hyperparameters constitutes another important research track. factors determining the diffusion of covid- and suggested strategy to prevent future accelerated viral infectivity similar to covid pooling data from individual clinical trials in the covid- era expected impact of lockdown in ile-de-france and possible exit strategies estimating the burden of sars-cov- in france the impact of a nation-wide lockdown on covid- transmissibility in italy measurability of the epidemic reproduction number in data-driven contact networks mathematical models in epidemiology a new framework and software to estimate time-varying reproduction numbers during epidemics the r package: a toolbox to estimate reproduction numbers for epidemic outbreaks improved inference of time-varying reproduction numbers during infectious disease outbreaks convex analysis and monotone operator theory in hilbert spaces image restoration: total variation, wavelet frames, and beyond proximal splitting methods in signal processing proximal algorithms. foundations and trends ® in optimization wavelet-based image deconvolution and reconstruction different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures epidemiological parameters of coronavirus disease : a pooled analysis of publicly reported individual data of cases from seven countries epidemiological characteristics of covid- cases in italy and estimates of the reproductive numbers one month into the epidemic nonlinear denoising for solid friction dynamics characterization sparsest continuous piecewise-linear representation of data the emerging field of signal processing on graphs: extending high-dimensional data analysis to networks and other irregular domains a first-order primal-dual algorithm for convex problems with applications to imaging proximal splitting algorithms: relax them all! fonctions convexes duales et points proximaux dans un espace hilbertien. comptes rendus de l'acadé mie des sciences de paris a douglas-rachford splitting approach to nonsmooth convex variational signal recovery on the definition and the computation of the basic reproduction ratio r in models for infectious diseases in heterogeneous populations reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission figs and are produced using open ressources from the openstreetmap foundation, whose contributors are here gratefully acknowledged. mapdata©openstreetmap contributors. conceptualization: patrice abry, pablo jensen, patrick flandrin. key: cord- -zisujjsx authors: sabat, iryna; neuman-böhme, sebastian; varghese, nirosha elsem; barros, pedro pita; brouwer, werner; van exel, job; schreyögg, jonas; stargardt, tom title: united but divided: policy responses and people's perceptions in the eu during the covid- outbreak date: - - journal: health policy doi: . /j.healthpol. . . sha: doc_id: cord_uid: zisujjsx to understand the public sentiment toward the measures used by policymakers for covid- containment, a survey among representative samples of the population in seven european countries was carried out in the first two weeks of april . the study addressed people's support for containment policies, worries about covid- consequences, and trust in sources of information. citizens were overall satisfied with their government's response to the pandemic; however, the extent of approval differed across countries and policy measures. a north-south divide in public opinion was noticeable across the european states. it was particularly pronounced for intrusive policy measures, such as mobile data use for movement tracking, economic concerns, and trust in the information from the national government. considerable differences in people's attitudes were noticed within countries, especially across individual regions and age groups. the findings suggest that the epidemic acts as a stressor, causing health and economic anxieties even in households that were not directly affected by the virus. at the same time, the burden of stress was unequally distributed across regions and age groups. based on the data collected, we draw lessons from the containment stage and identify several insights that can facilitate the design of lockdown exit strategies and future containment policies so that a high level of compliance can be expected. the outbreak of covid- triggered a wide range of responses from governments in the european union. given that the disease was new and effective medical countermeasures did not exist in early , governments had to adopt non-medical measures aiming at the containment and mitigation of covid- . with the aim of "flattening the curve," these policies included bans on public gatherings, closures of academic institutions and public places, national and international mobility restrictions, confinement, and several others [ ] . italy was the first country in europe to apply intervention measures from the beginning of march in response to the severity of the covid- outbreak. other eu countries followed soon afterward, using similar countermeasures around mid-march [ ] . the adoption of these policies varied in their scale, stringency, and pace across countries. while most european states implemented confinement measures, the extent of limitations of people's freedoms differed across individual countries. lockdowns were usually strictest where the pandemic was deadliest (italy, spain, and france), imposing severe limitations on population movements. some governments chose less stringent versions of confinement or no lockdown at all, for instance, "an intelligent lockdown" in the netherlands or "freedom under responsibility" in sweden [ ] . forced to react swiftly to the unfolding epidemic situation, policymakers in every country tried to balance the implementation of containment policies against numerous important factors with the priority mostly given to the protection of the population's health. consequently, there has been a lot of debate in every society about whether measures taken by the government were appropriate or not. some parts of the population have been voicing support for more severe containment policies to minimize the spread of the virus. such attitudes were likely fueled by people's worries about their health and the potential of their national healthcare system to withstand the epidemic. meanwhile, others expressed their concerns about the social and economic consequences of such policies, thereby advocating for less severe containment measures [ ] . as the pandemic began to abate, governments started designing the lockdown exit strategies and restarting their economies. however, the risk that the new wave of the epidemic may happen did not disappear, especially given that the vaccine development takes a long time, and herd immunity was not achieved [ ] . in this light, the issue of lifting lockdowns has become a new subject of public debate across and within european countries raising discussions about the appropriateness of timing, risks, and potential consequences of ending the confinement [ ] . lifting lockdown restrictions creates acute dilemmas to the policymakers since the economic and human costs of any exit strategy seem to be closely linked together. taking a utilitarian approach in this situation could backfire if the society's understanding is not preliminarily secured or expectations are not fulfilled. policymakers and public health experts have to persuade their citizens to make behavior changes and respect future containment interventions while facing the difficulty of enforcing such regulations. therefore, it becomes crucial to understand people's worries about the pandemic and their perceptions of the effects of containment policies, so that the design of further policies and contingency measures is well-informed, and a high level of compliance can be expected from the population. moreover, trust in the government and social institutions may become central to achieving a successful implementation of future measures, whereas lack of it may turn detrimental to the fight against the pandemic. hence it is of paramount importance to understand who people trust most so that public health messages can be amplified using correct means of communication. we provide a timely description of the current situation and draw lessons from the containment stage to inform the design and implementation of the lockdown exit policies. in order to understand the public sentiment towards the covid- containment measures and to inform future policy development, we collected information on people's support for these policies, their worries in relation to the unfolding epidemic, and their trust in different sources of information. we surveyed over , people representative of the adult population in seven european countries: denmark, france, germany, italy, portugal, the netherlands, and the uk. the fieldwork was conducted online during april - , , using multi-sourced online panels provided by the market research company dynata. to ensure that the sampling frame was representative given the online nature of the study, the company applied diverse recruiting procedures to reach the general population (through open recruitment, loyalty programs, affiliate networks, mobile apps). it then used quotas to match the national census shares in each country. the questionnaire was designed by the authors of the study except for the worry items that were adopted from the world health organization (who) covid- snapshot monitoring project [ ] . the questionnaire was carefully translated into six other languages by native speakers and then implemented using the qualtrics platform first as a pilot ( % of the sample in every country) and next as a large-scale survey. the data from the pilot study were included in the total sample. in each country, we collected data from a sample of , respondents representative of the national population in terms of region, age, gender, and education. given that the italian region lombardy was the most severely hit by the covid- outbreak, we collected additional responses in this region representative in terms of age and gender. learning about perceptions and attitudes of people who reside there could provide essential insights to researchers and policymakers. the extra data collected from lombardy were not included in the representative sample of italy. thus, no weighting was used as the additional lombardy sample was analyzed separately and denoted as lombardy in the results section. we assessed people's approval of policy measures that were taken (or were likely to be taken) by their national government in response to the covid- outbreak. in particular, we covered such issues as school closures, bans on public gatherings, border closures, bans imposed on the export of medical equipment, fines for quarantine violations, random temperature checks, curfews, public transport suspensions and utilization of mobile phone data for tracking covid- cases and their contacts. on average, % of people in the seven european countries approved of the policies taken in their country in response to the pandemic, implying considerable public support. nevertheless, the extent of approval differed by country and by policy measure. the most approved measures were fining -day quarantine violations, ban of public gatherings, and border closures (each supported by % of respondents). by the time of the survey's fieldwork, restrictions on public gatherings had been adopted in all countries covered by the study, whereas international travel controls had been imposed to a certain extent everywhere, except the uk [ ] . prior to complete border closures in mid-march , some countries (for example, italy, france, germany, denmark) had been requiring screening and -day quarantine for arrivals from high-risk regions already since february. in contrast, other countries, such as portugal and the netherlands, started later and turned directly to strict measures, such as banning arrivals from high-risk areas and imposing partial border closures. the latter typically implied either limitation on entries of nonresidents or closure of only certain types of borders (land, sea, air), while ensuring "green lanes" for freight vehicles transporting goods. however, complete border closures occurred haphazardly and led to disrupted commerce and stranding citizens. among countries covered in our study, denmark was the first to close all borders in mid-march, whereas the uk did so only in the second half of may . moreover, at the time of fieldwork, the uk did not have routine screenings at its airports or quarantine requirement for travelers [ , ] . thus, the results for the uk showed the extent of public support that these measures would have received, had they been implemented earlier. meanwhile, the most opposed containment policies were public transport suspension ( % of respondents against it), ban of medical export, use of mobile phone data for tracking, and the imposition of a curfew (each disapproved by approximately % of respondents). these trends might reflect within-country regional and age structure of the population. for example, older individuals and those living in remote areas tended to be the most strongly opposed to public transport suspension. in fact, among countries covered by the survey, public transport suspension was implemented only in italy, whereas its volume was reduced in all other states except for germany [ ] . the stay-at-home orders were most significantly opposed by the youngest respondents aged below . this measure was enforced in all countries covered by the survey except for denmark, where it was introduced as a recommendation [ ] . overall, a north-south gradient could often be noticed in the eu regarding policy support: people living in the southern states (portugal, italy, and france) tended to approve of the containment policies more than residents in the northern countries (denmark, germany and the netherlands). noteworthy, the largest share of supporters for every containment measure was noticed among the residents of italy and particularly in lombardy. here, on average, % of the population approved of the government's response to the pandemic. interestingly, the most significant share of the population who explicitly opposed each of the containment policies taken by their government was identified in denmark. here, for example, % of respondents disapproved of school closures and % disapproved of the imposition of a curfew. in comparison, the average disapproval of these measures in other countries was around % for schools and % for curfews. the most polarizing opinions were observed concerning the use of mobile data for tracking covid- cases and their contacts. the most significant share of people explicitly opposing such policy was identified in denmark ( %), the netherlands ( %), and germany ( %). it was particularly disfavored by the youngest age group ( % of respondents aged below against it). this policy received significant media attention as some countries and the european commission started the collaboration with telecom providers to access individual geolocation data for prediction and surveillance of covid- spread [ , ] . as of march , deutsche telekom provided german authorities with the anonymized data on the movement of its users. in italy, vodafone, windtre and telecom italia offered aggregated user data provision to the government for the same purpose. authorities in the lombardy region used mobile phone data to check compliance with the lockdown restrictions [ , , ] . other countries either initiated the development of their own mobile phone tracking apps or cooperated on the creation of common software, such as the pan-european privacy-preserving proximity tracing (pepp-pt) project led by germany. however, the launch of the pepp-pt was delayed at the end of april due to the data protection concerns voiced by experts and even some of the project participants [ ] . while proponents of the contact-tracing measures claim that using mobile data is of paramount importance in response to the covid- pandemic, many people worry about the government's use of technology due to possible privacy violations, thereby raising debates about the appropriateness of such social control measures [ , , ] . according to our data, people in some european countries expressed considerable reluctance about supporting such policy, which therefore makes future compliance questionable. moreover, such privacy disputes, as in the case of the pepp-pt project launch, might trigger higher reluctance among the potential users to use any contact-tracing app in the future, which could be detrimental for the implementation of a viable tracing technology [ ] . to better understand public opinion on certain policies, it is essential to look at the big picture and place obtained results into the national contexts. people's attitudes were likely based on their perceptions of the general state of affairs in their country, particularly in terms of the epidemic situation and restrictions they were subject to at that moment. in view of that, table summarizes the scale of the pandemic and the stringency of government's response in seven european countries at four points of time spaced around april (when the survey's fieldwork was % complete in every country). the public health situation in each state is described using total confirmed cases of covid- and total deaths attributed to covid- , both measured per million people and reported by the european centre for disease prevention and control [ ] . the stringency of government's response is measured with the covid- government response stringency index, a composite measure of containment policies ranging from to , where a higher value denotes a stricter response [ ] . at the time of the survey's fieldwork, the epidemic situation was worst, and the stringency index was highest in italy and france [ , ] . clearly, there was a north-south gradient in the stringency of government response: italy, france and portugal imposed more demanding policies than denmark, germany, the netherlands and the uk. nevertheless, although people in southern countries were exposed to more severe containment measures, they approved of them more than people residing in northern states, who experienced less stringent restrictions. turning now to within-country variations, we observed considerable heterogeneity of attitudes towards many policy responses within individual countries with particularly marked differences between regions and age groups in italy, france, and the netherlands. hereinafter, we grouped regions based on the severity of the covid- outbreak distinguishing between the most and the least affected areas. noteworthy, lombardy denotes the extra sample collected in italy and was analyzed separately from the representative italian sample. overall, we did not find significant differences in policy support between lombardy and the rest of italy. to illustrate within-country differences, fig. . reflects regional and age-related heterogeneity of public opinions in france and italy toward banning the export of medical equipment, such as masks. in fact, this measure was briefly undertaken by germany and france at the onset of the pandemic in early march , leading to political tensions between the eu member states. germany declared that the reason was to avoid shortages of masks, gloves and safety glasses within the country, whereas france argued that the ban was needed for the assessment of inventory and storage capacity [ ] . following the call for solidarity, both countries lifted the within-eu export ban on equipment in mid-march [ ] . while support for this policy tended to be similar in the most and the least severely affected parts of italy and france, the approval of the export ban conspicuously differed across age groups. older individuals approved more of this policy than younger people, which, besides other factors, may be related to the levels of worry people in these age categories have about the risks that covid- poses to their health. we found that % of french and % of italian respondents aged above perceived risks to their health from covid- as high or very high, while the corresponding share among people aged below equaled % in france and % in italy. to address the mental health implications of the covid- outbreak and subsequent containment measures, we assessed levels of worry prevailing in european societies over several domains (health, economic, emotional, work, and future). more specifically, we addressed concerns about losing a close person, becoming unemployed, health system getting overloaded, school closures, small companies running out of business, recession, restricted access to food supplies, blackouts, and society getting more egoistic. these items were adopted from the who covid- snapshot monitoring project, which will allow future comparisons with similar data collected for other countries and at different points in time [ ] . we found that the mean trend was similar in all countries: people worried most of all about the health system getting overloaded so that the capacities could become insufficient to cope with the surge in covid- cases. we observed that even in case of households that had not been directly hit by the novel coronavirus (above % of respondents in the total sample), the pandemic might have acted as a stressor causing health and economic anxieties. fig. presents people's worry about selected issues across seven eu countries (measured on a likert scale from -not worry at all to -worry a lot), where the higher intensity of color reflects a larger share of the population who worry "quite a bit" or "a lot". cross-country differences look substantial, and a north-south divide in the worry caused by the covid- outbreak is conspicuous. fig. . the proportion of respondents who worry "quite a bit" or "a lot" for instance, % of respondents in portugal and % in italy mentioned that they worried "quite a bit" or "a lot" about the national health system becoming overloaded, while the corresponding shares in denmark and germany were % and %, respectively. these health concerns might have reflected the development of the pandemic. as showed in table , the progress of the epidemic had a north-south pattern with more covid- cases and deaths per million of the population in southern states than in northern. the exception was the uk, where the epidemic was third deadliest after italy and france, but government response was less strict than in countries with a better epidemiological situation [ , ] . similarly, more people in portugal and italy were concerned with the economic consequences of the pandemic than in other european countries. for example, % of portuguese and % of italians were worried about losing their jobs, while respective shares in the netherlands and denmark were % and %, correspondingly. these cross-country differences in economic anxieties may be related to people's perceptions of the economic and financial countermeasures taken by their national government and the eu. during the pandemic, european countries implemented several fiscal and monetary measures to mitigate the economic impact of the covid- outbreak. these policies typically included support of wages under the reduced-hour scheme, postponement of tax payments for companies, direct financial supports and grants to small enterprises and self-employed, the extension of unemployment benefits, provision of capital buffers to banks, etc. [ ] . nevertheless, there were substantial variations in the timing and specific content of these countermeasures across the states. to briefly overview the scale of economic support provided by the government in each of the seven countries, table summarizes values of the economic support index, a composite measure reflecting income support and debt/contract relief provided by the national government to households [ ] . it is measured on a to scale, where a higher value refers to a more substantial economic assistance. at the time of the survey's fieldwork, all countries provided some type of economic relief to their residents. nevertheless, the extent of such support was conspicuously different: france and the uk ranked highest, while denmark, germany, and italy ranked lowest [ ] . hence, it may be possible that higher levels of economic concerns in some countries indicated people's beliefs in the insufficiency of the government's response, which will be subject to the analysis in the next waves of the survey. moreover, the composition of employment varies across the eu, especially in terms of informal and temporary employment. temporary contracts provide lower levels of social protection and job security to employees, but their prevalence has increased over the last years, particularly in the netherlands, italy, and france. as of , the share of temporary employees in the total number of employed was highest in southern european countries: portugal ( . %), france ( . %), and italy ( . %). in contrast, it was significantly lower in northern states: the uk ( . %), denmark ( . %), and germany ( . %). the only exception was the netherlands, where temporary workers constituted . % of all employees [ ] . thus, such differences in the employment composition may be in part responsible for the cross-country dissimilarities in economic concerns. we also observed differences in the levels of concern within individual countries. fig. shows the extent of worry about the health system and a recession in italy. we grouped regions based on the severity of the covid- outbreak and distinguished the levels of anxiety across age categories. higher intensity of the color reflects a greater extent of worry. overall, the level of worry in the highly affected regions of the country was not higher than elsewhere in italy, except for the youngest age group. however, economic concerns tended to be unequally distributed across the age groups. for instance, worries about the recession and small companies running out of business were higher among older individuals than younger age cohort. this pattern was similar in all countries covered by the survey. we asked people about the main sources of information from where they received news about covid- . the data show that overall % of respondents closely followed the news on the situation with covid- , implying a high level of public awareness. regarding the sources of information, % of respondents mentioned receiving updates from the tv and % additionally searched for information on the internet. presumably, reliable information presented through the television emerged as the best channel to reach the population at large. next, we assessed the extent of people's trust in the information received from various sources in the context of the covid- situation. the trust in the following information sources was addressed: national government, the eu, the who, hospitals and gps, national news channels and newspapers, social media, relatives and friends. fig. shows mean values of trust in information from six selected sources across seven european states (measured on a likert scale from -no trust at all to -trust very much). higher intensity of the color reflects a higher level of trust in the information from a specific source. the data show that overall people had the highest levels of trust in information from hospitals, family doctors, and the who, followed by information from the national government and main national news channels. this ranking of sources by trust was similar in all countries covered by the survey, except for france, where citizens had a high level of confidence only in healthcare providers and placed relatively little trust in all other sources. moreover, a north-south divide could be noticed in the level of trust in information from the national government. trust was highest in denmark and the netherlands (more than % of respondents trusted "much" or "very much"), whereas it was lowest in france ( % of respondents had a high level of trust). furthermore, a similar north-south gradient was observed concerning the trust in the eu: trust was highest in denmark ( %), germany ( %), the netherlands ( %) and the uk ( %), whereas it was lowest in italy ( %) and france ( %). portugal was an exception to this case since the corresponding value here constituted %. finally, we also observed considerable regional heterogeneities in levels of trust within countries with particularly noticeable differences across individual regions in italy, france, and germany. fig. shows people's trust in information from the national government in the context of covid- in germany and france as an example, where the higher intensity of the color indicates a greater extent of trust. while trust did not differ significantly between regions grouped with respect to the covid- severity, it was heterogenous across the age groups. although the survey asked about the level of trust in information from different sources in the context of the covid- situation and not about the overall trust in institutions, these two are likely to be related. generally, trust reflects people's perceptions of whether institutions are doing what is right. thus, trust in the information they provide can be considered an indicator of the confidence that citizens have in these institutions [ ] . the covid- pandemic raised new challenges for policymakers across the eu. the imminent threat to public health at the onset of the pandemic led most governments to impose a lockdown on society. however, as the peak of the pandemic abated, the focus of attention turned to the social and economic consequences of the containment measures. given that without acquired herd immunity the risk of a new wave of the epidemic remains high, and the production and distribution of vaccines may take to months [ ] , governments must try to strike the right balance between effects on public health, social life and the economy when considering possible exit-strategies from the current lockdown situation. in the absence of medical intervention, policymakers and public health officials must resort to non-medical behavioral interventions. lifting the lockdown requires that citizens support and adhere to the policy measures that aim to contain the spread of the virus as social and economic activity gradually restarts. given the difficulty of enforcing such regulations, future measures need to be both well-designed and well-communicated to the public. the more people are willing to comply voluntarily with the new measures, the less enforcement and supervision will be needed to achieve high compliance. for this, people's perceptions and attitudes need to be factored in at the policy-design and implementation stages. our survey sought to capture the public sentiment toward measures previously taken by policymakers to contain covid- and addressed people's support for policies, worries about the consequences of covid- , and trust in different sources of information. the first insights obtained from the data showed that containment and mitigating policies undertaken by national governments in response to the initial stages of the covid- pandemic were generally wellreceived by the population in all countries covered by the survey. nevertheless, the extent of approval varied across states and specific policy measures. several lessons can be drawn for the design and implementation of policies for the prolongation or gradual removal of lockdown restrictions. first, we observed a north-south divide in people's perceptions, worries and trust across the european countries. this finding suggests that further containment measures and lockdown exit strategies need to be balanced against the factors that worry people in each specific country. one noteworthy example is the level of importance that people in european countries attribute to the concepts of individual freedom and privacy. using mobile data for tracking covid- cases and their contacts may be a controversial decision to take even though it is believed by many experts to be a useful tool to manage the covid- outbreak. the effectiveness of this policy critically depends on a sufficient level of adoption of the technology by the population [ ] . our data suggest that this may not be achieved easily in some european countries. a clear takeaway is that an open dialogue with society on this matter is needed. explaining the need for and the advantages of such intrusive policies through trusted means of communication, while addressing people's concerns explicitly and being open about the risks of using such policy measures may help raise the support and compliance in society to a sufficient degree. another critical issue is the balance between saving lives and saving livelihoods. according to the survey, people in southern european countries are substantially more concerned about the economic aspects of the covid- outbreak than people in northern european countries. economic anxieties, if left unaddressed, may have adverse effects on the mental health and wellbeing of the population, as well as cause downward adjustments in consumption behavior, thereby exacerbating the economic situation in a country if the recession indeed happens. second, we found considerable heterogeneities in people's approval of policies within individual countries. this tendency was particularly noticeable in france and italy. one possible determinant of regional differences in public support could be the extent of the devolution of decision-making in the country. on the one hand, devolution could enable regional or local authorities to make better decisions due to their better awareness of region-specific circumstances. on the other hand, it could harm the coordination of policy responses between the central and regional authorities within individual countries. thus, it is crucial to understand the determinants of such differences and address them to secure public support of future policies and ensure high compliance with government measures. furthermore, our results showed that the burden of stress tended to be unequally distributed across and within countries. even in case of households that were not directly hit by covid- , the pandemic may have acted as a stressor causing health and economic anxieties. such worries may be detrimental to individual mental health and wellbeing, and they may become further exacerbated by the imposition of self-isolation policies. thus, it may be reasonable to consider an asymmetric approach to the design of exit strategies taking region-specific levels of support and worry into account. this includes the identification of vulnerable categories of the population not only in terms of health risks but also with respect to social and economic activities, and addressing their concerns satisfactorily. third, during a pandemic, public trust in the government and the information it provides is of paramount importance. to expect high compliance over extended periods of time, policymakers need to adopt effective strategies and means of communication whereby securing a sufficient level of trust and confidence from the society. as our results suggest, some countries were more successful in this respect than others. society needs to be well-informed about the dilemmas faced by policymakers, and for this, the communication between the government and the citizens must be clear and transparent. the data showed that % of respondents closely followed the news on the situation with covid- mainly using television to keep themselves updated. thus, television emerged as the best channel to reach the population at large, suggesting that presenting reliable information through this means is an effective strategy to follow. nevertheless, given that the data show regional and age-related heterogeneities in trust and policy support, it may be worth tailoring messages and means of communication to specific groups of the society. for example, cooperation with public figures and well-known experts can be used to deliver government and public health messages in a simple language, or local voices could be used to amplify such messages in individual regions of the country. overall, information provision, public education and effective communication strategies should be among the key guidelines for policymakers when implementing exit strategies and designing future containment measures so that these policies have public support and high compliance. additional waves of the survey are scheduled in june and august . this will allow us to investigate in more detail how the population copes with the health, social and economic consequences of the covid- pandemic as the situation evolves. declarations of interest: none estimating the number of infections and the impact of non-pharmaceutical interventions on covid- in european do low-trust societies do better in a pandemic? lockdown fatigue hits as europe enforces coronavirus restrictions answering the right questions for policymakers on covid- . the lancet global health the new york times covid- snapshot monitoring (cosmo): monitoring knowledge, risk perceptions, preventive behaviours, and public trust in the current coronavirus outbreak oxford covid- government response tracker. blavatnik school of government temporary reintroduction of border control tracking and tracing covid: protecting privacy and data while using apps and biometrics how will governments know when to lift restrictions? european mobile operators share data for coronavirus fight deutsche welle on the responsible use of digital data to tackle the covid- pandemic complete our world in data covid- dataset seeks solidarity as nations after intense discussions, i welcome that de and fr now allow for export of #covid protective equipment. i will continue to follow supply to it closely. no single eu country can win this battle alone. #cooperation #solidarity temporary employees as percentage of the total number of employees trust in government, policy effectiveness and the governance agenda the race against covid- this project has received funding from the european union's horizon research and innovation programme under the marie skłodowska-curie grant agreement no , the work was supported by funding under the excellence strategy by the german federal and state governments, as well as by the university of hamburg, erasmus university rotterdam, and nova school of business & economics lisbon -chair bpi | "fundação la caixa" on health economics. we thank our colleagues for their feedback and work on the adoption of the survey to national contexts: helen banks, joana pestana, maarten husen, laurie rachet jacquet, nicolai fink simonsen. key: cord- -cbfyydi authors: pierron, denis; pereda-loth, veronica; mantel, marylou; moranges, maëlle; bignon, emmanuelle; alva, omar; kabous, julie; heiske, margit; pacalon, jody; david, renaud; dinnella, caterina; spinelli, sara; monteleone, erminio; farruggia, michael c.; cooper, keiland w.; sell, elizabeth a.; thomas-danguin, thierry; bakke, alyssa j.; parma, valentina; hayes, john e.; letellier, thierry; ferdenzi, camille; golebiowski, jérôme; bensafi, moustafa title: smell and taste changes are early indicators of the covid- pandemic and political decision effectiveness date: - - journal: nat commun doi: . /s - - -y sha: doc_id: cord_uid: cbfyydi in response to the covid- pandemic, many governments have taken drastic measures to avoid an overflow of intensive care units. accurate metrics of disease spread are critical for the reopening strategies. here, we show that self-reports of smell/taste changes are more closely associated with hospital overload and are earlier markers of the spread of infection of sars-cov- than current governmental indicators. we also report a decrease in self-reports of new onset smell/taste changes as early as days after lockdown enforcement. cross-country comparisons demonstrate that countries that adopted the most stringent lockdown measures had faster declines in new reports of smell/taste changes following lockdown than a country that adopted less stringent lockdown measures. we propose that an increase in the incidence of sudden smell and taste change in the general population may be used as an indicator of covid- spread in the population. f ollowing similar decisions in china and italy, a strict lockdown was enforced in france beginning on march , to block the progression of covid- and alleviate pressure on hospitals. one issue currently faced by governments is how to conduct the progressive relaxation of the lockdown , which needs to be conducted systematically and carefully to prevent subsequent outbreaks while facilitating economic activity and recovery. on may , , the french government categorized each geographical area as being red or green, depending on their covid- prevalence. compared to green areas, red areas were characterized by: (i) higher active circulation of the virus, (ii) higher level of pressure on hospitals (i.e., ccru occupancy), and (iii) reduced capacity to test new cases (fig. a) . in each area, red/ green labels were used to define steps associated with the local relaxation of lockdown. the french ministry of health used the ratio of consultations for suspected cases of covid- to general consultations at the emergency room (er) in hospitals as an indicator to assess the active circulation of the virus (detailed in "methods" section). concurrently, changes in smell and taste are prominent symptoms of covid- [ ] [ ] [ ] [ ] , as has consistently. been demonstrated in many countries (e.g., iran , spain , france , italy , germany , and the uk , among others). more critically, these chemosensory changes generally occur earlier than other symptoms and may constitute more specific symptoms than fever or dry cough , . accordingly, monitoring self-reported changes in smell and taste could thus provide early and specific information on the spread of covid- in the general population and support health system monitoring to avoid daily ccru admission overflows. using data from a global, crowd-sourced study deployed in + languages (global consortium for chemosensory research survey, gccr, see "methods" section), we tested whether changes in smell/taste at the population level could be used as an early indicator for local covid- outbreaks. as pre-registered (see "methods" section), our primary aim was to test the association between self-reported smell and taste changes and indicators of pressure in hospitals (covid-related hospitalizations, ccru admissions, and mortality rates) for each french administrative region over the last months. our secondary aim was to examine temporal relationships between the peak of smell and taste changes in the population and the peak of covid- cases and the application of lockdown measures. the potential for self-reported smell and taste loss to serve as an early indicator of the number of covid- cases-and hence hospital stress-was tested in a natural experiment by comparing france with italy and the uk, which implemented lockdown with different timing and levels of stringency. here, we show that self-reports of smell/ taste changes are closely associated with hospital overload and are early markers of the spread of infection of sars-cov- . changes in smell and taste are associated with overwhelmed healthcare systems. the relationship between self-reported changes in smell and taste by french residents (diagnosed as covid- + or not, see "methods" section and supplementary table ) and estimators of local healthcare system stress was evaluated geographically. figure a depicts the geographical distribution in red and green regions (as defined by the french government) and participants who self-reported changes in their smell and taste. red areas of france account for . % of the population. green areas are clustered into a group with both a low number of self-reported chemosensory changes and a low number of admissions to ccrus (fig. b) . red areas show an opposite trend (chi-square < × − and biserial correlations p < . × − ). a strong relationship exists between self-reported changes in smell and taste and the number of admissions to ccrus (r smell = . , p = . × − ). this correlation remained significant even after removing the two most impacted areas (alsace and ile de france, r smell = . ; p < × − ), indicating that the significant relationship is not driven solely by these two regions. strikingly, use of self-reported chemosensory changes produced a stronger correlation than the current governmental indicator of virus circulation (fig. c) . overall, smell/taste changes are better correlated with the number of covid- admissions to hospitals than the current governmental indicator i.e., the ratio of er consultations for suspicion of covid- to general er consultations (r smell = . , p = . × − vs. r gov = . , p = . × − ); the same pattern was found for the number of covid- related deaths (r smell = . , p = . × − vs. r gov = . , p = . × − see supplementary table ). further, when smaller geographical areas were considered (france is divided into administrative units, called departments), these correlations remained highly significant (e.g., admissions to ccrus: r smell = . , p < × − ) (fig. c) . moreover, the three relationships (change in smell/taste versus covid- -related hospitalization, resuscitations, and death) also remained highly significant when considering only individuals who were not clinically diagnosed by a medical professional but considering themself showing some symptoms of covid- (e.g., admissions to ccrus: r smell = . , p = . × − ). potential sampling bias due to regional media coverage of our survey (supplementary table ) and self-reported chemosensory changes by region was ruled out by confirming these variables were not correlated (r < . , p > . ). notably, relationships between pandemic markers and online searches related to chemosensation were also significant in france. google queries related to smell or taste loss ("perte odorat," "perte goût" in french) were correlated with the three measures of an overwhelmed healthcare system described above (e.g., ccru admissions: r smell = . , p < × − , see supplementary table ). changes in smell and taste are early markers of the effectiveness of political decisions. next, we examined the temporal dynamics in france of self-reported changes in smell/taste, the current governmental indicator (ratio of er consults), and the number of ccru admissions due to covid- before and after the lockdown period. as shown in fig. d , the peak of the onset of changes in smell/taste appeared days after the lockdown and for these individuals, the first reported covid- symptoms occur even earlier. conversely, the governmental indicator of er consults only peaked days after the lockdown, while the peak of ccru admissions was shifted later by days. this is consistent with emerging data showing that covid- -related changes in smell and taste occur in the first few days after infection , [ ] [ ] [ ] . the robustness of smell and taste changes over time was assessed in two ways. first, we showed the peak of smell/taste changes remained the same regardless of our survey's completion date ( supplementary fig. a) . second, we observed the exact same peak when analyzing a separate french survey performed on individuals and focusing on smell alterations in the french population independently of covid- (see "methods" section): the peak of olfactory changes again occurred days after the lockdown decision, and this was independent of survey completion dates ( supplementary fig. b) . the robustness of smell and taste changes was also observed over age (supplementary fig. a ) and gender ( supplementary fig. b ). finally, we also show that the observed peak does not correspond to seasonal occurrence of allergies in france based on the ratio of consultations for allergy to general consultations at the emergency room ( supplementary fig. ) . further, analyses of google searches confirm this temporal relationship: on the same days where survey participants report experiencing their first symptoms (around march , ), there was a peak of google queries for terms associated with early covid- symptoms (fever, cough, aches, supplementary fig. a ). a few days later, the peak of online queries for "taste loss" and "smell loss" is synchronized with the report of smell and taste changes ( supplementary fig. b ). one week later, queries for shortness of breath preceded the peak of ccru admissions ( supplementary fig. c ). collectively, these results indicate a significant fraction of french covid- patients followed the same symptom time course, experiencing initial symptoms at the very start of the lockdown, which might be representative of a peak of infection a few days before the lockdown. this is consistent with the ultimate goal of the lockdown, which was to decrease the number of new infections following implementation. thus, the period immediately prior to lockdown represents the expected peak of new infections. in france, a large population may have been infected two days before lockdown because that weekend was crowded and sunny and occurred over the course of election day. further, there were busier train stations and supermarkets in anticipation of a shortage of supplies during lockdown . these data suggest that the short-term efficacy of a lockdown could be monitored by tracking changes in smell and taste in the population. to assess whether such a prediction might generalize to other countries, we performed parallel analyses with data from fig. changes in smell and taste as indicators of overwhelmed healthcare systems: geographic and time-related approaches. a french regions were assigned a green or red status by the french government to guide local relaxation of lockdown protocols. dots represent people self-reporting smell and taste changes in a web-based survey. base map is from openstreetmap and openstreetmap foundation. b the number of covid- -related ccru admissions (as of may , ) correlated with the number of self-reported chemosensory changes (between march and may , , total n = ). green dots correspond to regions with a post-lockdown level labeled green, and red triangles indicate regions considered red. values are standardized based on the number of inhabitants (inhab.) for each regions. the two red triangles with ccru admissions > are alsace and ile de france. the gray band represent the confidence interval of the linear smooth (formula 'y~x') r and p represent value of the test for association between paired samples, using one of pearson's product moment correlation coefficient, without correction for multiple comparisons. c colored bar represent the value of computed correlation coefficients (confidence intervals are depicted as thin black bars) between the number of ccru admissions per area and i) the number of people reporting smell and taste changes (n = , blue), and ii) the governmental indicator (gov. indicator), ratio of er consults for covid- (orange). analyses were done both at the level of metropolitan regions (reg) and departments (dep). d temporal relationships in france between smell/taste change symptom onset (blue solid line, n = ), the governmental indicator (orange dashed line), and covid- cases in ccrus (gray bars) around the lockdown period (vertical dashed line). data are -day running averages, normalized to the day with the highest value. with different levels of severity (see fig. ). we monitored the dynamics of confirmed covid- cases, self-reported first symptoms, and self-reported taste and smell changes, and compared them as a function of the governmental stringency index. immediately after lockdown, we found that the two countries with the higher stringency index experienced a more rapid decrease in both self-reported smell and taste changes and covid- symptoms. further, as expected, the evolution of confirmed covid- cases differs according to the stringency index. the governments of italy and france rapidly increased their stringency index, which led to a sharp decrease in covid- symptoms and cases. in contrast, in the uk, the number of people in the uk reporting symptoms showed a slower decrease, presumably due to a less severe lockdown policy, and the number of confirmed cases remained high during the observation window. in each country, self-reported smell and taste changes can be regarded as a useful metric to predict the dynamics of confirmed covid- cases. that is, when the number of new onsets of chemosensory changes decreases sharply (france and italy), the number of confirmed covid- cases also decreases, albeit with a lag of two weeks. on the contrary, a slow decrease in the number of new onset chemosensory changes is associated with a plateau of confirmed cases (uk). the present analyses reveal a strong spatial and temporal relationships between self-reported smell and taste changes and multiple indices of health care system stress, such as admissions to ccrus. this is consistent with cumulative evidence showing a high prevalence of chemosensory alterations in patients affected by covid- in europe (france , , italy , uk , , ). participants endorsed smell and taste changes only - days after their first symptoms. such early chemosensory estimators may represent a cost-effective and easy way to implement alternative surveillance methods to large-scale virology tests, which are difficult to perform, costly, and time-consuming, especially during a pandemic. a prominent question raised by these findings is whether the smell and taste changes observed in our study are solely related to covid- or whether they can be explained by other temporal patterns, like seasonal illnesses or allergies. to the best of our knowledge, there are no existing studies that have explored the dynamics of sudden anosmia (as in throughout the year in france. relationship between olfactory disturbances and seasons have been reported in korea, germany or us with a moderate increase of anosmia prevalence in spring [ ] [ ] [ ] overlap, the amplitude of reported changes (either due to allergy or viral affection) were very limited compared to the present report. to further rule out the possibility, we examined whether the annual peak of allergies in france could explain the peak of smell and taste changes observed here. in analyzing existing french governmental data, we found that the annual peak of allergies in france occurred around week (beginning of summer), multiple weeks after the observation window of the present study (from week to week , supplementary fig. ) . further, the french national aerobiological surveillance network (rnsa, https://pollens.fr), which follows pollen concentration in the atmosphere, has also indicated the first week of lockdown was very low risk for seasonal allergies. in addition, when considering google trends data, we did not observe any similar peaks in queries for smell/taste loss in the corresponding time period in previous years. finally, a comparative study in israel showed that in covid- suspected patient the frequency of smell change is almost ten time higher in a covid- positive patients ( %) than in covid- negative ( %). considering that most of the participants of the present study are diagnosed with covid- and that their description of a sudden loss of smell/ taste is consistent with the now typical presentation of covid- symptoms, it is highly probable that covid- infection is the main reason of their smell and taste change. collectively, these data suggest the peak of smell and taste changes studied here are more consistent with sudden covid- viral infections rather than an artifact due to seasonal illnesses. the time lag between the onset of covid- -related symptoms and their declaration by the respondents of our study also deserves comment. although immediate reporting of symptoms would have been ideal, such reporting is not possible within the context of the sudden first wave of a new viral pandemic. a similar time lag has been observed in other large-scale studies focusing on olfaction and covid- . indeed, this time lag is inevitable given the preparation time required for scientists and clinicians design and launch such a survey, with appropriate ethics approval, once anosmia and ageusia began to emerge as cardinal symptoms of covid- . the vast majority of participants completed the survey between april th and april th, , and most of them declared a date of onset of their symptoms roughly a month earlier (although a small fraction of participants did indicate onset prior to ). a possible consequence of a time lag between survey completion and the effective date of symptom onset is that subjects' statements may have been influenced by major societal events such as the lockdown decision, potentially creating some recall bias. to examine whether the date of a major event like the lockdown might bias dates of reported smell and taste loss, we explored narrative descriptions provided by our participants. by analyzing responses to the optional open-ended question "please describe the progression or order you noticed your symptoms", we observed that, for france, a mere of people (who have filled the optional question) used the term "confinement" ("lockdown") in their description of the onset date. separately, another factor that mitigates concerns about a potential recall bias is the stable nature of participant's statements, regardless of their date of completion. that is, logic suggests, the longer the time between the onset date of smell and taste loss and the reporting date, the greater the recall bias should be. however, our data clearly show that regardless of the date of completion, the onset date falls within the same period ( supplementary fig. ) . finally, other evidence against a potential recall bias comes from google trends data. analyzing real-time google queries in march, we observed a very particular trend in france (supplementary fig. ) . we first observed a peak of queries for terms associated with early covid- symptoms (fever, cough, aches) synchronized with the declared onset of the first symptoms in the survey (around march th). a few days later, a peak of online queries for "taste loss" and "smell loss" was seen, and this was synchronized with the date reported of smell and taste changes in our survey. the striking concurrence between google queries and reports in our survey argues against the idea that a recall bias could be driving the effects described here. another important factor to consider in our survey is the way the press and media might have influenced our findings. indeed, when the survey was launched, smell and taste changes were reported as symptoms of covid- in the national and local media, which might have influenced respondents to remind themselves of such symptoms and to then report these changes on the survey. such an emphasis on smell and taste loss would have biased attempts to explore the prevalence of chemosensory deficits in covid- . however, the primary aim of the present investigation was not to focus on the prevalence of anosmia and ageusia with covid- , but rather to explore use of reported smell and taste loss as indicators of covid- pandemic. still, the media coverage of our survey could also have biased the selection of participants geographically, as some french regions received more media coverage than others. however, as reported above, there was no correlation between the number of participants in a given region and the intensity of media and press coverage for the survey in that same region. finally, when participants were asked to describe the chronology of their symptoms, they did not refer to the media coverage as a prominent element influencing their awareness of their smell/taste changes. while this does not exclude an implicit and non-verbalized bias due to media coverage, this pattern suggests a genuine report of symptoms with a high occurrence of covid symptoms just after the lockdown. an interesting question raised by our findings is what impact they might have on government strategies in a pandemic. following lockdown, the rapid decrease of self-reported changes in smell and taste in france may be representative of the effectiveness of this decision in reducing infection rates. similarly, data from italian participants show highly similar patterns, but with a one-week difference compared to the french data. this might reflect highly similar responses by the italian and french governments. conversely, the prevalence of chemosensory changes in the uk shows a more gradual decrease. the uk government began with advice to avoid pubs, clubs and theaters, and to work from home from march , with restrictions around march . however, a lockdown was not declared until march , and this was less stringent than those in france or italy. notably, new covid- cases in the uk showed a plateau phase which is not observed in either france or italy. accordingly, we conclude that collecting online information about changes in smell and taste from residents (even retrospectively) may be a valuable metric of the effectiveness of reopening strategies related to the covid- pandemic. practically, in areas where smell and taste changes are notable covid- symptoms, the proportion of individuals who selfreport changes in their ability to smell or taste might be an early indicator of subsequent demand for healthcare. if confirmed, continuous monitoring of changes in smell and taste perception would then be a highly cost-effective, minimally invasive, and reliable way to track future covid- outbreaks. when used this way, we caution that particular attention must be paid to potential selection bias. that is, self-report studies online can be impacted by multiple selection biases, including socioeconomic status, fluency with technology and willingness and interest in participating in scientific research. when considering the present data, at least parameters may contribute to a selection bias in our sample: ( ) the age, ( ) the gender of the participants, and ( ) the format and the advertising of the survey. regarding participant' age, our study cohort (mean . years, sd = . )) was quite similar to the french population mean ( . years, according to insee, https://www.insee.fr/fr/statistiques/ ); however, we did only include individuals over due to issues of consent, and administrative reasons, and seniors were also less represented. for gender, our sample contained a greater proportion of women ( %) compared to men, which might influence the results. however, additional analysis showed no differences in peaks of smell/taste changes across age or gender, minimizing concerns that such selection biases may have influenced present results (see supplementary fig. ) . we also tested the potential selection bias due to format and the advertising of the survey, by comparing the gccr dataset with an independent second study performed on french residents (see "methods" section). remarkably we observed highly similar results across studies where advertising, inclusion criteria, and survey format were different. based on the present findings, we highlight the paramount importance and robustness of associations between smell/taste changes and covid- and we strongly endorse the need for additional large-scale validation studies to assess the causality between the observed association between smell/taste changes and indicators of the covid- pandemic. this could be achieved by setting up a simplified interface where selection biases are controlled for (age, gender, motivation, media coverage, socioeconomic level, etc.) through both traditional and online media-and whereby real time information about changes in smell and taste in the general population may be available to decision-makers. subjects' participation in the questionnaire and the reliability of the answers should also be considered. in particular, if a participant knows how their answers may influence enforcement of lockdown, their answers might become less truthful. this motivation can be expressed through different forms of behavior. whereas some individuals may tend to provide statements that minimize their symptoms in order to avoid strict containment measures, others will maximize their declaration to maintain the lockdown, or will provide honest answers in order to participate in the collective effort to better understand the covid- pandemic. these motivational factors are a recurrent risk in online studies and different strategies should be held to control for them in future predictive studies. based on the above, a large implementation of the study of smell and taste changes in institutional models should allow for monitoring of covid- spread. this might be especially relevant in in areas in which testing proves difficult or delayed and for future outbreaks that may overlap with other seasonal viral diseases which share many of the symptoms (fever, cough etc.) but whose treatment or prevention (vaccination) are less demanding in terms of critical care than covid- . we advocate that self-report surveys should be used to enhance other strategies such as large-scale pcr tests and covid- symptom assessments (including anosmia and ageusia) in primary/secondary care. in summary, we propose that an increase in the incidence of sudden smell and taste change in the general population may be used as a valuable minimally invasive indicator of coronavirus spread in the population. to formally test the temporal relationship between chemosensory changes and spread of the disease, we recommend that a large-scale causal study in different countries be conducted on real-time monitoring of self-reported changes in the ability to smell or taste. such a prospective study will allow for the creation of statistical models that can assist in prediction of future hospital admissions for covid- . further, it could also help decision-makers take important measures at the local level, either in catching new outbreaks sooner, or in guiding the relaxation of local lockdowns, given the strong impact of lockdown on economic and social activities. online survey. this study is mainly based on data from the global consortium for chemosensory research survey (gccr, https://gcchemosensr.org/)a global, crowd-sourced online study deployed in + languages . the data analyzed here were collected from april to may , . the protocol complies with the revised declaration of helsinki and was approved as an exempt study by the office for research protections at the pennsylvania study university (penn state) in the u.s.a. (study ; pi hayes). participants in the gccr questionnaire were recruited by word of mouth, as well as through social and traditional media (flyers, social media, television, radio) during the covid- pandemic. it was well covered by the french press, as over articles mentioned the project, at both the regional and national level (see supplementary table ). respondents received no monetary incentive for their participation. inclusion criteria were as follows. (i) questionnaire completion was allowed only to participants who indicated they had suffered from a respiratory disease in the past two weeks, whether they noticed a change in their taste/smell or not. (ii) participants aged years old or younger were excluded. for the analyses conducted in this article, only individuals reporting a change in smell and/or taste perception were included, based on the question "have you had any of the following symptoms with your recent respiratory illness or diagnosis?". moreover, to exclude unreliable entries, participants must have reported a quantitative decrease of at least on a -to- rating scale between their ability to smell and/or taste before and during their recent respiratory illness or diagnosis. therefore, due to this inclusion criteria, "smell/taste change" is equivalent to a quantitative decrease of participant ability to smell and/or taste. we then extracted individuals from the full dataset who reported living in france, italy or the uk. as the country of residence was completed as a text entry, we allowed for typical variations (e.g., "united kingdom" or "uk"), spelling mistakes, use of different languages (e.g., "italie" or "italia"), as well as subdivisions (e.g., "scotland") and major cities ("paris"). metropolitan france was split into so-called "regions" in . however, we considered the former system where france was split into regions here, since the organization of the health system mostly remains based on the structure built before . an alternative, finer granularity, splits metropolitan france into so-called "departments." to retrieve the french department and region of the participants, we used the city of residence they reported in the questionnaire and combined them with the french public website (data.gouv.fr, after a semi-manual correction of spelling). participants came from all metropolitan departments but three (mayenne, creuse, cantal). consequently, the number of responses analyzed in france was between n = and depending on the analysis conducted (i.e., on whether the information of interest was present or missing and the date range of analysis, see supplementary table complementary and independent french survey. the data of another online survey were used to evaluate the robustness of the temporal evolution of smell and taste changes. this survey was conducted in the french population between april and may , and aimed at characterizing chemosensory disorders in people with and without covid- , as well as their consequences on quality of life. the data of respondents were eligible for comparison with data from the gccr survey, i.e., responses where both the date of completion and the date of smell loss onset were provided. only responses that were complete and from people who were responding to the questionnaire for the first time and were over age were included. this survey was approved by the cnrs ethics committee. data collection was strictly anonymous. the protocol complies with the revised declaration of helsinki and the study was approved by the ethics committee of the institute of biological sciences of the cnrs on the rd of april (dpo #trrech- ). all individuals provided informed consent when participating in the survey. online trends. trends of online queries by french region were performed using google trends, a tool returning the popularity of a search term in a specific state or region. google is by far the most used search engine in france (> % of internet searches, according to statcounter global stats). we looked for the popularity of terms (listed in supplementary fig. , using default selection of "all categories" and "web search"), within the timeframe of february , to may , (from the month of the first official covid-related death in europe to the end of lockdown in france). it should be noted that google trends does not provide the actual numbers of searches but rather a relative score from to ( corresponding to the day with the greatest number of searches during the specified time period). to compare google trends scores between french regions, we transformed them by computing the relative number of queries per day in the region of interest. for example, despite a value of , the peak day might represent only % of the total number of queries related to the topic across the timeframe of interest (see above). healthcare system data. the french governmental indicator to estimate the circulation of the virus was calculated from the ratio of consultations for suspected covid- to general consultations at the emergency room (er) in hospitals. this ratio corresponds to the medical diagnostic for covid- suspicion (codes cim : u . , u . , u . , u . , u . , u . , u . , b . , b . ). the definition of covid- has evolved rapidly during the lockdown period but the diagnosis is principally based on symptoms of covid- considered as common such as fever, cough, and dyspnea (difficulty breathing). to the best of our knowledge, anosmia and ageusia were officially considered in france as putative symptoms of covid- from a letter of the direction générale de la santé (april st) and communication of the haut conseil de la santé publique (a letter dated april , published online april , following a letter from the cnp-orl dated march ) . areas with values of the french governmental indicator higher than % are considered having a high virus circulation. this indicator contributes to the assignment of a red/green label. allergies incidence in previous years were calculated from the ratio of consultations for allergy to general consultations at the emergency room (er) in hospitals. data dealing with the health status across countries (number of covid- cases and deaths for each day) were downloaded on may , from the european centre for disease prevention and control databank (ecdc, https:// www.ecdc.europa.eu/en). data regarding healthcare system stress in france (hospitalizations, ccru entries and deaths) were also downloaded on may from the french public health website (géodes, santé publique france, https:// geodes.santepubliquefrance.fr/#c=home). here, we use the term ccru (critical care resuscitation unit) to translate the french hospital service of "réanimation." raw data were normalized across regions with regard to their number of inhabitants as estimated by insee. the temporal evolution of the stringency of government response was retrieved from the oxford covid- (https://www.bsg. ox.ac.uk/research/research-projects/coronavirus-government-response-tracker). here, the stringency level of a country is computed according to which measures of a list of items (e.g., school closures, cancellation of public events, international travel controls, etc.) are undertaken. for the post-lockdown situation, the color assigned by the french government to each department was downloaded on may from the government website. only data before may (the initial lift of the lockdown) were included in the analyses. statistical analyses. statistical analyses were pre-registered at the open science framework (osf). data were analyzed using r software ( . ) and its standard packages (maps, ggplot, etc.). data were grouped at the national level (france, italy, uk). in france they were also grouped at the regional level (according to the division into regions in place prior to the reform). the rationale behind this is that the healthcare system is still structured following this organization, with university hospitals in regional main cities serving patients of the surrounding departments. participants from overseas french territories were not included in the geographical analysis because of too few data (n < ). the relationship between ( ) gccr responses (or online queries), and ( ) public health data was determined using parametric (e.g., pearson correlations) statistics as allowed by the normal distribution of the variable of interest. the association between gccr participant and red/green post-lockdown status was tested using chi-square tests and biserial correlations. complementary analyses not planned in the pre-registration included: (i) the analysis using the independent french online survey (see section "complementary and independent french survey" of the methods), (ii) the correlation between regional media coverage and the number of responses to the online survey per region, (iii) the correlation at the level of department, (iv) the correlation excluding extreme points, and (v) the correlation with the government indicator. pre-registered statistical analyses not presented here include: (i) mann-kendall trend test and change-point detection test to detect time series changes, and (ii) part of the google trends analysis. preparing for a responsible lockdown exit strategy real-time tracking of self-reported symptoms to predict potential covid- sudden and complete olfactory loss function as a possible symptom of covid- a new symptom of covid- : loss of taste and smell corona viruses and the chemical senses: past, present, and future coincidence of covid- epidemic and olfactory dysfunction outbreak in iran acute-onset smell and taste disorders in the context of covid- : a pilot multicenter pcr-based case-control study utility of hyposmia and hypogeusia for the diagnosis of covid- self-reported olfactory and taste disorders in sars-cov- patients: a cross-sectional study predictive value of sudden olfactory loss in the diagnosis of covid- selfreported olfactory loss associates with outpatient clinical course in covid- estimating the burden of sars-cov- in france anosmia and dysgeusia in patients with mild sars-cov- infection features of anosmia in covid- olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid- ): a multicenter european study smell dysfunction: a biomarker for covid- a data science-based analysis of seasonal patterns in outpatient presentations due to olfactory dysfunction olfactory dysfunction from acute upper respiratory infections: relationship to season of onset epidemiology of anosmia in south korea: a nationwide population-based study self-rated smell ability enables highly specific predictors of covid- status: a case control study in israel relationship between odor intensity estimates and covid- prevalence prediction in a swedish population more than smell-covid- is associated with severe impairment of smell, taste, and chemesthesis data acquisition and curation reporting summary. further information on research design is available in the nature research reporting summary linked to this article. the authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files. (source data file). source data are provided with this paper. r scripts are available on the osf server (https://osf.io/gew p/). the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - -y.correspondence and requests for materials should be addressed to d.p., j.g. or m.b.peer review information nature communications thanks micael widerstrom and the other, anonymous reviewer(s) for their contribution to the peer review of this work. peer review reports are available.reprints and permission information is available at http://www.nature.com/reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/ . /. key: cord- -y nherse authors: lepelletier, didier; andremont, antoine; choutet, patrick title: risque d’introduction et voies d’importation par l’homme de maladies infectieuses exotiques : cas particulier de l’émergence de bactéries pathogènes multirésistantes aux antibiotiques, importées en france à l’occasion de voyages internationaux ou du rapatriement de patients hospitalisés à l’étranger date: - - journal: bulletin de l'académie nationale de médecine doi: . /s - ( ) - sha: doc_id: cord_uid: y nherse summary the spread of multidrug-resistant bacteria has become a major problem in france in recent years, owing to increasing antibiotic exposure, growing international exchanges, repatriation of hospitalized french patients, and treatment of french and foreign travelers in french hospitals. this article examines how different pathogens may become endemic in france. the spread of multidrug-resistant bacteria has become a major problem in france in recent years, owing to increasing antibiotic exposure, growing international exchanges, repatriation of hospitalized french patients, and treatment of french and foreign travelers in french hospitals. this article examines how different pathogens may become endemic in france. les êtres humains jouent un rôle important dans l'introduction de maladies infectieuses. en toutes ces infections, très médiatisées, ne doivent pas sous-estimer d'autres risques comme l'introduction en france de bactéries résistantes. en effet l'apparition de bactéries pathogènes devenues résistantes aux antibiotiques et leur diffusion constituent un des phénomènes émergents majeurs de ces trente dernières années. certaines espèces bactériennes sont devenues résistantes à plusieurs antibiotiques et parfois à l'ensemble des antibiotiques disponibles : on parle alors de bactéries multirésistantes, de bactéries extrêmement résistantes ou de bactéries panrésistantes [ ] [ ] [ ] . ces terminologies expriment l'évolution de la mulitrésistance et concernent des bactéries cause d'infections associées aux soins pouvant, dans certains cas, aboutir à des impasses thérapeutiques [ ] . les niveaux très élevés de la résistance qui sont observés actuellement résultent de l'exposition massive aux antibiotiques à laquelle ont été soumis les humains et les animaux au cours des cinquante dernières années. les résistances aux antibiotiques affectent non seulement les bactéries pathogènes mais aussi, et probablement même beaucoup plus, les bactéries commensales qui colonisent les individus (humains et animaux) et qui sont beaucoup moins facilement repérables car le portage ne s'associe à aucun signe clinique. toutefois, selon les spécificités locales de la pression de sélection et des modes de vie des populations, les niveaux de résistance ne sont pas égaux dans tous les pays. a partir d'une zone où elle a émergé, la diffusion de cette résistance est ensuite facilitée par l'intensité des échanges internationaux et la mondialisation. si les déplacements des animaux, voire de produits de l'agriculture jouent un rôle dans la dissémination mondiale de la résistance, nous allons ici analyser des évènements infectieux liés aux déplacements des populations humaines. en france, les brassages de population sont importants. notre pays est le numéro un mondial des arrivées de touristes avec plus de millions de voyageurs étrangers chaque année. dans la même période, , millions de français voyageaient à l'étranger [ ] . par ailleurs, , million de français vivent à l'étranger dont % en europe, % en amérique, % en afrique, , % en asie-océanie et , % au proche et moyen orient [ ] . le rapatriement sanitaire de français hospitalisés à l'étranger, mais aussi les simples retours de voyage et la prise en charge sanitaire d'étrangers en voyage en france, quelle que soit leur nationalité, expose donc la population française à des bactéries multirésistantes aux antibiotiques qui auraient pu être acquises dans des zones de haute prévalence de résistance. ce risque d'émergence et de diffusion à partir des brassages de population est mal évalué quantitativement à l'heure actuelle en france. on sait toutefois qu'il est réel et des évènements sporadiques ou épidémiques ont été observés concernant des pathogènes tels que mycobacterium tuberculosis multirésistant, clostridium difficile de ribotype , klebsiella pneumoniae productrice de carbapénèmase, acinetobacter baumannii multirésistant, staphylococcus aureus résistant à la méticilline commu-bull. acad. natle méd., , , n o , - , séance du novembre nautaire producteur de la leucocidine de panton-valentine ou entérocoques résistant à la vancomycine. dans ce contexte, la question se pose de savoir quel degré de priorité de sécurité sanitaire doit être attribué à la surveillance et au contrôle de la diffusion de ces pathogènes multirésistants aux antibiotiques importés en france, à l'occasion du retour d'un voyage ou du rapatriement de patients hospitalisés à l'étranger. habituellement isolées [ ] . [ ] . si la souche de c. difficile était la souche prédominante lors des épidémies survenues au québec, elle n'était cependant pas la seule souche cause de l'augmentation de l'incidence observée dans les différents pays.. en particulier, une souche toxine a négative/toxine b positive a causé des épidémies d'icd dans plusieurs hôpitaux d'abord au canada [ ] , aux pays-bas [ ] et en irlande [ ] , associant également une résistance aux fluoroquinolones et aux macrolides. il est probable que la forte augmentation de l'utilisation des fluoroquinolones dans les années qui ont précédé a favorisé ce phénomène. aussi, la surveillance et le contrôle des icd est devenue une priorité nationale depuis afin d'identifier les cas liés à la souche épidémique mondiale et de maîtriser sa diffusion en france. l'émergence d'entérobactéries résistantes aux carbapénèmes depuis le début des années est inquiétante, laissant entrevoir des impasses thérapeutiques [ ] . les carbapénèmes étant utilisés dans le traitement des infections sévères à entérobactéries productrices de β-lactamases à spectre étendu (e-blse), l'explosion de l'épidémiologie des e-blse est ainsi à l'origine de l'émergence des bactéries résistantes aux carbapénèmes. leur large utilisation crée une pression de sélection qui favorise ensuite l'émergence des souches d'entérobactéries qui y sont résistantes. les souches d'entérobactéries résistantes aux carbapénèmes ainsi sélectionnées appartiennent essentiellement à l'espèce klebsiella pneumoniae mais aussi à d'autres espèces comme escherichia coli. la première souche de klebsiella pneumoniae a été isolée aux etats-unis en , en caroline du nord et dénommée kpc- . par la suite, d'autres souches de kpc ont été décrites à travers les etats-unis (kpc- à kpc- ) sur des modes sporadiques ou épidémiques [ ] [ ] [ ] . la première épidémie de kpc en dehors du territoire américain a été rapportée en israël, à partir de voyageurs et/ou de patients ayant transité entre les deux pays [ ] . depuis, de nombreux continents ont rapporté l'émergence de kpc, comme l'amérique du sud, et l'asie. en europe, le phénomène semble rare mais les kpc ont été isolées de manière sporadique en suède, en irlande, au royaume-uni [ ] et en grèce qui représente (figure ). en france, plusieurs cas sporadiques ont été isolés chez des voyageurs, rapatriés des États-unis après avoir été hospitalisés [ , ] , mais d'autres cas ont été importés en provenance d'autres pays de la communauté européenne, notamment de grèce [ ] . une attention toute particulière doit être portée à l'importation en france de ce type de bactéries multirésistantes, n'ayant pas encore diffusé sur un mode épidémique, à partir de voyageurs rapatriés et ayant été hospitalisés à l'étranger, a fortiori dans un pays de haute prévalence. le haut conseil de santé publique s'est saisi de ce problème. les sarm communautaires ont émergé aux États-unis à la fin des années , dans des populations jeunes, sans facteur de risque. ces souches étaient génétiquement différentes de celles provenant des hôpitaux et provoquaient principalement des infections cutanées et des pneumopathies nécrosantes [ ] , souches dont la virulence est liée à la présence de la toxine de panton-valentine. la diffusion des différents clones de sarm communautaire est complexe et mal élucidée. le clone le plus répandu est le clone américain usa , particulièrement épidémique. cette souche usa est présente en france, mais le clone principalement détecté sur notre territoire est le clone européen st . si le clone st n'était détecté, en europe, seulement en france et en suisse avant [ ] , on le retrouve maintenant dans de nombreux pays, comme la belgique, le royaume-uni, l'Écosse, la suède, la norvège, la finlande, la grèce, la roumanie, l'allemagne, la croatie, les pays-bas, le danemark, la slovénie, mais aussi en algérie et à singapour [ ] . d'autres clones sont également présents en france (st , st , st , st depuis une dizaine d'années, les autorités sanitaires internationales ont dû faire face à l'émergence et à la diffusion rapide à travers le monde de nouvelles souches de virus grippal, du syndrome de détresse respiratoire aiguë, du chikungunya, et de la tuberculose multirésistante aux antibiotiques... les transports modernes et l'augmentation du tourisme, les voyages d'affaires et l'immigration ont contribué à la dissémination de ces pathogènes à haut impact épidémique [ , ] . les bactéries multirésistantes aux antibiotiques représentent aussi un risque important [ , ] . l'augmentation des voyages internationaux de populations à haut risque infectieux, nécessitant une prise en charge médicale ou chirurgicale, et de migrants recherchant des soins spécifiques n'existant pas dans leur pays d'origine, a déjà des implications internationales dans l'émergence et la diffusion de la résistance bactérienne aux antibiotiques [ ] . les données de la littérature sur le dépistage systématique de patients hospitalisés à l'étranger et rapatriés dans leur pays d'origine sont peu nombreuses et relativement anciennes. cependant, elles apportent des éléments de réflexion intéressants sur la prise en charge des patients hospitalisés à l'étranger et rapatriés et sur la diffusion de souches mulitrésistantes de pays à pays [ , ] . cette réflexion doit être intégrée dans les politiques nationales de diminution et de contrôle de la diffusion de la résistance bactérienne aux une large utilisation de céphalosporines favorise l'émergence d'entérocoques. il est probable que l'interdiction de l'utilisation des dérivés des glycopeptides comme promoteurs de croissance en élevage depuis et l'utilisation plus parcimonieuse de la vancomycine, notamment orale, en médecine humaine ont protégé la france d'une explosion des entérocoques résistants aux glycopeptides. de plus des recommandations ont été rédigées par le comité technique des infections nosocomiales et des infections liées aux soins (ctinils) en pour stopper la diffusion de la résistance à partir d'un cas sporadique ou de réduire son importance en cas d'épidémie installée. -pandrug resistance (pdr), extensive drug resistance (xdr), and multidrug resistance (mdr) among gram-negative bacilli: need for international harmonization in terminology emergence of extensively drug-resistant and pandrug-resistant gram-nagative bacilli in europe a. -the diversity of definitions of multidrugresistant (mdr) and pandrug-resistant (pdr) acinetobacter baumannii and pseudomonas aeruginosa has the era of untreatable infections arrived institut national de la statistique et des études économiques (insee) ministère des affaires etrangères -la tuberculose en france est-elle d'actualité ? les cas de tuberculose maladie déclarés en france en surveillance de la résistance aux antituberculeux en france : données récentes wordwilde emergence of extensively drug-resistant tuberculosis -le poids de la tuberculose en afrique et ses enjeux internationaux emergence of clostridium difficile toxinotype iii, pcr-ribotype -associated disease clostridium difficile infection in patients discharged from us short-stay hospitals a portrait of the geographic dissemination of the clostridium difficile north american pulsed-field type strain and the epidemiology of c. difficile-associated disease in quebec -outbreak of clostridium difficile infection in an english hospital linked to hypertoxin-producing strains in canada and the us van den broek p.j. -clostridium difficile ribotype , toxinotype iii in the netherlands first isolation of clostridium difficile pcr ribotype , toxinotype iii in belgium. eurosurveillance weekly -first cluster of c. difficile toxinotype iii, pcr-ribotype associated disease in france: preliminary report an outbreak of toxin a negative, toxin b positive clostridium difficile-associated diarrhea in a canadian tertiary-care hospital nosocomial outbreak of clostridium difficileassociated diarrhoea due to a clindamycin-resistant enterotoxin a-negative strain emergence and control of fluoroquinolone-resistant, toxin a-negative, toxin b-positive clostridium difficile the real threat of klebsiella pneumoniae carbapenemaseproducing bacteria novel carbapenem-hydrolyzing â-lactamase kpc- from a carbapenem-resistant strain of klebsiella pneumoniae molecular epidemiology of kpc-producing klebsiella pneumoniae isolates in the united states: clonal expansion of multilocus sequence type emergence of kpc- and kpc- in carbapenem-resistant klebsiella pneumoniae strains in an israeli hospital arrival of klebsiella pneumoniae producing kpc carbapenemase in the united kingdom kpc- -producing klebsiella pneumonia infections in greek hospitals are mainly a hyperepidemic clone intercontinental travels of patients and dissemination of plasmid-mediated carbapenemase kpc- associated with oxa- and tem- plasmid-mediated carbapenem-hydrolyzing β-lactamase kpc in a klebsiella pneumoniae isolate from france plasmid-mediated carbapenem-hydrolyzing β-lactamase kpc in klebsiella pneumoniae isolate from greece veb- extended-spectrum β-lactamase-producing acinetobacter baumannii multi-resistant infections in repatriated patients after natural disasters: lessons learned from the tsunami for hospital infection control multidrug-resistant organisms in military wounds from iraq and afghanistan community-acquired methicillin-resistant staphylococcus aureus carrying panton-valentine leukocidin genes: worldwide emergence global distribution of panton-valentine leukocidin-positive methicillin-resistant staphylococcus aureus antimicrobial resistance and molecular epidemiology of vancomycin-resistant enterococci from north america and europe: a report from the sentry antimicrobial surveillance program emergence ans spread of vancomycin resistance among enterococci in europe rapide control of an outbreak of vancomycin-resistant enterococci in a french university hospital successful control of a hospital-wide vancomycin-resistant enterococcus faecium outbreak in france -les entérocoques résistants aux glycopeptides (erg) : situation épidémiologique, mesures de contrôle actuelles et enjeux à venir debate-guidelines for control of glycopeptide-resistant enterococci (gre) have not yet worked human migration and infectious diseases globally mobile populations and the spread of emerging pathogens evaluation of repatriation parameters: does medical history matter? international aeromedical evacuation population mobility, globalization, and antimicrobial drug reistance bacterial colonization of patients undergoing international air transport: a propsective epidemiologic study vanderbroucke-grauls c. -carriage of resistant microorganisms in repatriates from foreign hospitals to the netherlands godeau quelle est la responsabilité d'une antibiothérapie préalable inadaptée dans la sélection d'entérocoques résistants ? key: cord- -p dg jw authors: bigault, lionel; brown, paul; bernard, cécilia; blanchard, yannick; grasland, béatrice title: porcine epidemic diarrhea virus: viral rna detection and quantification using a validated one-step real time rt-pcr date: - - journal: j virol methods doi: . /j.jviromet. . sha: doc_id: cord_uid: p dg jw since , porcine epidemic diarrhea virus (pedv) has reemerged in europe. rt-pcr methods have been described for the detection of pedv, but none have been validated according to a norm. in this study we described the development and validation of a sybr™ green one-step rt-qpcr according to the french norm nf u - , for the detection and quantification of pedv viral rna. the method was validated from sample preparation (feces or jejunum) through to nucleic acid extraction and rt-qpcr detection. specificity and sensitivity, limit of detection (lod), limit of quantification (lq), linearity, intra and inter assay variability were evaluated using transcribed rna and fecal and jejunum matrices spiked with virus. the analytical and diagnostic specificities and sensitivities of this rt-qpcr were % in this study. a lod of genome copies/ µl of extract from fecal matrices spiked with virus or rna transcript and genome copies/ µl of extract from jejunum matrices spiked with virus were obtained. the lower lq (llq) was genome copies/ µl and the upper lq (ulq) ( ) copies/ µl. this method is the first, validated according a norm for pedv and may serve as a global reference method to harmonize detection and quantification of pedv viral rna in both field and experimental settings. porcine epidemic diarrhea (ped) was first described in europe in . it is characterized by watery diarrhea, vomiting, dehydration, and is most notable in young piglets. the etiologic agent, porcine epidemic diarrhea virus (pedv) which was first identified by electron microscopy (em) in (chasey and cartwright, ; debouck and pensaert, ) is now characterized as an enveloped virus with a single stranded positive sense rna genome, member of the order nidovirales, suborder cornidovirinae, family coronaviridae, subfamily orthocoronavirinae, genus alphacoronavirus, subgenus pedacovirus (walker et al., ) . in the 's, pedv was detected for the first time in asia whilst in europe it was endemic. during the 's only few sporadic cases were reported in europe and most of these were reported in italy were it remains endemic (martelli et al., ) . during the last two decades new pedv strains have appeared in china and some of these strains have caused extremely severe outbreaks characterized by a morbidity of % and a mortality of - % on suckling piglets (sun et al., ) . this has led to the naming of pedv as either s-non-indel or s-indel genotypes. in general the more virulent viruses belong to the s-non-indel group. in the last decade both s-non-indel and s-indel viruses have emerged in the usa with serious consequences for the industry. throughout europe, the predominant types are now closely related to the viruses circulating in asia and north and central america (boniotti et al., ) . furthermore, all viruses reported in europe since belong to the s-indel group (grasland et al., ; stadler et al., ; steinrigl et al., ; theuns et al., ) except for one in the ukraine (dastjerdi et al., ) . this data highlights the importance of pedv diversity across several continents. in france, since , ped caused by s-non-indel is a notifiable disease. for territory monitoring purpose, all pedv suspicions have to be notified to french ministry of agriculture and the pedv genotype has to be confirmed by the national reference laboratory at the french agency for food, environmental and occupational health safety (anses). until today, no official method has been validated for the detection and quantification of the pedv viral rna. since the s, real-time pcr emerged as a tool of choice for the detection and quantification of viral rna and has multiple benefits: i) these tests are highly specific ii) are easily standardized compared to "classical" virology procedures, iii) are much less time consuming, and iv) are highly reproducible. several rt-pcrs have been described for the detection of pedv rna (kim et al., ; miller et al., ) . for a rapid, accurate and reliable diagnosis of ped in the veterinary laboratory, a method for the detection of pedv viral rna has been developed and more importantly validated according to the "association francaise de normalisation" (afnor) french nf u - norm entitled "requirement and recommendation for the implementation, development and validation of pcr in animal health" (afnor, a; afnor, b) . this validated sybr tm green one-step rt-qpcr was based on a previously published taqman® probe real time rt-qpcr (kim et al., ) and targeted the same zones of sequence in the conserved n open reading frame (orf) as this had previously allowed for broad range detection and the capability to differentiate between the closely related virus transmissible gastro-enteric virus (tgev). the method developed in the current study under nf u - , unlike other molecular tests developed for pedv, evaluates the whole process from sample preparation through to the detection and quantification by rt-qpcr. this method should help harmonize detection and quantification of viral rna from pedv belonging to both s-non-indel and s-indel strains in both field and experimental settings. all commercial methods were performed according to the manufacturers' recommendations unless otherwise stated. an alignment of pedv n orf sequences that were available on the data base at the time of the study ( ) was made using mafft (katoh and standley, ) and the probabilities of the nucleotides at the priming zones defined by kim et al. ( ) (pednf : '-cgcaaagactgaacccactaattt- ', and pednr : '-ttgcctctgttgttactt-ggagat- ') were calculated using r (wagih, ) (fig. ). based on these probabilities forward primer mpednf ( '-cgcaaagactgaacccactaa- ') and reverse primer pednr were chosen (fig. ) . these primers were subsequently checked against n orfs of the s-indel and s-non-indel pedv strains circulating in europe (dastjerdi et al., ; grasland et al., ; hanke et al., ; martelli et al., ; stadler et al., ; steinrigl et al., ; theuns et al., ) . original cv , the pedv reference strain isolated in , was collected from perfused jejunum performed in and kept at - °c. this stock was named wtcv . wtcv was propagated in cell culture as previously described (hofmann and wyler, ) and was named cccv . a stock of cccv was produced as follows: x mm confluent monolayer of vero cells (atcc® ccl- ) were infected each with µl of . x tcid of cccv in infection media; emem (thermofisher scientific, france) supplemented with . % tryptone phosphate broth, . % yeast extract, % penicillin/streptomycin and µg/ml trypsin. after hours of infection, cells were subjected to three freeze thaw cycles and the culture medium was clarified by centrifugation at g for minutes. a total volume of l of supernatant was then centrifuged for four hours at g to pellet the virus. the pellet was then resuspended in ml of pbs. the infectious viral titer of cccv was determined by immuneperoxidase monolayer assay according kärber's method (kärber, ) . the virus stock solution was titrated by immuno-peroxidase monolayer assay to . x tcid /ml. four other pedv strains were used: three french field strains (pedv/fr/ / genbank accession number (gb acc) kr , pedv/fr/ / and pedv/fr/ / gb acc mn ), and one american strain (pedv/usa/ /iowa gb acc mf , kindly provided by dr p.gauger from iowa state university). nine other 'non-pedv' rna viruses were also used: one pig alpha-coronavirus (porcine respiratory coronavirus, prcv), and two gamma-coronaviruses (infectious bronchitis virus (ibv) gb acc fj ), turkey coronavirus (tcov) gb acc kr ) as well as other pig viruses: a pig artevirus (porcine reproductive and respiratory syndrome virus (prrsv), gb acc ky ), a pestivirus (classical swine fever virus (csfv)), three pig ortomyxoviruses (swine influenza viruses h ni, h n , h n ), and two swine dna virus, one circovirus (porcine circovirus type (pcv ) gb acc af ), and an asfavirus (african swine fever virus (asfv) bankit anses-mada ). jejunum and fecal samples were collected from both specific pathogen free (spf) pigs confirmed negative for coronavirus rna by deep sequencing and from pedv infected pigs positive for pedv rna. the pedv positive samples had been collected during previous experimental studies (gallien et al., a; gallien et al., b; gallien et al., ) . spf samples were used as negative controls or were spiked with pedv produced in vitro as described in section . . spiked spf samples were used for the validation of the method and are later referred to as 'infectious reference materials'. for each jejunum sample, mg were homogenized in ml of phosphate buffered saline (pbs) (merck, france) with mm stainless steel beads in a tissuelyserii (qiagen, france). samples were then clarified by centrifugation at g for minutes. for each fecal sample, ml was diluted in ml of pbs and vortexed for minutes before clarification by centrifugation as describe above. to determine the limit of quantification (lq) of the pcr and produce standard for quantification, a rna transcript was produced by in vitro transcription of the pedv wtcv n orf sequence. wtcv rna was extracted using trizol (thermofisher scientific, france). viral rna extract was subjected to reverse transcription using hexanucleotide primers and superscript iii reverse transcriptase (thermofisher scientific, france). reverse transcription was performed at °c for hour followed by enzyme inactivation at °c for minutes. to amplify the n orf, µl of rt were subjected to pcr amplification in µl reaction containing nm of primers ogvb -f (gtcggatccactttatggcttct) and ogvb -r (gtcctcgagatt gtttaatttccterror! reference source not found.), . units of platinum taq hifi (invitrogen, france), µl of x high fidelity pcr buffer, and mgso at a final concentration of mm. the pcr was performed as follows: °c for minutes for initial denaturation, cycles of °c for seconds, seconds at °c decreasing by . °c per cycle and then °c for minutes, follow by cycles of °c for seconds, °c for seconds and °c for minutes. amplified pedv n cdna was separated on % agarose gel and extracted using montage gel extraction kit (millipore, france). ng of extracted product were cloned in pcr -topo vector (invitrogen, france). plasmid dna was prepared using nucleospin® plasmid kit (macherey nagel, france). in vitro transcription was performed with maxiscript tm t transcription kit (thermofisher scientific, france) using µg of precipitated spei linearized n orfs plasmid. rna was purified with agencourt® rnaclean xp kit (beckmancoulter, france), and quantified using qubit® fluorometer (life technology, france, saint aubin). stock of in vitro transcribed rna was stored at - °c. number of molecular copies was calculated according the following formula: ) × . × rna transcript was diluted to molecules/ µl, aliquoted in µl, supplemented with µl of rnastable® (m, france) and dried in speedvac® vacuum concentrator (thermoelectron, france). the standard transcript was resuspended in ml in deionized nuclease-free water and then log serially diluted from to copies/ µl and stored at - °c. all rna extractions were performed using rneasy® mini kit (qiagen, france) with the following modifications. µl of sample mixture containing µl of sample, µl of an external exogenous control (eec) and µl of proteinase k were used as opposed to µl of sample alone as recommended by the kit. rna was eluted with µl of nuclease-free water and stored at - °c until use. eec used in this study was viral rna genome (mengovirus). reactions were carried out in an applied biosystems real-time pcr system, with power sybr tm green rna-to-ct tm -step kit (applied biosystems, saint aubin, france). the final pcr mix volume was composed of . µl of master mix ( x), . µl of enzyme mix, µl of rna template, primers mpednf and pednr at nm or nm, h o to final volume of µl. rt-pcr cycles were as follows: reverse transcription at °c for minutes, followed by °c for minutes, then cycles of °c for seconds, °c for minute, and a final melting curve analysis step as defined by the applied software v . . all sample amplifications with a melting temperature corresponding to the standard with a viral rna concentration equal to, or above to the limit of detection (lod) were considered positive. all of the following tests were performed using primers at nm. j o u r n a l p r e -p r o o f the analytical sensitivity and specificity were determined as described in the nf u - norm. all nucleic acid extractions from viruses listed in . were tested. five strains of pedv were tested for inclusivity, and eleven other virus for exclusivity, among which, four coronaviruses, five other rna viruses, and two dna virus, all known as pathogens in pigs. the diagnostic sensitivity and specificity were determined as described in the nf u - according to nf u - , lod is the last dilution of reference material that allows a detection of the target with a confidence level of %. n rna transcript dilutions were tested for the lod of the pcr. six points of a two-fold dilution series ranging from to . genome copies/ µl were analyzed in eight replicates. three independent assays were performed for rna transcripts (lodpcr). to determine the lod of the method, spf jejunum and fecal samples spiked with cccv from to - tcid /ml, were tested in two independent assays on a hundredfold serial dilution ranging from to and n transcripts equivalent/ µl, as infectious reference materials (lodjejunum or lodfeces). lod's were determined by probit calculation (finney and stevens, ) . according to nf u - , lq is defined as the lowest (lower lq, llq) and highest level (upper lq, ulq) between which, for each dilution, the statistical bias is under or equal to . log . the bias is the difference between the measured value and the theoretical value calculated by linear regression on all dilutions. uncertainty is calculated as the variance of calculated point plus the medium bias value. the statistical bias is defined as the medium of uncertainty. for the lq, seven points of a ten-fold serial dilution of n rna transcript were tested ( to ). ten independent assays were performed on four independent serial dilutions. the lq for organic matrices were calculated on results obtained for the lod assessment (hundredfold dilution from to ). pcr efficiency was evaluated by plotting the ct against an expected rna copy number in respect to the tcid /ml (data not shown) for infectious reference material or by qubit j o u r n a l p r e -p r o o f quantification for rna transcript. in agreement with the nf u - norm, an efficiency of - % was accepted. the forward primer of kim et al. (kim et al., ) (pednf) had perfect base pairing with of the ( . %) n orfs sequences. the forward primer designed in the current study (mpednf) which did not contain the last three bases of kim et al. ( ) had perfect base pairing with of ( , %) and of those that did not match at % only one had a mismatch at the last ' position ( fig. a) . sequence of the reverse primer (pednr) had perfect base pairing with of sequence ( . %) and those that did not match at % did not have any mismatches in the last three nucleotides of the ' end ( fig. b) . concerning the alignments with the european strains available after may , pednf had perfect base pairing with of n orfs sequences (fig. c) . mpednf had perfect base pairing with of sequences, those sequences that did not match at % only contained one mismatch and these were localized close to the ' end ( fig. c) . pednr had perfect base pairing with of sequences and only one single miss-match with the remaining sequence at the ' end. amongst the different viruses strains listed in . , only the pedv strains (cv , american field strain, and three french field strains) were positive. wtcv (ct = ), cccv (ct = ), all with a tm of . ± . °c which is the expected tm for the pedv sequence amplicon according to the in vitro transcription control. all the other viruses were negative. the analytical specificity and sensibility were both %. efficiency of the method, calculated by linear regression, was . % ± . ( . ) for rna transcripts, . % ± . ( . ) for spiked jejunum and . % ± . ( . ) for spiked feces. different concentrations of primers had no effect on the efficiency of the method (data not shown), however melting curve analysis showed the presence of primer dimers at nm and not at nm (figure ). the lod was determined at copies/ µl for the rna transcript, copies/ µl ( . x . tcid /ml for the spiked feces and copies/ µl ( . tcid /ml) for spiked jejunum (table ) . for every selected rna dilution tested, from to copies/ µl, bias enlarged of uncertainty were included in the norm limits (- . to . ) and statistical bias (mean of uncertainty) were < . log ( table ). the ulqs and llq were and copies/ µl respectively for all matrices. calculations were done when a minimum of out of results were positive for the lod and for all replicates for lq. all coefficients of variation (cv) were below the . limit given by the norm nf u - with . - . , . - . , . - . , for rna transcript, jejunum and feces intra-assay cvs respectively and . - . , . - . , . - . for rna transcript, jejunum and feces inter-assay cvs respectively (table ) . the diagnostic sensitivity was % at two and fourteen dpi, pedv viral rna were detected in all true positive pigs. the diagnostic specificity was % as all non pedv infected pigs were found negative all along all experiments. pedv is of global importance to the pig industry with many different strains and genotypes existing in different continents. after and the introduction of both s-indel and s-non-indel strains to north america and the resulting huge economic losses, the french ministry for agriculture classified ped caused by the s-non-indel virulent strains as a notifiable disease. thus there was a need for a reliable method for rapid, accurate and specific detection and quantification of a broad range of pedv strains and one that was completely validated according to french norm nf u - . many methods have been developed and used for pedv detection and quantification as previously reviewed (diel et al., ) such as direct viral isolation, but it is laborious, time consuming, and requires a reliable model for all possible strains. furthermore, many pedv strains cannot be isolated in vitro. many immuno-assay tests have been developed to detect viral proteins (ifa, blotting, elisa) but all these methods are time consuming, have a low sensitivity and reaction, and are subject to cross reactivity decreasing the specificity. for these reasons the current study focused on developing and validating a specific and rapid diagnostic test for the detection of pedv viral rna. basing this test on a taqman® multiplex rt-qpcr, published by kim et al. ( ) , we developed and validated a sybr tm green one-step rt-qpcr method. the development and validation of the complete method, including the steps of sample preparation, rna extraction, and rt-qpcr, were done according to the french standard nf u - . this norm is an adaptation to the french context of the manual of diagnostic tests and vaccines for terrestrial animals (international office of epizootics, ) and respects the criteria stated by the world organization for animal health (oie). these standards describe the validation criteria for a pcr method in animal health and allows the characteristics not only of rt-qpcr to be determined, but also of the complete method, including sample preparation and extraction. for this, fecal and jejunum samples were used as this material has previously been described as the best matrices for detection of pedv rna in animals (gallien et al., a) . validating the complete method in this way means that the method is applicable for both experimental and diagnostic purposes. in the current study the primers used by kim et al. in were refined by in silico analysis. n orf alignments of the priming site showed that the pednf forward primer of kim et al. ( ) had mismatches with several different pedv n orfs and that the last three nucleotides at the ' end only matched with . % of the sequences. removing these three nucleotides in primer mpednf allowed a % match with . % of international sequences and with . % of european strains. the method using the new coupled primers demonstrated sufficient sensitivity to detect all tested pedv strains (historical, s-indel and s-non-indel strains). although sybr tm green pcrs are characteristically less specific than probe based pcrs, the specificity of the method was % against all viral types tested. primer dimer formation, which are problematic for fluorescent dye based methods as they interfere dramatically with quantification, were eliminated by optimizing the primer concentration to nm. during validation, the sample preparation and rna extraction step were optimized by the addition of a proteinase k treatment step which allowed the statistical bias to be maintained in acceptable limits (< . log ). the statistical bias obtain with the proteinase k treatment confirms a correct reproducibility at all quantification points, and guarantees a near or equivalent lod ( and copies/ µl for feces and jejunum) for the different matrices than for the transcribed rna ( copies/ µl). in addition, the detection limit determined in this study ( . tcid /ml) is very similar to other rt-qpcrs ( . tcid /ml) (miller et al., ) . in conclusion, many pcrs have been developed to detect and monitor the presence of pedv, but, as yet to the authors' knowledge none have been developed with a complete validation according to a norm such as the french nf u - . this fully validated method is the first of its kind for pedv and should help harmonize detection and quantification of pedv viral rna in both field and experimental settings. nucleotide probabilities at each position are shown as coloured text above the alignments. red text in the alignment sequences represent a mismatch. sequences of primers are shown above the alignment (pednf, mpednf or pednr). pednr is shown as reverse complement. each line represents a hybridization sequence, the number of strains presenting this sequence is indicated to the left of the sequence. j o u r n a l p r e -p r o o f afnor nf u - - méthodes d'analyse en santé animale -pcr (réaction de polymérisation en chaîne) -partie : exigences et recommandations pour la mise en oeuvre de la pcr en santé animale afnor nf u - - méthodes d'analyse en santé animale -pcr (réaction de polymérisation en chaîne) -partie : exigences et recommandations pour le développement et la validation de la pcr en santé animale porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus virus-like particles associated with porcine epidemic diarrhoea porcine epidemic diarrhea virus among farmed pigs experimental infection of pigs with a new porcine enteric coronavirus, cv porcine epidemic diarrhea virus: an overview of current virological and serological diagnostic methods a table for the calculation of working probits and weights in probit analysis better horizontal transmission of a us non-indel strain compared with a french indel strain of porcine epidemic diarrhoea virus evidence of porcine epidemic diarrhea virus (pedv) shedding in semen from infected specific pathogen-free boars limited shedding of an s-indel strain of porcine epidemic diarrhea virus (pedv) in semen and questions regarding the infectivity of the detected virus complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france porcine epidemic diarrhea in europe: in-detail analyses of disease dynamics and molecular epidemiology propagation of the virus of porcine epidemic diarrhea in cell culture manual of diagnostic tests and vaccines for terrestrial animals : (mammals, birds and bees) beitrag zur kollektiven behandlung pharmakologisher reihenversuche mafft multiple sequence alignment software version : improvements in performance and usability multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy evaluation of two real-time polymerase chain reaction assays for porcine epidemic diarrhea virus (pedv) to assess pedv transmission in growing pigs first detection, clinical presentation and phylogenetic characterization of porcine epidemic diarrhea virus in austria outbreak of porcine epidemic diarrhea in suckling piglets complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium ggseqlogo: a versatile r package for drawing sequence logos changes to virus taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses the authors wish to thanks ms. cherbonnel-pansart for her help with afnor validation methodology, phd. le guyader for the furniture of the mengovirus and phd p.gauger for the s-indel strain furniture. this work was partially funded by "direction générale de l'alimentation" of the french ministry of agriculture (project n° - ). key: cord- - a pviol authors: kamilia, chtara; regaieg, kais; baccouch, najeh; chelly, hedi; bahloul, mabrouk; bouaziz, mounir; jendoubi, ali; abbes, ahmed; belhaouane, houda; nasri, oussama; jenzri, layla; ghedira, salma; houissa, mohamed; belkadi, kamal; harti, youness; nsiri, afak; khaleq, khalid; hamoudi, driss; harrar, rachid; thieffry, camille; wallet, frédéric; parmentier-decrucq, erika; favory, raphaël; mathieu, daniel; poissy, julien; lafon, thomas; vignon, philippe; begot, emmanuelle; appert, alexandra; hadj, mathilde; claverie, paul; matt, morgan; barraud, olivier; françois, bruno; jamoussi, amira; jazia, amira ben; marhbène, takoua; lakhdhar, dhouha; khelil, jalila ben; besbes, mohamed; goutay, julien; blazejewski, caroline; joly-durand, isabelle; pirlet, isabelle; weillaert, marie pierre; beague, sebastien; aziz, soufi; hafiane, reda; hattabi, khalid; bouhouri, mohamed aziz; hammoudi, driss; fadil, abdelaziz; harrar, rachid al; zerouali, khalid; medhioub, fatma kaaniche; allela, rania; algia, najla ben; cherif, samar; slaoui, mohamed taoufik; boubia, souhail; hafiani, y.; khaoudi, a.; cherkab, r.; elallam, w.; elkettani, c.; barrou, l.; ridaii, m.; mehdi, rihi el; schimpf, caroline; mizrahi, assaf; pilmis, benoît; le monnier, alban; tiercelet, kelly; cherin, mélanie; bruel, cédric; philippart, francois; bailly, sébastien; lucet, jc; lepape, alain; l’hériteau, françois; aupée, martine; bervas, caroline; boussat, sandrine; berger-carbonne, anne; machut, anaïs; savey, anne; timsit, jean-françois; razazi, keyvan; rosman, jérémy; de prost, nicolas; carteaux, guillaume; jansen, chloe; decousser, jean winoc; brun-buisson, christian; dessap, armand mekontso; m’rad, aymen; ouali, zouhour; barghouth, manel; kouatchet, achille; mahieu, rafael; weiss, emmanuel; schnell, david; zahar, jean-ralph; artiguenave, margaux; sophie, paktoris-papine; espinasse, florence; sayed, faten el; dinh, aurélien; charron, cyril; geri, guillaume; vieillard-baron, antoine; repessé, xavier; kallel, hatem; mayence, claire; houcke, stéphanie; guegueniat, pascal; hommel, didier; dhifaoui, kaouther; hajjej, zied; fatnassi, amira; sellami, walid; labbene, iheb; ferjani, mustapha; dachraoui, fahmi; nakkaa, sabrine; m’ghirbi, abdelwaheb; adhieb, ali; braiek, dhouha ben; hraiech, kmar; ousji, ali; ouanes, islem; zaineb, hammouda; abdallah, saousen ben; ouanes-besbes, lamia; abroug, fekri; klein, simon; miquet, mattéo; thouret, jean-marc; peigne, vincent; daban, jean-louis; boutonnet, mathieu; lenoir, bernard; merhbene, takoua; derreumaux, celine; seguin, thierry; conil, jean-marie; kelway, charlotte; blasco, valery; nafati, cyril; harti, karim; reydellet, laurent; albanese, jacques; aicha, narjess ben; meddeb, khaoula; khedher, ahmed; ayachi, jihene; fraj, nesrine; sma, nesrine; chouchene, imed; boussarsar, mohamed; yedder, soumaya ben; samoud, walid; radhouene, bousselmi; mariem, bousselmi; ammar, asma; cheikh, asma ben; lakhal, hend ben; khelfa, messaouda; hamdaoui, yamina; bouafia, nabiha; trampont, timothée; daix, thomas; legarçon, vincent; karam, henri hani; pichon, nicolas; essafi, fatma; foudhaili, nasreddine; thabet, hafedh; blel, youssef; brahmi, nozha; ezzouine, hanane; kerrous, mahmoud; haoui, saad el; ahdil, soufiane; benslama, abdellatif; abidi, khalid; dendane, tarek; oussama, ssouni; belayachi, jihane; madani, naoufal; abouqal, redouane; zeggwagh, amine ali; ghadhoune, hatem; chaari, anis; jihene, guissouma; allouche, hend; trabelsi, insaf; brahmi, habib; samet, mohamed; ghord, hatem el; habiba, ben sik ali; hajer, nouira; tilouch, najla; yaakoubi, sondes; jaoued, oussama; gharbi, rim; hassen, mohamed fekih; elatrous, souheil; arcizet, julien; leroy, bertrand; abdulmalack, caroline; renzullo, catherine; hamet, maël; doise, jean-marc; coutet, jérôme; cheikh, chaigar mohammed; quechar, zakaria; joris, magalie; beauport, dimitri titeca; kontar, loay; lebon, delphine; gruson, bérengère; slama, michel; marolleau, jean-pierre; maizel, julien; gorham, julie; ameye, lieveke; berghmans, thierry; paesmans, marianne; sculier, jean-paul; meert, anne-pascale; guillot, max; ledoux, marie-pierre; braun, thierry; maestraggi, quentin; michard, baptiste; castelain, vincent; herbrecht, raoul; schneider, francis; couffin, severine; lobo, david; mongardon, nicolas; dhonneur, gilles; mounier, roman; le borgne, pierrick; couraud, sophie; herbrecht, jean-etienne; boivin, alexandra; lefebvre, françois; bilbault, pascal; zelmat, setti-aouicha; batouche, djamila-djahida; mazour, fatima; chaffi, belkacem; benatta, nadia; sik, ali habiba; talik, i.; perrier, maxime; gouteix, eliane; koubi, claude; escavy, annabelle; guilbaut, victoria; fosse, jean-philippe; jazia, rahma ben; abdelghani, ahmed; cungi, pierre-julien; bordes, julien; nguyen, cédric; pierrou, candice; cruc, maximilien; benois, alain; duprez, frédéric; bonus, thierry; cuvelier, grégory; ollieuz, sandra; machayekhi, sharam; paciorkowski, frédéric; reychler, gregory; coudroy, remi; thille, arnaud w.; drouot, xavier; diaz, véronique; meurice, jean-claude; robert, rené; turki, olfa; ben, hmida chokri; assefi, mona; deransy, romain; brisson, hélène; monsel, antoine; conti, filomena; scatton, olivier; langeron, olivier; ghezala, hassen ben; snouda, salah; ben, chiekh imen; kaddour, moez; armel, anwar; youness, lafrikh; abdelhak, bensaid; youssef, miloudi; najib, al harrar; mustapha, amouzoun; noufel, mtioui; mohamed, zamd; salma, el khayat; ghizlane, medkouri; mohamed, benghanam; benyounes, ramdani; montini, florent; moschietto, sébastien; gregoire, emilien; claisse, guillaume; guiot, julien; morimont, philippe; krzesinski, jean-marie; mariat, christophe; lambermont, bernard; cavalier, etienne; delanaye, pierre; benbernou, soumia; ilies, sofiane; azza, abdelkader; bouyacoub, khalida; louail, meriem; mokhtari-djebli, houria; arrestier, romain; daviaud, fabrice; francois, xavier laborne; brocas, elsa; choukroun, gérald; peñuelas, oscar; lorente, josé-angel; cardinal-fernandez, pablo; rodriguez, josé-maria; aramburu, josé-antonio; esteban, andres; frutos-vivar, fernando; bitker, laurent; costes, nicolas; le bars, didier; lavenne, franck; devouassoux, mojgan; richard, jean-christophe; mechati, malika; gainnier, marc; papazian, laurent; guervilly, christophe; garnero, aude; arnal, jean michel; roze, hadrien; richard, jean christophe; repusseau, benjamin; dewitte, antoine; joannes-boyau, olivier; ouattara, alexandre; harbouze, nadia; amine, a. m.; olandzobo, a. g.; herbland, alexandre; richard, marie; girard, nicolas; lambron, lucile; lesieur, olivier; wainschtein, sarah; hubert, sidonie; hugues, albane; tran, marc; bouillard, philippe; loteanu, vlad; leloup, maxime; laurent, alexandra; lheureux, florent; prestifilippo, alessia; cruz, martin delgado maria; romain, rigal; antonelli, massimo; blanch, torra lluis; bonnetain, franck; grazzia-bocci, maria; mancebo, jordi; samain, emmanuel; paul, hebert; capellier, gilles; zavgorodniaia, taissa; soichot, marion; malissin, isabelle; voicu, sebastian; garçon, pierre; goury, antoine; kerdjana, lamia; deye, nicolas; bourgogne, emmanuel; megarbane, bruno; mejri, olfa; hmida, marwa ben; tannous, salma; chevillard, lucie; labat, laurence; risede, patricia; fredj, hana; léger, maxime; brunet, marion; le roux, gaël; boels, david; lerolle, nicolas; farah, souaad; amiel-niemann, hélène; kubis, nathalie; declèves, xavier; peyraux, nicoals; baud, frederic; serafini, micaela; alvarez, jean-claude; heinzelman, annette; jozwiak, mathieu; millasseau, sandrine; teboul, jean-louis; alphonsine, jean-emmanuel; depret, françois; richard, nathalie; attal, pierre; richard, christian; monnet, xavier; chemla, denis; jerbi, salma; khedhiri, wafa; necib, hatem; scarfo, paolo; chevalier, charles; piagnerelli, michael; lafont, alexandre; galy, antoine; mancia, claire; zerhouni, amel; tabeliouna, kheira; gaja, ali; hamrouni, bassem; malouch, abir; fourati, sami; messaoud, rihab; zarrouki, youssef; ziadi, amra; rhezali, manal; zouizra, zahira; boumzebra, drissi; samkaoui, mohamed abdennasser; brunet, jennifer; canoville, bertrand; verrier, pierre; ivascau, calin; seguin, amélie; valette, xavier; du cheyron, damien; daubin, cedric; bougouin, wulfran; aissaoui, nadia; lamhaut, lionel; jost, daniel; maupain, carole; beganton, frankie; bouglé, adrien; dumas, florence; marijon, eloi; jouven, xavier; cariou, alain; poirson, florent; chaput, ulriikka; beeken, thomas; maxime, leclerc; haikel, oueslati; vodovar, dominique; chelly, jonathan; marteau, philippe; chocron, richard; juvin, philippe; loeb, thomas; adnet, frederic; lecarpentier, eric; riviere, antoine; de cagny, bertand; soupison, thierry; privat, elodie; escutnaire, joséphine; dumont, cyrielle; baert, valentine; vilhelm, christian; hubert, hervé; leteurtre, stéphane; fresco, marion; bubenheim, michael; beduneau, gaetan; carpentier, dorothée; grange, steven; artaud-macari, elise; misset, benoit; tamion, fabienne; girault, christophe; dumas, guillaume; chevret, sylvie; lemiale, virginie; mokart, djamel; mayaux, julien; pène, frédéric; nyunga, martine; perez, pierre; moreau, anne-sophie; bruneel, fabrice; vincent, françois; klouche, kada; reignier, jean; rabbat, antoine; azoulay, elie; frat, jean-pierre; ragot, stéphanie; constantin, jean-michel; prat, gwenael; mercat, alain; boulain, thierry; demoule, alexandre; devaquet, jérôme; nseir, saad; charpentier, julien; argaud, laurent; beuret, pascal; ricard, jean-damien; teiten, christelle; marjanovic, nicolas; palamin, nicola; l’her, erwan; bailly, arthur; boisramé-helms, julie; champigneulle, benoit; kamel, toufik; mercier, emmanuelle; le thuaut, aurélie; lascarrou, jean-baptiste; rolle, amélie; de jong, audrey; chanques, gérald; jaber, samir; hariri, geoffroy; baudel, jean-luc; dubée, vincent; preda, gabriel; bourcier, simon; joffre, jeremie; bigé, naïke; ait-oufella, hafid; maury, eric; mater, houda; merdji, hamid; grimaldi, david; rousseau, christophe; mira, jean-paul; chiche, jean-daniel; sedghiani, ines; benabderrahim, a.; hamdi, dhekra; jendoubi, asma; cherif, mohamed ali; hechmi, youssef zied el; zouheir, jerbi; bagate, françois; bousselmi, radhwen; schortgen, frédérique; asfar, pierre; guérot, emmanuel; fabien, grelon; anguel, nadia; sigismond, lasocki; matthieu, henry-lagarrigue; gonzalez, frédéric; françois, legay; guitton, christophe; schenck, maleka; jean-marc, doise; dreyfuss, didier; radermacher, peter; frère, antoine; martin-lefèvre, laurent; colin, gwenhaël; fiancette, maud; henry-laguarrigue, matthieu; lacherade, jean-claude; lebert, christine; vinatier, isabelle; yehia, aihem; joret, aurélie; menunier-beillard, nicolas; benzekri-lefevre, dalila; desachy, arnaud; bellec, fréderic; plantefève, gaëtan; quenot, jean-pierre; meziani, ferhat; tavernier, elsa; ehrmann, stephan; chudeau, nicolas; raveau, tommy; moal, valérie; houillier, pascal; rouve, emmanuelle; lakhal, karim; gandonnière, charlotte salmon; jouan, youenn; bodet-contentin, laetitia; balmier, adrien; messika, jonathan; de montmollin, etienne; pouyet, victorine; sztrymf, benjamin; thiagarajah, abirami; roux, damien; de chambrun, marc pineton; luyt, charles-edouard; beloncle, françois; zapella, nathalie; ledochowsky, stanislas; terzi, nicolas; mazou, jean-marc; sonneville, romain; paulus, sylvie; fedun, yannick; landais, mickael; raphalen, jean-herlé; combes, alain; amoura, zahir; jacquemin, aemilia; guerrero, felipe; marcheix, bertrand; hernandez, nicolas; fourcade, olivier; georges, bernard; delmas, clément; makoudi, sarah; genton, audrey; bernard, rémy; lebreton, guillaume; amour, julien; mazet, charlotte; bounes, fanny; murat, gurbuz; cronier, laure; robin, guillaume; biendel, caroline; silva, stein; boubeche, samia; abriou, caroline; wurtz, véronique; scherrer, vincent; rey, nathalie; gastaldi, gioia; veber, benoit; doguet, fabien; gay, arnaud; dureuil, bertrand; besnier, emmanuel; rouget, antoine; gantois, guillaume; magalhaes, eric; wanono, ruben; smonig, roland; lermuzeaux, mathilde; lebut, jordane; olivier, andremont; dupuis, claire; radjou, aguila; mourvillier, bruno; neuville, mathilde; d’ortho, marie pia; bouadma, lila; rouvel-tallec, anny; rudler, marika; weiss, nicolas; perlbarg, vincent; galanaud, damien; thabut, dominique; rachdi, emna; mhamdi, ghada; trifi, ahlem; abdelmalek, rim; abdellatif, sami; daly, foued; nasri, rochdi; tiouiri, hanene; lakhal, salah ben; rousseau, geoffroy; asmolov, romain; grammatico-guillon, leslie; auvet, adrien; laribi, said; garot, denis; dequin, pierre françois; guillon, antoine; fergé, jean-louis; abgrall, gwénolé; hinault, ronan; vally, shazima; roze, benoit; chaplain, agathe; chabartier, cyrille; savidan, anne-charlotte; marie, sabia; cabie, andre; resiere, dabor; valentino, ruddy; mehdaoui, hossein; benarous, lucas; soda-diop, marième; bouzana, fouad; perrin, gilles; bourenne, jeremy; eon, béatrice; lambert, dominique; trebuchon, agnes; poncelet, géraldine; le bourgeois, fleur; michael, levy; camille, guillot; naudin, jérôme; deho, anna; dauger, stéphane; sauthier, michaël; bergeron-gallant, krystale; emeriaud, guillaume; jouvet, philippe; tiebergien, nicolas; jacquet-lagrèze, matthias; fellahi, jean-luc; baudin, florent; essouri, sandrine; javouhey, etienne; guérin, claude; lampin, marie; mamouri, ouardia; devos, patrick; karaca-altintas, yasemin; vinchon, matthieu; brossier, david; eltaani, redha; teyssedre, sonia; sabine, meyet; bouchut, jean-christophe; peguet, olivier; petitdemange, lucie; guilbert, anne sophie; aoul, nabil tabet; addou, zakaria; aouffen, nabil; anas, benqqa; kalouch, samira; yaqini, khalid; chlilek, aziz; abdou, rchi; gravellier, perrine; chantreuil, julie; travers, nadine; listrat, antoine; le reun, claire; favrais, geraldine; coppere, zoe; blanot, stéphane; montmayeur, juliette; bronchard, régis; rolando, stephane; orliaguet, gilles; leger, pierre-louis; rambaud, jérôme; thueux, emilie; de larrard, alexandra; berthelot, véronique; denot, julien; reymond, marie; amblard, alain; morin-zorman, sarah; lengliné, etienne; pichereau, claire; mariotte, eric; emmanuel, canet; poujade, julien; trumpff, guillaume; janssen-langenstein, ralf; harlay, marie-line; zaid, noorah; ait-ammar, nawel; bonnal, christine; merle, jean-claude; botterel, francoise; levesque, eric; riad, zakaria; mezidi, mehdi; yonis, hodane; aublanc, mylène; perinel-ragey, sophie; lissonde, floriane; louf-durier, aurore; tapponnier, romain; louis, bruno; forel, jean-marie; bisbal, magali; lehingue, samuel; rambaud, romain; adda, mélanie; hraiech, sami; marchi, elisa; roch, antoine; guerin, vincent; rozencwajg, sacha; schmidt, matthieu; hekimian, guillaume; bréchot, nicolas; trouillet, jean louis; besset, sébastien; franchineau, guillaume; nieszkowska, ania; pascal, leprince; loiselle, maud; sarah, chemam; laurence, dangers; guillemette, thomas; jacquens, alice; kerever, sebastien; guidet, bertrand; aegerter, philippe; das, vincent; fartoukh, muriel; hayon, jan; desmard, mathieu; fulgencio, jean-pierre; zuber, benjamin; soufi, a.; khaleq, k.; hamoudi, d.; garret, charlotte; peron, matthieu; coron, emmanuel; bretonnière, cédric; audureau, etienne; audrey, winters; christophe, duvoux; christian, jacquelinet; daniel, azoulay; cyrille, feray; aissaoui, wissal; rghioui, kawtar; haddad, wafae; barrou, houcine; carteaux-taeib, anna; lupinacci, renato; manceau, gilles; jeune, florence; tresallet, christophe; habacha, sahar; fathallah, ines; zoubli, aymen; aloui, rafaa; kouraichi, nadia; jouet, emilie; badin, julie; fermier, brice; feller, marc; serie, mathieu; pillot, jérôme; marie, william; gisbert-mora, chloé; vinclair, camille; lesbordes, pierre; mathieu, pascal; de brabant, fabienne; muller, emmanuel; robaux, marie-aline; giabicani, mikhael; marchalot, antoine; gelinotte, stéphanie; declercq, pierre louis; eraldi, jean-pierre; bougerol, françois; meunier-beillard, nicolas; devilliers, hervé; rigaud, jean-philippe; verrière, camille; ardisson, fanny; kentish-barnes, nancy; jacq, gwenaëlle; chermak, akli; lautrette, alexandre; legrand, matthieu; soummer, alexis; thiery, guillaume; cottereau, alice; canet, emmanuel; caujolle, marie; allyn, jérôme; valance, dorothée; brulliard, caroline; martinet, olivier; jabot, julien; gallas, thomas; vandroux, david; allou, nicolas; durand, arthur; nevière, rémi; delguste, florian; boulanger, eric; preau, sebastien; martin, ruste; cochet, hélène; ponthus, jean pierre; amilien, virginie; tchir, martial; barsam, elise; ayoub, mohsen; georger, jean francois; guillame, izaute; assaraf, julie; tripon, simona; mallet, maxime; barbara, guilaume; louis, guillaume; gaudry, stéphane; barbarot, nicolas; jamet, angéline; outin, hervé; gibot, sébastien; bollaert, pierre-edouard; holleville, mathilde; legriel, stéphane; chateauneuf, anne laure; cavelot, sébastien; moyer, jean-denis; bedos, jean pierre; merle, philippe; laine, aurelie; natalie, de sa; cornuault, mathieu; libot, jérome; asehnoune, karim; rozec, bertrand; dantal, jacques; videcoq, michel; degroote, thècle; jaillette, emmanuelle; zerimech, farid; malika, balduyck; llitjos, jean-françois; amara, marlène; lacave, guillaume; pangon, béatrice; mavinga, josé; makunza, joseph nsiala; mafuta, m. e.; yanga, yves; eric, amisi; ilunga, jp; kilembe, ma; alby-laurent, fanny; toubiana, julie; mokline, amel; laajili, achraf; amri, helmi; rahmani, imene; mensi, nidhal; gharsallah, lazheri; tlaili, sofiene; gasri, bahija; hammouda, rym; messadi, amen allah; allain, pierre-antoine; gault, nathallie; paugam-burtz, catherine; foucrier, arnaud; chatbri, bassem; bourbiaa, yousra; thabet, lamia; neuschwander, arthur; vincent, looten; beck, jennifer; vibol, chhor; amelie, yavchitz; resche-rigon, matthieu; pirracchio, jean mantzromain; bureau, côme; decavèle, maxens; campion, sébastien; ainsouya, roukia; niérat, marie-cécile; prodanovic, hélène; raux, mathieu; similowski, thomas; dubé, bruno-pierre; demiri, suela; dres, martin; may, faten; quintard, hervé; kounis, ilias; saliba, faouzi; andré, stephane; boudon, marc; ichai, philippe; younes, aline; nakad, lionel; coilly, audrey; antonini, teresa; sobesky, rodolphe; de martin, eleonora; samuel, didier; hubert, noemie; nay, mai-anh; auchabie, johann; giraudeau, bruno; jean, reignier; darmon, michaël; ruckly, stephane; garrouste-orgeas, maïté; gratia, elisabeth; goldgran-toledano, dany; jamali, samir; dumenil, anne sylvie; schwebel, carole; brisard, laurent; bizouarn, philippe; lepoivre, thierry; nicolet, johanna; rigal, jean christophe; roussel, jean christian; cheurfa, cherifa; abily, julien; lescot, thomas; page, isaline; warnier, stéphanie; nys, monique; rousseau, anne-françoise; damas, pierre; uhel, fabrice; lesouhaitier, mathieu; grégoire, murielle; gaudriot, baptiste; gacouin, arnaud; le tulzo, yves; flecher, erwan; tarte, karin; tadié, jean-marc; georges, quentin; soares, m.; jeon, kyeongman; oeyen, sandra; rhee, chin kook; gruber, pascale; ostermann, marlies; hill, quentin; depuydt, peter; ferra, christelle; muller, alice; aurelie, bourmaud; niles, christopher; herbert, fabien; pied, sylviane; loridant, séverine; françois, nadine; bignon, anne; sendid, boualem; lemaitre, caroline; dupre, celine; zayene, aymen; portier, lucie; de freitas caires, nathalie; lassalle, philippe; le neindre, aymeric; selot, pascal; ferreiro, daniel; bonarek, maria; henriot, stépahen; rodriguez, julie; taddei, mara; di bari, mauro; hickmann, cheryl; castanares-zapatero, diego; deldicque, louise; van den bergh, peter; caty, gilles; roeseler, jean; francaux, marc; laterre, pierre-françois; dupuis, bastien; machayeckhi, sharam; sarfati, celine; moore, alex; mendialdua, paula; rodet, emilie; pilorge, catherine; stephan, francois; rezaiguia-delclaux, saida; dugernier, jonathan; hesse, michel; jumetz, thibaud; bialais, emilie; depoortere, virginie; michotte, jean bernard; wittebole, xavier; jamar, françois title: proceedings of réanimation , the french intensive care society international congress date: - - journal: ann intensive care doi: . /s - - - sha: doc_id: cord_uid: a pviol nan introduction the study of the bacterial cartography in thoracic surgery is extremely important for the treatment of post-operative infections due to the severity of the underlying pathology, the fragility of patients after surgery in addition to the choice of the empiric antibiotic therapy. we led a prospective study following all the patients who underwent a pulmonary resection surgery for a period of months from january to july , jointly with the microbiology department, chu ibn rochd, casablanca. the bronchial secretions were collected by a protected distal bronchial sample using a (combicath) after the intubation. results during the period of the study, patients underwent a pulmonary resection, % for a neoplastic pathology. the medium age was years ± and % of our sample were male. % of our patients had smoking habits and of them had pulmonary tuberculosis, had repeated respiratory infections. the antibiotics used in pre-operative: % of beta-lactams; % of fluoroquinolones; % of macrolides. moreover, % of our patients were classified asa . of the obtained samples, were positive ( . %). the most frequently observed germs were the acinetobacter baumannii ( . %), pseudomonas aeruginosa ( . %), klebsiella pneumoniae ( . %), staphylococcus aureus ( . %). the acinetobacter baumannii was the most resistant germ ( % sensibility to carbapenem). these patients were followed until their d after surgery, of them developed a post-operative pneumonitis with cases of multi-resistant acinetobacter baumanii, of which deceased. conclusion pneumonitis after pulmonary resection are common and severe that's why it is necessary to establish a global prevention strategy mainly based on general patricians and pneumologists' awareness concerning the choice of the prescribed antibiotics, in order to avoid the spread of multi-resistant germs. introduction carbapenemase-producing enterobacteriaceae (cpec) are increasingly reported worldwide and constitutes a real challenge antibiotic for clinicians to preserve the bacterial ecology. its incidence has remarkably increased in our intensive care unit during the last years. the esbl spread has a major consequence in term of antibiotic choices. carbapenem antibiotic are regarded as the most effective treatment. however numbers of authors suggest that alternatives antibiotics (i.e. noncarbapenems) could be used in esbl-pe infections. there are some conflicting data regarding the use of alternatives in case of esbl-pe infections. moreover as far as we know, there are no data in icu. objectives the aim of this study was to describe esbl-pe infections in icu and therapeutic options chosen in these specific situations. patients and methods prospective multicentric observational cohort study conducted in volunteers icu. all consecutive patients hospitalized in icu with esbl-pe infection according to cdc definitions were included. severity of illness was defines according to bone criteria, saps ii and sofa. demographic datas, empirical and definitive antibiotic therapy (et and dt), clinical evolution, and outcome were recorded. in vitro antimicrobial susceptibility testing was performed by the disk diffusion method or the vitek system according to the guidelines of the antibiogram committee of the french microbiologic society. results during the study period patients with esbl-pe infection met eligibility criteria with respectively a median age and saps ii score of ( - ) and ( - ). the median sofa score at first day of antibiotic therapy and icu admission were ( - ) and ( ) ( ) ( ) ( ) ( ) ( ) ( ) respectively. the most frequent site of infection were respiratory tract ( %), urinary tract ( %) and abdominal ( %). the most frequent isolated species were: escherichia coli ( %), klebsiella sp ( %) and enterobacter sp ( %). respectively , and % patients had septic shock, severe sepsis and sepsis according to bone criteria. among esbl-pe, . % were carbapenem and . were blbi sensitive. among the whole population, ( %) patients received a carbapenems as et. ( %) received a dt with carbapenems and ( %) patients received an alternative dt. the most frequent reasons for maintaining carbapenems as dt were: antibiotic susceptibility tests ( % of cases), severity level ( % of cases) immunosuppression ( % of cases). the median length of icu stay after infection was respectively ( - ) and ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) days for carbapenems and alternatives dt (p = . ). the d mortality was % for patients with carbapenems dt and % for patients with alternatives dt (p = . ). surprisingly, there were no differences between the groups (carbapenems vs alternatives) in term of severity. conclusion alternatives are frequently used for esbl-pe infections in icu. in our cohort ( %) patients received antibiotics other than carbapenems regardless of the severity. introduction bacterial resistance to antibiotics is a common problem worldwide. in south america, this prevalence is reported to be the highest in the world. however, in french guyana, there is no data on the epidemiology of colonization and infection caused by extended spectrum b-lactamase producing enterobacteriaceae (esbl-pe). we conducted this study to investigate the prevalence of colonization with esbl-pe and subsequent icu acquired infection in french guiana. introduction the implementation of hemofiltration (hf) as a renal replacement therapy in septic shock patients requires the supply of large quantities of replacement solutions. these solutions are either industrially prepared in autoclaved expensive plastic bags (conventional hemofiltration, chf) or continuously provided in unlimited amounts at the dialysis machine directly from the water treatment plant to form the replacing solutions (on-line hemofiltration, olhf).the aim of our study was to evaluate the safety and effectiveness of on-line hemofiltration compared to conventional hemofiltration in septic shock patients. the investigative protocol was approved by the institutional ethics authorities and all patients or their legally authorized representatives provided written informed consent. it was a prospective, randomized, clinical study, including septic shock patients with acute renal failure. patients were randomized to receive either on-line hemofiltration (n = ) or conventional hemofiltration (n = ) for renal replacement therapy during days. hemodynamic monitoring was conducted by conventional devises, including: electrocardiogram and a radial arterial catheter for invasive arterial pressure every h during period study. we collected serum samples also every h (urea, potassium and sodium levels, troponin, hemoglobin, platelets, c-reactive protein and lactates). results the evolution of heart rate (hr), mean arterial pressure (map), biological markers were comparable between the two groups over time except a significant decrease in map in the olhf group compared to chf group only at h (p = . ) and h (p = . ) and a significant decrease in c-reactive protein level in the olhf group at h (p = . ). conclusion on-line hemofiltration seems to be a safe and reliable method of renal replacement therapy in septic shock patients. it may be associated with attenuated pro-inflammatory cytokine profile (c-reactive protein). none. introduction therapeutic plasma exchange (tpe) is crucial for the management of auto-immune diseases like thrombotic thrombocytopenic purpura or myasthenia gravis. tpe is performed either by centrifugation, with specific machines which are not routinely available in icus, or by using specific plasma separation membranes with widely spread in icus hemofiltration machines. regional citrate anticoagulation for tpe is well established with centrifugation but has been seldom described for membrane tpe. we are reporting the experience of our icu in this field. patients and methods retrospective study including all patients who received tpe with citrate regional anticoagulation between and in an -bed icu. tpe is performed solely in the icu in our institution. results patients were included. tpe was required for thrombotic microangiopathy ( patients), vasculitis ( patients), hyperviscosity syndrome ( patients), guillain-barré syndrome ( cases) and others ( patients) . mean saps score was [standard deviation (sd) . ] . tpe were performed, with a mean number of . (sd . ; range - ) tpe per patients. coagulation of the circuit of tpe occurred in ( %) patients. coagulation of the circuit occurred in . % ( / ) of the tpe. minor adverse events have been reported in two patients: one had a rash during the first tpe (no recurrence during the next tpes) and the other had paresthesia during the first two tpes (the calcium infusion was increased and there had been no recurrence during the next tpes). no serious adverse events related to citrate were observed. conclusion regional anticoagulation with citrate allowed us to perform tpe in patients, without significant adverse events. the rate of circuit coagulation was . % per tpe. none. introduction a reduced incidence of membrane thrombosis after injection of anti-thrombin (at) has been reported in septic patients with acquired deficit in at undergoing continuous hemofiltration. as this strategy was routinely performed in our unit until , we investigated its cost-effectiveness. patients and methods data about the use of hemofiltration, the consumption of at and hemofiltration devices during (period with routine use of at) and (period with use of at only if a membrane thrombosis occurred) were extracted from the administrative database of the institution. a decisional tree was built to modelize the impact of at on the consumption of hemofiltration devices and blood products. the decisional tree took into account the probability of membrane thrombosis with and without at and the probability of transfusion after membrane thrombosis. costs were obtained from the pharmacy of the institution (at, hemofiltration devices) and from the literature (blood products). results during , days of hemofiltration were performed, with the use of doses of at ( , €) and hemofiltration devices ( , €) . during , (− %) days of hemofiltration were performed, with the use of (− %) doses of at ( €) and (+ %) hemofiltration devices ( , €) . the mean cost of day of hemofiltration decreased from € to € with the diminution of the use of at. according to the decisional tree, at was almost never cost-effective. the only circumstances associated with a benefit for the use of at was the association of a probability of thrombosis with at inferior to . , of a probability of thrombosis without at equal , of a probability of transfusion after thrombosis equal and a cost of transfusion of €. in these extremely favorable circumstances, at could decrease the daily cost of hemofiltration of . - . €. discussion the model has several limits: the losses of utility related to transfusion and to interruption of hemofiltration due to thrombosis were not taken into account; the cost of at measurement was not estimated; the work load of changing a membrane and of transfusion after membrane thrombosis was not analyzed. conclusion our results suggest that anti-thrombin is not costeffective to reduce the costs of hemofiltration related to membrane thrombosis. none. introduction in intensive care unit (icu), some patients suffering from acute kidney injury need renal replacement therapy (rrt). it requires the circuit anticoagulation, this could be done by a regional citrate method. today, this is a recommended approach for the everyday care, even if the technique isn't widespread yet [ ] . the ionized calcemia dosing through the filter ("post-filter" ionized-calcemia) is used to monitor the technique efficacy, with a target of . - . mmol/l showing a good filter anticoagulation. the objective of our study was the assessment of efficacy and safety of our regional citrate anticoagulation protocol, with a less restrictive post-filter ionized calcemia target ( . - . mmol/l). the main goal was the analysis of the circuit lifespan, considering a lifespan above h, as well as the search of some clinical and biological factors affecting the technique efficacy. moreover, we analyzed the side effects incidence of the protocol (hypernatremia, metabolic alcalosis), and their consequences. the study received the scientific ethical agreement of university hospital of toulouse, and is registered with number - . patients and methods patients, admitted to one of the two university hospital icus of toulouse, needing a continuous rrt method, without any need for systemic heparin anticoagulation, and without severe hepatocellular failure, were included in the study. filters included over a -year period were analyzed. results results show a mean filter lifespan of h, with a lifespan above h for . % of all filters. coagulation was the cessation reason for . % of filters, most of them before h of the filter use. a value of post-filter ionized calcemia at day below . mmol/l was the main factor influencing a filter lifespan above h. an age older than and a saps ii severity score below were other factors conditioning a filter lifespan of more than h. side effects of citrate were rare and didn't have any clinical impact among our patients. discussion these results suggest that citrate used for anticoagulation in rrt could have an additional anti inflammatory effect through the induced hypocalcemia, as well as an energetic gain which could lead to a renal protection against ischemia-reperfusion mechanism [ ] . moreover, these results call into question the need of post-filter ionized calcemia dosing for the monitoring of citrate anticoagulation efficacy, since the method safety is monitored by the total-to-ionized calcium ratio. conclusion during continuous rrt in icu, a regional citrate anticoagulation protocol with a non-restrictive post-filter ionized calcemia target seems to be efficient and could reduce side effects. these results need to be confirmed with a randomised control study. introduction continuous veno-venous haemofiltration (cvvh) is used to treat acute kidney injury in critically ill patients. to optimize its efficiency, cvvh requires effective anticoagulation. systemic anticoagulation with standard heparin, the most used, can lead to major bleeding complications. hemofilters that are able to adsorb heparin molecules on their surface such as an st and oxiris membranes represent an alternative. the objective of this study was to compare these two types of filters in terms of duration, efficiency, dysfunctions and cost. materials and methods from october to may , we conducted a retrospective, observational, and non-interventional study. all patients admitted in the intensive care unit needing cvvh were included. the primary endpoint was the filter lifespan: an st versus oxiris. the secondary endpoint was the filter efficiency (urea reduction ratio: urr). the main analysis did not consider the anticoagulation type. we conducted a subgroup analysis taking into account the use or not of an anticoagulation. results sessions in patients were carried out using filters representing , h of treatment. the mean an st filter lifespan was ± h and ± h for oxiris filters (p > . ). there is no significant difference in terms of duration between the two filters. the subgroup analysis taking into consideration the use or not of anticoagulation did not show any difference either. the mean urr was ± % in the an st group and ± % in the oxiris group (p > . ). concerning the dysfunctions, there were no significant difference between the two filters. one hundred and seventy-six an st filters were used for a total cost of , euros. two hundred and ten oxiris filters were used for a total cost of , euros. conclusion the an st and oxiris lifespans are not significantly different. they were as efficient in terms of blood epuration and had as many dysfunctions. the use of an oxiris filter rather than an an st to extend the circuit's lifespan in the same clinical conditions is not justified considering the extra cost generated. introduction because oliguria is a poor prognostic sign in patients with acute renal failure (arf), diuretics are often used to increase urine output in patients with or at risk of arf. from a pathophysiological point of view there are several reasons to expect that loop diuretics could have a beneficial effect on renal function. however, a review of literature shows that the use of loop diuretics in patients with arf has been associated with inconclusive results despite the theoretical benefits [ ] . to assess the adjunctive effect of diuretics, to alter the progression to kidney injury or failure, in patients at risk for acute renal failure. patients and methods this is a retrospective chart review of consecutive patients who developed arf with oliguria in the intensive care unit. chart abstractors were well trained residents. two chart reviewers (senior intensivists) studied all the charts. an explicit protocol was used to precise all needed definitions. uniform handling of data was ensured especially for conflicting, missing or unknown data. oliguria was defined as urine output lower than . ml/kg/h for at least h. rifle score was assessed before and after urinary output normalisation. therapeutic intervention to optimize pre-renal perfusion was described. mean arterial blood pressure (mbp) before and after therapeutic initiation, oliguria duration, delay from oliguria onset to diuretic administration, delay from diuretic administration to urinary output normalisation were measured. results patients were studied over a years period. ] h. the delay from diuretic administration to urinary output normalization was [ . , ] h. after resumption of diuresis, rifle score was assessed as (patients without risk, %; r, %; i, %; f, % l, zero; e, zero) (fig. ) . increased serum creatinine level, above . fold normal range, was observed only in ( %) patients. conclusion rapid optimization of pre-renal hemodynamic disturbances associated with short delay administration of diuretics could significantly alter the progression to kidney injury or failure in at risk acute renal failure icu patients. the ventilator associated pneumonia (vap) is a common and severe complication of assisted ventilation. it's the leading cause of nosocomial infections in intensive care unit and remain responsible for a high morbidity and mortality because of the emergence of multidrug resistant (mdr) bacterial agent such us acinetobacter baumannii (ab). the aim of this study was to determine the incidence, risk factors and prognosis of ab vap. patients and methods retrospective study extending over a year period (january -january ) that included all patients over patients were divided into two groups: one consisting of patients who developed vap to ab and the second developed vap to another bacterial pathogen. results one hundred and forty patients developed vap. the incidence rate of ab vap was . % with a density of incidence of . per ventilator days. age, male gender, the time between hospitalization and mechanical ventilation and the medical pathology were risk factors for developing ab vap. ab was resistant to ceftazidime in %, to imipenem in %, tobramycin in % and netilmycin in . %, rifampin in % with a sensitivity to colistin in % of cases. the resistance of this germ to imipenem increased from % in to . % in . the evolution of patients with ab vap developed frequently septic shock compared to other patients ( vs . %; p = . ). the ab vap mortality was higher ( vs %; p = . ). conclusion the increasing incidence of multi-drug resistant ab vap is responsible for a high morbidity and mortality. so we need to identify risk factors and to strengthen the means of prevention of hand contamination and cross transmission during invasive procedures. introduction central line associated bloodstream infections (clabsi) are among the serious hospital-acquired infections. the aim of this study is to determine the incidence of clabsi, the pathogens and the risk factors that play a role in the development of bsi among patients followed in a tunisian medical intensive care unit. patients and methods all patients admitted for more than h were included in the study over a -year period in an -bed medical icu. the enrollment was based on clinical and laboratory diagnosis of bsi. blood samples were collected from catheter hub of all patients for culture, followed by identification and antibiotic sensitivity testing of the isolates. was higher compared with the mean rate of clabsi in icu reported by the nnis system surveillance for , which is . / catheter.days [ ] . duration of catheterization, frequent manipulation of catheter, catheter location, catheter type, underlying diseases, suppression of immune system, and types of fluids administered through the catheter are significant risk factors in development of bsis [ ] . in our study both duration of catheterization and number of attempts are independent factors for clabsi. conclusion in a monocenter cohort, clabsi had a moderate density rate but are associated with poor outcome. identifying the risk factors is necessary to find solutions for this major health problem. introduction according to some studies, field-intubated patients have . - times greater risk of ventilator associated pneumonia (vap). endobronchial intubation (ei) can be unrecognized by the physicians and may result in complications such as atelectasis which in turn could increase the risk of vap. the aim of our study was to confirm this hypothesis. patients and methods this monocentric retrospective study included all consecutive patients > years who underwent an out-of-hospital tracheal intubation before their admission to the intensive care unit (icu) between january and december . exclusion criteria were suspected aspiration or pneumonia on admission, patients who died within the first days of icu stay, extubation in less than h and underlying disease making radiological interpretation difficult for vap diagnosis. vap were divided into early onset (< days) and late onset (≥ days) events and were independently diagnosed by two experienced intensivists who had no access to the initial chest x-ray performed to check the position of the tracheal tube, based on the clinical pulmonary infection score. onset of ventilator associated tracheobronchitis (vat) was also noted. inadvertent endobronchial intubation was determined by another independent physician based on the interpretation of admission chest x-ray. results patients were intubated out-of-hospital. of the patients excluded, had an extubation in less than h, were died within the first days, had a suspicion of pneumonia, a suspicion of aspiration and an underlying disease making radiological interpretation difficult. of the patients included, ( . %) had an ei upon admission. no significant difference was observed between the ei and non-ei group for gender, age, saps , comorbidities and diagnostic category (cardiorespiratory arrest, trauma, coma and cardiorespiratory failure). early-onset vap were diagnosed in % in the ei group and in % of non-ei patients (p = . ). adding early onset vat, the respiratory infection rate was % in the ei group and % in the non-ei group (p = . ) (fig. ). late-onset vap were observed in . % in the non-ei group and . % in the ei group, without difference between groups (p = . ). there was no inter-group difference in the duration of ventilation, duration of icu stay and icu mortality. staphyloccocus aureus was the most prevalent pathogen in patients with early-onset vap ( . %, only one strain was methicillin-resistant). conclusion this study found a high rate of inadvertent prehospital endobronchial intubation with a higher incidence of early-onset vap. these results support the implementation of specific procedures to decrease the incidence of ei. introduction ventilator-associated pneumonia (vap) is associated with increased hospital stay and high morbidity and mortality in critically ill patients. the classic dichotomy between early and late onset vap is no longer helpful available. the aims of this study were to determine the incidence of multidrug-resistant pathogens in the first episodes of vap and to assess potential differences in bacterial profiles of subjects with early-onset versus late-onset vap. patients and methods retrospective cohort study over a period of months including all patients who had a first episode of vap confirmed by positive culture. subjects were distributed into groups according to the number of intubation days: early-onset vap (< days) or late-onset vap (≥ days).the primary endpoint was the nature of causative pathogens and their resistance profiles. results sixty patients were included, men and women. the average age was ± years. the igs at admission was . [ ; ] apache [ ; ] . monomicrobial infections were diagnosed in of patients ( %).two different bacteria were isolated in cases ( %). a. baumannii was the most frequently isolated in % (n = ) of patients; followed by p. aeruginosa in % (n = ), enterobacteriaceae in % (n = ) and s. aureus in % (n = ). the isolated bacteria were multidrug-resistant in most cases ( / ). the vap group comprised episodes ( %) of early-onset vap and episodes ( %) of late-onset vap. a. baumannii was isolated in % of early vap (n = ) versus % of late vap (n = ) (p = ns), p. aeruginosa in % of early vap (n = ) versus % of late vap (n = ) (p = ns) and enterobacteriaceae in % of early vap (n = ) versus % of late vap (n = ) (p = ns). for the resistance profile of the different pathogens isolated, there was no difference between early and late onset vap. conclusion according to new data from the literature, there were no microbiological differences in the prevalence of potential multidrugresistant pathogens or in their resistance profiles associated with early-onset versus late-onset vap. the bacterial nosocomial infection is a major cause of morbidity and mortality in burned. the bacterial ecology in an icu has a major impact in terms of morbidity and mortality, particularly in the center of burned or length of stay of patients is increased compared to a general intensive care. we conducted an observational study spread over months in icu for severe burned burnt including any who have spent more than h with nosocomial infection (modified cdc criteria), and in which all biological and bacteriological samples were taken. the different types of infections studied were: skin, urinary, lung and bloodstream infections. they excluded all patients belatedly supported or having stayed in other healthcare facilities. results one hundred twenty ( ) patients showed nosocomial infection during this period. the sex ratio (m/f) was . and the mean age was ± years. bacteremia was present in . % of cases, followed by the urinary tract infection that was present in . % of cases, followed by the cutaneous infection in . % of cases, and last pulmonary infection in % of cases. infection was polymicrobial in . % of cases. the main bacteria identified were: acinetobacter baumanii ( . %) of which % is resistant to imipenem, enterobacteriaceae ( . %), pseudomonas aeruginosa ( %) of which . % is resistant to ceftazidime and . % is resistant to imipenem, enterococcus ( %) and staphylococcus aureus ( . %). conclusion the incidence of nosocomial infection is very high compared to literature. the rate of resistance to common antibiotics is very high. a drastic management of antibiotics in our context, the selection of patients and the frequent use in the operating room for skincare allow a better management of these patients. introduction acinetobacter baumannii (ab) ventilator-associated pneumonia (vap) is common in critically ill patients. the aims of this study were to describing the epidemiological characteristics of ab-vap, to identify risk factors for acquisition and factors predictive of a poor outcome. materials and methods a retrospective-prospective study was conducted at the medical intensive care unit of the university hospital ibn sina, rabat-morocco from january to december . they were included in the study that all patients developed vap with identified germ. for identification of risk factors of acquisition of ab vap, two groups of patients were compared: patients with ab vap versus patients with vap caused by other germs. to identify factors associated with mortality, two other groups were compared: survivors versus died. results patients presented vap among which were caused by acinetobacter baumannii. among isolates of ab, . % were drug susceptible, and . % were multidrug-resistant while % were extensively drug-resistant. they were independent risk factors for acquisition of ab vap in multivariate analysis: the presence of a central venous catheter before the occurrence of vap, duration of prior hospitalization ≥ days and icu duration of stay ≥ days. the mortality rate of ab vap was %. the independent risk factors for poor outcome in multivariate analysis were: duration of antibiotic treatment > days, the reintubation and the presence of a previous hospitalization. discussion our data were similar to those of the literature with a high incidence of vap due to the ab ( %) and a high rate of resistance to this bacterium particularly to carbapenems. however, and compared to the literature, the vap ab were responsible for a death rate much higher ( %). conclusion our data were similar to those of the literature with a high incidence of vap due to the ab ( %) and a high rate of resistance to this bacterium particularly to carbapenems. however, and compared to the literature, the vap ab were responsible for a death rate much higher ( %). introduction ventilator-associated pneumonia (vap) is common in critically-ill patients. in fact, - % of patients requiring invasive mechanical ventilation develop this complication. the onset of vap has been reported to be associated with increased mortality. however, data related to critically-ill elderly patients are scarce. the aim of this study is to assess the prognostic impact of vap in critically-ill elderly patients. patients and methods mono-center, retrospective study conducted from / to / / . all old patients (age ≥ years) requiring mechanical ventilation were included. two groups were compared: patients who developed vap (vap (+) group) and those who did not develop vap (vap (−) group). results during the study period, patients were included. the causes of admission in the intensive care unit (icu) were shock (n = ), acute respiratory failure (n = ) and disturbed level of consciousness (n = ). diabetes mellitus, hypertension and chronic obstructive pulmonary disease were the most common comorbidities ( . , . and . % respectively). mean age was . ± . years. sex-ratio (m/f) was . . mean apache(ii) score was ± . the mean duration of mechanical ventilation was ± days. thirty patients ( . %) developed vap. icu-mortality was significantly higher in the vap (+) group ( vs . %; p = . ). multivariate analysis identified two independent factors predicting icu mortality: shock on admission (or = . , ci % [ . - . ], p < . ) and vap (or = . , ci % [ . - . ], p = . ). conclusion vap is common in critically-ill elderly patients and is associated with worse outcome. therefore, preventing its onset is of paramount importance. increased health-care costs. among pathogens responsible of vap, acinetobacter baumannii which is characterized by its ability to spread in the hospital environment and to acquire resistance leading sometimes to therapeutic impasses is associated with a particularly high mortality reaching - %. objective to describe the epidemiological characteristics of a. baumannii vap, to determine their prognosis and identify factors associated with mortality. patients and methods it is a monocentric observational study conducted over a period of years in a tunisian intensive care unit (icu) including mechanical ventilated patients for more than h with confirmed a. baumannii vap. results one hundred and twenty-three patients were included in the study. a. baumannii was responsible for % of vap in our icu. the vap were late in % of cases. more than % of isolates pathogens were resistant to ticarcillin, piperacillin, piperacillintazobactam, ceftazidime and ciprofloxacin. sixty percent of germs were sensitive to imipenem. resistance to imipenem has increased consistently from % at the beginning of the study to % in . all pathogens were susceptible to colistin. a. baumannii vap was complicated by septic shock in % of cases. the median duration of mechanical ventilation and of icu stay were (iqr: [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and days (iqr: - ) respectively. the use of parenteral nutrition was the only factor associated with the occurrence of a. baumannii vap resistant to imipenem (odds ratio . , % ci [ . - . ], p = . ). icu mortality was %. it was higher in patients with a. baumannii vap resistant to imipenem ( vs %, p > . ). in the multivariate analysis, the age, the use of renal replacement therapy and the occurrence of vap relapse have been identified as factors associated with mortality. conclusion a. baumannii resistance to imipenem became threatening. the use of parenteral nutrition was the only factor associated with the occurrence of a. baumannii vap resistant to imipenem. the choice of empiric antimicrobial for vap caused by this pathogen must take in consideration the epidemiologic data of each country and each icu. a. baumannii vap was associated with high mortality. the age, the use of renal replacement therapy and the occurrence of vap relapse have been identified as predictive of poor outcome. none. admission in intensive care unit for severe adverse drug event: what finding? julien arcizet , bertrand leroy , caroline abdulmalack , catherine renzullo , maël hamet , jean-marc doise , jérôme coutet introduction adverse drug events (ade) remain a serious public health problem. they represent between . and . % of hospital admissions and between . and . % of intensive care unit (icu) admissions. they are defined as any injury related to a drug, and include both adverse drug reactions, expected or not, but also underuse, overuse and misuse, unintended or undesired, preventable or not. indeed, mortality from iatrogenic event would rise between . and . %, whereas these ade that resulted in icu hospitalization could be prevented in . - . % of cases. these unplanned admissions overload icu, limit access to health care for other patients and have serious economic consequences for the health system. it is therefore necessary to study these ade to know their main causes and attempt to find a solution to avoid them. the main objectives of our study were to clinically and pharmaceutically analyze and stratify the different ade leading to hospitalization in our icu. this is a monocentric prospective study, between june to january , in medico-surgery icu. from all admissions, we had included patients admitted in our hospital for involuntary ade (plausible, likely and very likely causal). we had collected clinical aspects (failure mode, igsii score, mortality in icu) and pharmaceutical aspect (number of drug, offending drugs) at daily medical staff meeting. conclusion hospitalizations in icu for ade are still too common despite their preventability for most cases. many patients with known cognitive disorder manage their treatment themselves and this is probably one of the reasons of iatrogenic events. anticoagulants and antiplatelet agents, by side effects, misuse, underuse or overuse are very often involved. the onset of kidney failure from dehydration and the continuation of nephrotoxic and antidiabetic treatment also remain one of the most common causes. consequently, it is necessary to continue and develop primary, secondary and tertiary prevention strategies to prevent their appearance, to limit their consequences and to reduce recidivism. introduction intensive care unit (icu) is usually identified as a place of acute care, concentrated over a short period. for many reasons, a prolonged stay in the icu has a pejorative connotation for the intensivist physician. the aim of our study is to describe the epidemiological, clinical, paraclinical profile of patients hospitalized for a long time in icu (over days) and to identify the main prognostic factors and those that can predict the duration of stay in icu. we conducted a retrospective study, over a period of years and months (january to june ), enrolling patients whose length of stay was greater than or equal to introduction despite an improvement in prognosis of patients with hematologic malignancies for the last decade, mortality of such patients admitted to the intensive care unit (icu) remains high. yet, it seems that a first icu stay does not modify prognosis of the malignancy. until now, there is no data on readmission in the icu of such patients and its effect on short and long term prognosis impact. patients and methods this retrospective, single-center study conducted on a years period in the medical icu from our university hospital included patients with hematological malignancies admitted for a first stay. objectives were to evaluate the icu, day and months mortality, to identify prognostic factors associated with mortality within uni-and multivariate analysis, to evaluate readmission rate within the days after discharge, to indentify the admission risk factors associated with icu readmission and the prognosis factors associated with mortality during the second icu stay. multivariate analysis poor performance status, igs ii, hlh, mv and anti-fungal administration were associated with increased icu mortality, infections with pseudomonas were associated with higher day mortality. catheter related infections were associated with better icu survival and cr was associated with lower day mortality. of ( . %) candidate patients for icu readmission after a first stay were readmitted within the days following discharge. median overall survival was lower in readmitted versus non readmitted patients. months mortality was . % for readmitted versus . % for no readmitted patients (p < . ). the second icu stay mortality was . % and month mortality was . %. by multivariate analysis, only mv was associated with prognosis. the months mortality rate of patients who survived to the second icu stay was significantly higher than the patients who survived to the first admission but were not readmitted ( . vs . %, p = . ). conclusion main features, short and long term mortality and prognostic factors associated with icu admission are in lines with previous studies. early readmission rate was high with a negative impact on survival. despite admission in the icu of patients with hematologic malignancies seems not to affect long term prognosis, early readmission seems to have a pejorative impact on the course of the malignancy. introduction lung cancer is among all types of cancer, the most common solid tumour admitted in intensive care [ ] . recent studies showed that the prognosis of patients with lung cancer during intensive care unit (icu) stay has improved [ ] . the aim of our study was to determine the causes of icu admission of lung cancer patients, their prognosis and to identify factors predicting hospital mortality and survival after hospital discharge. in fact, temporary full-code icu management in patients with relapsed aml seems to be appropriate. none of the life-sustaining interventions at admission and on day were able to predict survival. an icu trial of days might not be enough to appraise precisely the outcome. bone marrow transplant was associated with a high mortality in our study. in case of relapsed aml with bmt, icu management is still challenging. the growing population of chronically critically-ill patients has a poor prognosis despite all the resources mobilised [ ] . our primary objective was to analyse the prognostic value of different definitions used to describe them. our secondary objective was to look for early clinical and biological factors that could be associated with the in-hospital mortality. we conducted an epidemiological prospective study in intensive care units (neurosurgical, cardiosurgical and medical) of a large french teaching hospital (henri mondor, créteil). we included all the patients hospitalized for at least days. we tested definitions: the prolonged mechanical ventilation, the definition taken up by kahn et al. [ ] , the prolonged length of stay, the persistent critical illness and the persistent inflammation-immunosuppression and catabolism syndrome. two biological examinations were performed: upon entering the study and week later. the study endpoint was the in-hospital mortality. results thirty patients were included between april and july . among them, only % matched the definition of prolonged mechanical ventilation, which is still the most used in the literature. further, it was not associated with the mortality, but the prolonged length of stay was, with % of these patients, that did not survive to their hospital stay. other parameters that were significantly different between the patients who died and those who survived were an advanced age, an elevated igs ii score at hospital admission, an elevated sofa score at study entry, a late healthcare-associated infection and several biological variables: a high c reactive protein, low albumin and prealbumin and a poor percent of monocytes expressing hla-dr, all measured at day . conclusion the in-hospital mortality of chronically critically-ill is still high. a prolonged length of stay is the only definition who may be helpful to identify the patients with the poorest outcome. among the early factors associated with mortality, we found a late healthcareassociated infection and a low percent of monocytes expressing hla-dr, pointing to the value of studying the immune system of these patients. introduction as a result of demographic transition, the proportion of «very elderly» (≥ years) patients is increasing worldwide and more of these patients are nowadays admitted to intensive care units (icu). among physicians the discussion about appropriateness of these icu admissions still remains controversial mostly due to questionable outcome, limited resources and costs. the aim of the study was to determine and evaluate the clinical characteristics and outcome in a very old population admitted to a medical icu in an urban teaching hospital. we present here a monocentric, retrospective and observational study. we reviewed the charts of all patients (≥ years) admitted to a medical icu between and ( years). we collected epidemiological, clinical and biological parameters and all therapeutic measures during the icu stay. a longterm survival follow-up was also performed. two hundred eighty-four patients were included for statistical analysis. multivariate cox regression was also performed to identify risk factors for -day outcome. results a total of patients were included, which represented . % of admissions to the icu during the period of the study. the mean age was . ± . years, the sex ratio was . . most of patients ( %) were admitted from the emergency department. % of these admitted patients suffered of previous dementia. the mean charlson comorbidity score was . ± . and the mean mccabe score was . ± . . the admission diagnosis in the icu was mainly respiratory distress ( %), septic shock ( %), cardiac arrest ( %) and coma ( %). the mean saps-ii score within h of icu admission was . ± . . half of these patients required support by mechanical ventilation (mean duration . days) and vasoactive drugs and % of patients received renal replacement. icu and in-hospital mortality rates were and % respectively. overall survival at months after hospital discharge was %. multivariate regression revealed necessity of catecholamines and mechanical ventilation as independent risk factors and urinary sepsis as protective factor for -day outcome. in fine, for % of these patients, a limitation of active treatment was decided (on average after days of stay). for all others there was no justification for limiting care because of a well-established treatment plan (with family, gp, icu team). conclusion the proportion of elderly patients remains low, but they are increasingly being treated in intensive care units. nevertheless, the in-hospital mortality is high compared to the average mortality in our icu over the same period ( %). the prognosis is often not as poor as initially perceived by physicians. the indication for icu treatment in our study was mostly justified; in the setting of consistent patient care and good clinical practice. it remains therefore appropriate to discuss every single icu admission of elderly patients without any restriction related to age. thus, the ongoing cluster-randomized trial of icu admissions for the elderly patients (ice-cub study) is deeply awaited to confirm or not these results [ ] . keywords intensive care; prognosis; outcome; elderly patients; over -years old. introduction regardless of the route of delivery, the postpartum hemorrhage (pph) is defined as blood loss ≥ ml after childbirth, and severe pph as blood loss ≥ ml. pph is the leading cause of maternal mortality in africa. the aim of this prospective study was to assess the quality of the initial management of pph in algeria in oran ehu and to determine the factors of care with the severity of this complication. we conducted a prospective cohort study between april and september at the ehu oran. all women who delivered vaginally and showed hpp including the suspected cause was uterine atony were included. the severe pph was defined as bleeding that required invasive surgical treatment (hysterectomy, arterial ligation), a transfusion, a transfer to an intensive care unit or death of the patient. the quality of care was evaluated using objective criteria defined by a delay of diagnosis and care and mortality. results among the women who delivered vaginally during the study period, had a pph, link with uterine atony alleged at diagnosis, of which presented signs of severity. in % of cases, the delay in diagnosis of pph was less than min; % of women received oxytocin within min after diagnosis. the tranexanique acid was used in case. the examination of the cervix, uterine exploration and uterine massage was performed in , and %, respectively. the failure of first line treatment involved % of patients. among them, the time between the diagnosis of pph and administration of blood derivatives was greater than h in a third of cases. the administration of oxytocin delay exceeds min multiplied by . the risk of severe pph. however we had deaths in our series. discussion in our study the optimal period of care was not adequate, obtaining blood derivatives in our institution remains among the factors aggravating among the main risk factors for pph, uterine atony was the main source of complication. bleeding postpartum aggravated in our two patients has led to the deaths from late diagnosis and care that was not optimal. these hemorrhages pp is the leading cause of mortality: % of obstetric deaths ( % in the confidential survey - ) [ ] . a hysterectomy was indicated after failure to conservative treatment. the death rate is estimated at % following a disorder complicated hemostasis of disseminated intravascular coagulation (dic). in some series, the mortality rate is estimated between and % [ ] . conclusion the management of pph in obstetrics gynecology service the ehu oran was not optimal. the issue of timing of diagnosis and initial treatment is crucial. solutions must be sought locally to ensure the administration of essential medicines in time, especially the injection of oxytocin within min after diagnosis. introduction chronic obstructive pulmonary disease (copd) is a common pathology that would represent the third cause of death worldwide by . its evolution is interspersed with episodes of acute exacerbations (aecopd) that may indicate an admission in intensive care unit in the most. objective to study the evolution of management modalities of patients admitted in our intensive care unit for aecopd, to determine their prognosis and to identify factors associated with mortality. patients and methods it is a retrospective, monocentric study, performed in a tunisian intensive care unit (icu) over a period of years. we including all patients admitted in icu for aecopd. parameters collected were demographic features, comorbidities, regular treatment, dyspnea assessed by the mrc scale, initial clinical severity reflected by saps ii and apache ii scores, modalities and icu admission deadlines, initial arterial blood gas analysis, management of patients in the icu (ventilation modalities, prescription of antibiotics, use of vasoactive drugs) and their outcomes (incidence of nosocomial infections and their sites, length of stay and icu mortality). results a total of patients, which represents . % of all hospitalizations, with mean age of years (iqr: - ) were admitted for aecopd during the study period. the mean saps ii and apache ii were respectively (iqr: - ) and (iqr: - ). of these, % were ventilated with niv whose overall failure rate was % with a significant decrease between the beginning and the end of the study ( vs % p = . ). sixty-four percent of patients received antibiotics at admission. the prescription rate of antibiotics has decreased significantly over the years from to %. the incidence of nosocomial infections was %. it remained steady between and %. their sites were pulmonary in % of cases. icu mortality was %. in multivariate analysis, icu admission deadlines, niv failure and the use of vasoactive drugs were identified as factors associated with mortality. conclusion our study showed the importance of aecopd in the activity of our icu. the management of these patients has evolved over the years, which was reflected by the significant decrease in the prescription of antibiotics and the enhancement of niv success rate. this result could be attributed to the combination of several factors: precocious management of patients, experience of the healthcare team and the use of efficient ventilators. icu admission deadlines, niv failure and the use of vasoactive drugs were identified as factors associated with mortality. introduction aim. investigate the effect of music therapy on the tolerance of non-invasive ventilation (niv) during its introduction. currently, % of the trauma are intubated. thirty-three percent of the patient admitted in intensive care suffers from acute respiratory distress syndrome (ards). the fmhs chose oxygen concentrator as oxygen source in addition to oxygen pressurized bottles. their supply can be uncertain in conflict areas. insufficient data are available concerning the use of oxygen concentrator in intensive care unit. the primary endpoint was to determine over the total duration of oxygen therapy, the number of days on which the use of pressurized oxygen was needed for patients oxygenated by oxygen concentrator. the secondary endpoints were to identify when pressurized oxygen was needed, describe the characteristics of the population with oxygen therapy and estimate the oxygen quantity economised thanks to the use of oxygen concentrator. the study took place in the forward surgical unit of bouffard. it's a french role located in djibouti republic in africa. all patients over admitted in the intensive care and needing oxygen therapy were included. all the patients were oxygenated with an oxygen concentrator. the oxygen concentrators used were sequaltm integra om, that could deliver up to l/min of normobaric oxygen. the ventilator used were pulmonetictm ltv and . results thirty-six patients were included over the months' study period. sixty percent of the patients were men with an average age of two hundred and fifty-one days represents the total number of days of oxygen therapy divided into days of invasive ventilation, days of noninvasive ventilation and days of oxygen mask. the use of pressurized oxygen was necessary times over the days of oxygen therapy which represents . % of the total time. the causes of its use were in ten cases ( . %) criteria of severe ards, in six cases an emergency intubation and in three cases a transfer. one dysfunction of an oxygen concentrator happened during our study. the oxygen concentrator produced m of oxygen over the study period, which represents oxygen pressurized bottles of litres. this enabled an economy of , euros. conclusion it is safe to use oxygen concentrator to take care of critically ill patients in limited resources environment. the use of pressurized oxygen is still compulsory in two situations: in case of electricity failure and in case of high fio (above %). oxygen concentrators are sufficient in . % of the time. they enable to deliver oxygen any time which is essential when supply is uncertain in conflict areas. none. table ). for the same mv and level of ofr, fdo was in our experiment, with an ofr of l/min, when ifr = l/min (mv = l/min and ti/ttot = . ), the fdo is equal to % (± %) (see table ). to this value of ifr, the fdo is in accordance with the formula of ats, but when ifr increase beyond l/min, the fdo decrease and the formula is not in accordance with ats. this can be explain because during inspiratory phase, air room (fractional oxygen = . ) entry in airway mixes with ofr (fo = ), which modifies the fdo . in this case, when ifr increase then fdo decrease and vice versa. medical and paramedical staff must be aware that with patients who receive ofr by nasal cannula, any change of ofr and/or inspiratory flow changes the fdo . in this case, for maintain the same fdo , it is necessary that modify the value of ofr. the actual fio delivered under oxygen mask in patients with acute respiratory failure and the factors that may influence the fio are poorly known. in clinical practice, different methods including formula or conversion tables based on oxygen flow can be used to estimate delivered fio . we aimed to assess first the factors influencing measured values of fio , and second the best method to estimate fio in patients breathing under oxygen mask. we included icu patients admitted for acute hypoxemic respiratory failure from a previous prospective trial [ ] in whom fio was measured under oxygen mask using a portable oxygen analyzer. we collected demographic variables and respiratory parameters that may influence measured fio . low fio was defined according to the median measured fio . for each patient, measured fio was compared to "calc + %" formula (fio = oxygen flow in liters per minute × . + . ) to "calc + %" formula (fio = oxygen flow in liters per minute × . + . ), and to a conversion table [ ] . a ± % limit of agreement for each estimation method was arbitrarily considered acceptable. results among the patients included, median measured fio was % [ - ]. after adjustment on oxygen flow, the three variables independently associated with low measured fio using multivariate analysis were patient's height, a low paco , and a respiratory rate greater than breaths/min. using paired analysis, each estimation methods differed significantly from measured fio (p < . for each). values outside the limits introduction acute hyperglycemia is common in intensive care. it was associated with poor prognosis and increased mortality. the purpose of our study is to investigate the frequency of hyperglycemia in our icu, to determine the main causes of high blood sugar and to analyze the impact of this hyperglycemia. our study is prospective during months. it was conducted in the intensive care unit of the university hospital habib bourguiba sfax-tunisia. were included in our study all patients admitted to the service during the period of the study. for each patient included were collected from the icu admission, clinical and biological data. results during the study period, patients were hospitalized in our icu and the diagnosis of hyperglycemia (> mmol/l) was admitted in patients ( %). the comparison between patients who developed hyperglycemia and those free hyperglycemia group showed that, the patients of the first group were significantly older (p < . ). additionally, hyperglycemic patients had more medical history including history of diabetes (p < . ), a higher saps ii (p < . ), a more significant frequency of active infections (p < . ). moreover, the presence of hyperglycemia was associated with shock (p < . ) and respiratory distress (p < . ). their evolution was marked by the significantly higher frequency of infectious complications (p < . ), thromboembolic complications (p < . ) and acute renal failure (p < . ). the average duration of mechanical ventilation and the length of stay were also significantly prolonged in hyperglycemia group patients (p < . for both). finally, the presence of hyperglycemia was significantly associated with a higher mortality rate. conclusion we concluded that hyperglycemia is correlated with poor prognosis of morbidity and mortality. but strict glycemic control remain controversial. thus, further studies on this subject will be recommended to define the exact place of glycemic control in intensive care. none. the rrt was prophylactic in four cases started when phophatemia was more than mmol/l, and therapeutic for renal failure and established tls in three cases. the median duration stay in icu was [ ] [ ] [ ] [ ] j. thirteen patients left the icu without major metabolic dysfunction. two patients deceased due to infectious complications. discussion monitoring of electrolytes was done on average, three times a day which is hard to do in onco-hematology unit. the early use of rasburicase and the aggressive iv hydration helped to prevent tls for seven patients. the aggressive iv hydration was made according to echocardiography data and close monitoring of vital signs and urine output which has allowed to avoid volume overload and acute pulmonary edema. the early prophylactic rrt prevented renal failure and metabolic complications. conclusion early management of tls in icu can prevent tls and most of its serious complications and should be considered in tls prophylaxis recommendations. none. the both urinary (expressed as the ratio of ngal on urinary creatinine) and plasma ngal were predictive of aki stage . predictive value of plasmatic measurements was higher than the urinary one (auc of . and . , respectively, p = . between auc), but not higher than either baseline serum creatinine (auc = . ) or h diuresis (auc = . ). backward multivariate regression showed that plasma ngal concentration was associated with serum creatinine, crp and albumin, whereas urinary ngal was associated with leucocyturia and baseline creatinine. discussion previous positive studies with ngal did not compare the performance of this costly biomarker with simple usual clinical parameters to predict aki. moreover, several parameters were associated with ngal concentrations with a high risk of collinearity (crp) and/or false positive results (leucocyturia). our data do not support any added value of ngal concentration over baseline serum creatinine or urine output to predict aki. introduction acute renal failure (arf) is a common entity in intensive care, concern that the heavy morbidity and mortality it is associated [ ] . early diagnosis of this entity remains difficult, neither diuresis and creatinine are early parameters in the diagnosis of arf. the kidney is an organ that suffers long to become faulty, the priority is to recognize renal aggression and to achieve a therapeutic allowing reversibility of the infringement. a number of markers have been developed for the diagnosis of the ira but costs remain high not allowing their routine use. the measurement of resistance index with the renal doppler could be a solution for the diagnosis of aggression and also of the etiology. the elevation of creatinine was seen later within h after the ir > . discussion in our series the resistance index has a value of early diagnosis of renal prognosis aggression in the occurrence and development of renal failure. renal doppler associated with a strictly applied standardized protocol achieves the two goals of monitoring who aid in the diagnosis and guide treatment. although the recommendations of experts to this tool provides that it should probably not use the resistance index measured by renal doppler to diagnose or treat an ira (grade ) [ ] . identifying the cause of kidney aggression is a prerequisite before any therapeutic action. hypovolemia and soda hydro overload are the causes principales. excess filling hyper intra thoracic pressure and hypoxia are the main causes of kidney congestion. conclusion doppler is an early renal medium in the diagnosis of renal aggression. a larger series could assert this observation. none. ), had significantly more pre-eclampsia, / ( %) versus / ( %) p = . . pe were started at an average of . days after foetal extraction, and with an average of sessions. patients of the pe group had significantly lower nadir of hemoglobin but also lower hemoglobin level at day and day . nadir of platelets count was also lower and level remain lower at days , , and . acute kidney injury (using kdigo classification) was more frequent with a higher rate of dialysis in icu, in the pe group ( / ( %) vs / ( %) p = . ) with a more frequent need for dialysis at the exit of icu. proteinuria was significantly higher in the pe group ( . mg/mmol vs . mg/mmol, p = . ). adamts dosage was done only in patients with pe. we find a diminution of adamts activity (before pe) with an average of % [ - ] in this group. there was no death, and adverse effects were not significantly different. discussion this study shows that pe was used when diagnosis was uncertain in the most severe form of pp-tma. low hemoglobin, low platelets, acute kidney injury and high level of proteinuria are the main factors associated with the decision to begin pe. this technique was safe and not associated with major adverse events. several studies show that there are physiopathological crossovers between diseases associated with pp-tma, for example low adamts activity in hellp or mutation in alternative complement pathway which induced hellp. moreover, studies and case reports show a benefit of pe in hellp syndrome. our study did not find significant difference in adverse events (maybe due to a lack of power), but this is another argument to discuss pe in the management of pp-tma in severe patients. the main limits of our study are that none of the patients who had a plasmatic exchange had a diagnosis of ptt and that diagnosis tests were not performed in all patients with pp-tma (complements level, adamts …). conclusion pp-tma treated with pe has lower hemoglobin, lower platelets, higher rate of kidney injury and proteinuria than those treated without pe. no difference were found for adverse events. begining of pe should be discussed for management of a pp-tma without amelioration after foetal extraction. none. introduction diffuse alveolar damage (dad) is the typical histological feature of acute respiratory distress syndrome (ards). however, in a previous study including patients with criteria for ards, we found that only % of them had dad at autopsy exanimation [ ] . it has been shown that patients with ards and dad on open lung biopsy had higher mortality than those without dad [ ] . thus, we aimed to identify markers associated with dad in patients with ards. we included the patients who met criteria for ards at time of death in our large database of clinical autopsies [ ] . we assessed the proportion of dad according to the severity of ards including the degree of hypoxemia and the ancillary variables from the berlin definition: use of high levels of positive endexpiratory pressure (peep at least cmh o), radiographic severity ( or quadrants on chest radiograph), altered respiratory system compliance (≤ ml/cmh o), and large dead space defined as a corrected expired volume per minute (≥ l/min). results dad was associated with all the severity markers abovementioned using univariate analysis. after multivariable logistic regression, the three markers independently associated with presence of dad were the gender with an odds ratio ( conclusion dad was significantly more frequent in females. in addition to the severity of hypoxemia, diffuse infiltrates involving the quadrants was a significant marker of dad. introduction ventilation induced lung injury (vili) is responsible for an increased mortality in ards [ ] . mechanical ventilation may trigger an inflammatory response, comprising alveolar macrophage activation and recruitment, which may be specifically, repeatedly and spatially assessed by functional imaging techniques such as positron emission tomography combined with computerized tomography (pet/ct) [ ] . c-pk is a pet radiotracer with potential to quantify macrophage inflammation. we aim to assess its performance to detect lung macrophage recruitment in an experimental highvolume vili model. materials and methods vili was performed in anesthetized pigs under neuromuscular blockade by rapidly increasing the tidal volume (vt) to obtain a transpulmonary pressure (tpp) between and cmh o under zero end-expiratory pressure. pet/ct acquisitions were performed before (t ) and after h of high-volume ventilation (t ), and image-derived measurements were realized on the whole lungs, and regionally on distinct lung regions (divided along the anteroposterior and the cephalocaudal axes). c-pk lung uptake was estimated using the standardized uptake value (suv), normalized to the ct-derived tissue fraction in the region of interest (roi). mechanical lung aggression was estimated by ct-derived dynamic and static strains, and tidal alveolar hyperinflation (expressed as a fraction of the tidal variation in the roi volume). after euthanasia, alveolar damage and macrophage recruitment were assessed in the lung regions, using semi-quantitative scores. results between t and t , vt and tpp significantly increased from . ± . to . ± . ml/kg and . ± . to . ± . cmh o, respectively. suv on the whole lung significantly increased from . ± . to . ± . between t and t and dynamic strain from . ± to . ± . , whereas static strain did not significantly vary. tidal alveolar hyperinflation significantly increased from ± to ± % on the whole lung between t and t . regionally, dynamic strain, and tidal alveolar hyperinflation significantly differed between regions, as well as between t and t . regional suv differed between t and t but not between regions. regional static strain did not differ between regions, nor between t and t . in multivariate analysis, regional suv was independently and significantly associated with dynamic strain and tidal alveolar hyperinflation. histologic analysis showed significant regional differences in alveolar damage but not in macrophage recruitment. suv was positively associated with macrophage recruitment but not with alveolar damage. discussion in this experimental vili model, c-pk suv was significantly increased after h of injurious ventilation, and was significantly and positively associated with high-volume ct-derived mechanical parameters, such as dynamic strain and tidal alveolar hyperinflation. the radiotracer's specificity for macrophages is confirmed by the suv significant association with macrophage recruitment and the lack of association with alveolar inflammatory edema. conclusion c-pk is a macrophage-specific pet radiotracer, with potential to dynamically and specifically assess alveolar macrophage inflammation induced by high-volume ventilation. research founded by the french society of intensive care medicine (srlf) and la fondation pour la recherche médicale (dea ). the reverse triggering (rt) is the term used to name the contractions reflexes of the muscle diaphragmatic provoked ("triggered") by the periodic insufflations, delivered by the ventilator, at sedated patients under mechanical ventilation [ ] . the rt constitutes a new form of patient-ventilator interaction clinically difficult to detect and little known. the rt could have potential implications during the management of acute respiratory distress syndrome (ards). at present, the management of severe ards consists among others, on the use of an early and systematic perfusion of neuromuscular blockade agents (nmba) during a h' period, continuation to the acurasys essay which showed a reduction of the mortality in the group of the severe ards patient receiving nmba. the reason of the beneficial effect of curare is not perfectly known. it is possible that the phenomenon of rt is a mechanism implied in the deleterious role of the mechanical ventilation during ards. the abolition of this phenomenon by nmba could explain the beneficial effect of nmba in ards [ ] . the objective was to look for the phenomenon of rt in two groups of ards patients: a group receiving nmba and a group not receiving nmba. patients and methods physiological observational and comparative study in intensive care units. we record continuous signals of airflow, airway pressure, and esophageal pressure during h of consecutives patients with ards criteria and pao /fio ratio ≤ at a positive end-expiratory pressure (peep) of cmh o evolving for less than h under mechanical ventilation. recording of esophageal pressure of consecutives moderate to severe ards patients were blinded analyzed (group nmba n = ; group unless nmba n = ). any phenomenon of rt was observed in the group of mild ards patients receiving nmba (fig. a) . we confirmed the existence of rt on patients of in the group of mild ards who not receiving nmba (p = . ) (fig b) . discussion one of the main limits was the quality of the collection of the signal of esophageal pressure. the monitoring of esophageal pressure is technically difficult, and can d influence the quality of the signal and the reliability of the results. conclusion this study confirms the existence of the phenomenon of reverse triggering among deeply sedated patients not receiving nmba with a % incidence. more research is needed to determine if the reverse triggering is a risk factor independent from vili, associated with the bad prognosis of severe sdra patients and, if a strategy of early treatment based on nmba, could improve the prognosis of reached patients. after ecmo removal had a significant median reduction of days in the bipap-aprv group, p = . (fig. ). we reported the feasibility of a protocol based on bipap-aprv aiming at resuming sv as soon as possible in ards patients under ecmo. the occurrence of spontaneous inspiratory efforts in ards patients can major variability of transpulmonary pressure and as result jeopardise vt and driving pressure control. this might be an issue if protective ventilation is not guaranteed anymore. vt with bipap-aprv remains within safe range when the ratio fig. circles are pac group, rhombus are aprv group. mv mechanical ventilation, psv pressure support ventilation. data are presented as median (iqr), comparison between the groups at each time mann-whithney test, *p < . of spontaneous minute ventilation to total minute ventilation is between and % [ ] . bipap-aprv is more efficient than psv to increase lung aeration in patients with ards [ ] . recruitment of dependent region is more likely to achieve if sv is not supported by synchronized positive airway pressure as during bipap-aprv [ ] . our strategy targeting a percentage of sv between and % with high peep could be viewed as a compromise in order to promote sv and protective ventilation at the same time. conclusion protective ventilation combined with sv under ecmo by using a specific protocol based on bipap-aprv is feasible and safe. it may facilitate weaning and thus reduce the time under mv after ecmo. to what extend this beneficial effect is directly due to the presence of sv deserve further investigations. introduction since the first transplant from a patient in a state of brain death conducted in at the university teaching hospital ibn rushd of casablanca, the number of transplants has increased. however, it is still inadequate meet the growing needs of organs. the refusal of families remains the main obstacle to the developpement of organ transplantation in morocco. the aim of our study is to monitor and analyse the evolution of family refusal to organ donation in a brain dead patient. patients and methods this is a retrospective and comparative study from august until december .the data were collected from records of brain dead patients candidates for organ donation at the intensive care units on ibn rushed hospital. the coordination registers were also studied. a questionnaire was distributed to families who refused organ donation to investigate the causes of the refusal. results during this period, patients with brain death have been identified and families had been approached. families ( %) refused organ donation. the main causes of refusal were: fear of body mutilation ( %), lack of will ( %) and religious causes in % of cases. the refusal rate for families decreased from % in to % in . only patients experienced cardiac arrest before transplantation. during this period, cornea transplants from braindead patient were conducted with kidney transplants and two liver transplants. discussion the evolution of the refusal of families saw a decline through awareness and communication campaigns for organ donation. conclusion improvements to our health care system must be proposed including strengthening detection of potential donors and relationships with the donor's family and effective communication policy. in the icu, three major actors are involved in the caring relationship: patient, relatives and caregivers. acting as spontaneous testimonials of the lived experience, thank-you letters from relatives may be considered by icu teams as a source of original information which could help in improving care for critically ill patients and families. this study aimed to investigate the qualitative content of thank-you letters from relatives of patients who stayed in the icu. specifically, our research questions were, with regards to the letters' content, ( ) how is the caring relationship tackled and characterized by relatives? ( ) to what extent does this relationship impact their experience of icu? materials and methods the study took place in a -beds icu during a -month period. the research team consisted in a care assistant, a nurse (also clinical research associate), a psychologist (not working in the icu) and an intensivist. the corpus consisted in twenty thankyou letters received in the icu. we conducted a qualitative study according to the thematic inductive approach. the process of coding was intended to create established meaningful patterns. results two main themes emerged as specific determinants of the caring relationship: ( ) the temporality, comprising the time dedicated to the patients and their family, the time spent with the icu team, the striking time corresponding to significant events for relatives needed to be shared with the staff, the extension of the link with caregivers by evocating a new life after icu stay, the writing time as a countergift to the caregivers; ( ) the caregivers behaviour, including human skills detailed in many core values (kindness, availability, devotion, attention, goodwill, sensitivity) psychological support, emotional sharing, capabilities to give informations. relatives feel to be "at the center of all attention" in the same way as their loved ones. through the narration of icu experience, the caring relationship is characterized as follows: ( ) the caregiver becomes a close person with an equal relationship (feelings of friendship, emotional closeness); ( ) the icu team becomes a new family (contrasting with the poor living environment of icus); ( ) the relative becomes a caregiver (with appropriation of medical terms or speaking of his loved one as a patient); ( ) the caregiver is seen as a "super-hero" through an asymmetrical relationship with an overstatement of personal dedication and investment of the staff members (abnegation, vocation, involvement). the caring relationship impacts relatives' experience of intensive care in several ways: ( ) relatives are deeply touched by caregivers' human behavior, emotional support being a source of solace and resilience in particular for bereaved families; ( ) relatives express the idea that taking care of humans is not a valued and rewarded task and the emerging awareness of hospital realities and difficulties of work in the icu; ( ) the most striking transformational change in relatives is the perception of their own vulnerability and humanity, leading them to exhibit an outward-looking attitude (for example filling out their organ-donation card), and encouraging the icu caregivers to continue their missions for the others. conclusion thank-you letters provide both encouraging and informative messages for icu teams about relational care for patients and families notably the indivisibility of the families and their critically ill loved ones. the relatives' experience of the icu appears strongly influenced by the caring relationship in the way they express an authentic revelation of their own humanity and altruistic thoughts. the thematic content of thank-you letters questions determinants and fundamental values at stake in the patient-relatives-caregivers relationship. introduction far from medical paternalism, the doctor-patient relationship has now evolved to respect "the autonomy and patients' rights". changing behavior has been gradual, while the law offered the patient the freedom to consent to care and then of expressing their wishes regarding the therapeutic intensity they would benefit, in critical situations where consent would not be possible, through advance directives (ad) [ ] . their use is of paramount interest for intensivist in many critical situations. unfortunately, the use of ad remains marginal because of the unfamiliarity of patients with their use and an appropriation default by clinicians [ ] . the aim of our study was to investigate the perspective of the coming family physician generation on advances directives. patients and methods population of interest was general practitioner fellow (gpf) from class of to . we built an online questionnaire survey about knowledge and the place they want to give to ad in their forthcoming daily clinical activity. this questionnaire was sent to gpf emails obtained by universities, unions and via the official mailing lists of different regionals classes provided by the first contacted. descriptive analysis of quantitative data was expressed as mean and standard deviation, qualitative data in number and percentage. the comparison of continuous variables was performed by the student t-test and the comparison of categorical variables by a chi test. analyzes were conducted on biostatgv website and microsoft excel ® . results gpf answered the survey, mainly from ile de france (n = ), toulouse (n = ) and lille (n = ). for gpf the majority of patients do not know the ad ( . %) and % think that those who know do not know how to use it. . % of gpf think writing ad by patients requires better information. according to them, the information should concern the support offered in the icu ( . %), the use of mechanical ventilation ( . %), dialysis ( . %) and the evolution of patients after hospitalization in icu ( . %). nevertheless information on the prognosis of chronic diseases or organ failure seems interesting for only and . % of them respectively. . % of gpf wish to propose the drafting of ad to their patients. however, only . % of them are willing to suggest ad to patients with cancer or hematologic malignancies, . % to patients with neurological and/or degenerative disorders, . % to elderly patients. discussion despite the low proportion of the population we think these observations to be of interest because we probably selected the gpf the most interested in ad as the participation was not mandatory. conclusion a large majority of young of future general practitioner is willing to be involved in the implementation of ad with their patients, however the target population remains very limited, considering that half of them do not want to discuss ad with patients suffering from diseases potentially associated with icu admission or therapeutic intensity discussion. this study was conducted in adult intensive care units in public or private hospitals in four countries: canada, france, italy, spain. in each country, health care professionals were solicited for an exploratory interview about the sources of stress in the work environment: senior physicians, residents, experienced nurses (with more than years of experience in the service) and inexperienced nurses (with less than years of experience in the service). all the interview transcripts were analysed using an inductive coding approach. results one hundred and sixty professionals ( physicians and nurses) were included in the study. eight themes emerged from the analysis, and they concerned the stress linked to ( ) patient ( ) care, ( ) team, ( ) family, ( ) institutional context, ( ) environment, ( ) organizational context, ( ) individual dimensions. in each theme, sub-themes have been identified and determine more precisely the difficulties at work. discussion our findings emphasize the complexity of work in icus and show the specifics factors not taken into account in the generic stress scales such as stress in relation with family relationships, the end of life decisions and inequity of health care. conclusion the specific stress scale should allow to better identified stress in icu and to develop measures of prevention and support and training programs. introduction intensive care units (icu) is a place where caregivers face many constraints that can affect their physical and mental health due to the use of specific care and strong emotional charge related to patient death and pain of the families. the aim of the present study is to detect anxiety disorders and/or depression among staff working in icus. on september , a questionnaire was distributed to staff (medical and paramedical) operating in icus in the university hospital fattouma bourguiba monastir, tunisia ( medical icu, surgical icu, cardiologic ccus and nephrologic intermediate care unit). this questionnaire included demographic data of participants (age, sex, marital status, length of service, psychiatric history, consumption of anxiolytic and/or antidepressant) and the hospital anxiety and depression scale (had: scale composed by items to screen the anxiety (a) and/or depression (d) among hospital staff ). results during the study period, participants completed the questionnaire ( %), % of them were women, the median age was years ± . . forty-nine participants were doctors (the majority of them residents: / ). . % of participants (all paramedics) worked on night shift, seniority of more than a year in the icu was found in % of participants. . % of staff interviewed were married and only . % of them reported consumption of anxiolytics and/or antidepressants. . and . % of the participants had respectively symptoms suggesting anxiety and depression. the median had score was (iqr = ); the medical function seems to be significantly associated with the occurrence of symptoms of anxiety and depression compared to paramedics, however the type of icu (medical/surgical icus vs cordiologic/nephrologic icus) does not appear to be related to the occurrence of symptoms of anxiety or depression (table ) . conclusion anxiety and depression are common symptoms among caregivers in icus. improved conditions of work in these units should be a target to avoid burn out syndrome. none. anxiety, n (%) depression, n (%) introduction carbon monoxide (co) poisoning is one of the common causes of poisoning specially in the cold season, which leads to a significant morbidity and mortality. we retrospectively reviewed the medical data of patients who presented to the toxicology emergency department with co poisoning during january to march . we analyzed patients' characteristics, management, and outcomes. results a total of six hundred and sixty-six patients ( female and male), aged of ± years, were included; poisoning occurred between december and february in % of cases, secondary to an indoor heating system exposure in the majority of cases ( %). the estimated duration of exposure was . ± h [ . - h], with a mean carboxyhaemoglobin (cohb) level on arrival at . ± %. neurological changes were the most presenting symptoms including headache (n = , %), dizziness (n = , %), seizure (n = , . %) and loss of consciousness (n = , . %). digestive disorders involving vomiting and nausea were observed in . % (n = ). one woman without cardiovascular risk factors developed non stsegment elevation myocardial infarction complicated by lung edema. the majority of patients (n = , %) received normobaric oxygen during h (n = ) and h (n = ). hyperbaric oxygen therapy was administered at . ata during h to patients for neurological changes (n = ), pregnancy (n = ) and elevated cohb ≥ % (n = ). mechanical ventilation was required for patients, and admission into intensive care unit in patients ( %). death occurred in cases ( . %). conclusion the carbon monoxide poisoning is a common reason for emergency department visits in winter. the physician should be aware of the serious neurological and cardiovascular complications, if symptomatic treatment and oxygen therapy regimens were not respected. none. neuro-respiratory toxicity of baclofen in the rat: study of the concentrations/effects relationships and role of gabaergic introduction baclofen, a gaba-b receptor agonist is used as muscle relaxant agent and recently for the treatment of alcohol dependence. the number of poisonings has significantly increased since this new indication. clinical presentation of poisoning mainly includes sedation, hypotonia, respiratory depression and seizures. to characterize the neurorespiratory toxicity of this molecule at high doses, we aimed at investigating alterations in sprague-dawley rat ventilation and brain electrical activity after baclofen administration and studied their reversal by gaba-receptor antagonists. materials and methods rat ventilation was investigated using plethysmography and arterial blood gas analysis while brain electrical activity was studied using eeg with one implanted frontal electrode. three baclofen doses were used including . mg/kg ( % lethal dose- %), . mg/kg ( %) and mg/kg ( %). baclofen concentrations were obtained using hplc-msms assay. we modeled baclofen pharmacokinetics and analyzed the doses/effects and effects/concentrations relationships. results baclofen induced early-onset and prolonged dosedependent sedation (p = . ), hypothermia (p = . ), eeg and respiratory depression ( . ) characterized by significant increase in the inspiratory (p = . ) and expiratory times (p = . ). significant increase in paco and decrease in arterial ph and pao were observed at mg/kg (p = . ), peaking at min. eeg showed signal slowing, burst-suppression aspects and spikes peaking at - h post-injection without normalization at the end of the experiment at h. we did reverse baclofen-induced decrease in tidal volume with saclofen (a gaba-b receptor antagonist) and interestingly no alteration of baclofen-induced respiratory depression was observed with flumazenil (a gaba-a receptor antagonist). pharmacokinetic parameters of baclofen were obtained at the three doses and were dose-dependent. significant but non-linear relationships were observed between baclofen-induced effects and concentrations. conclusion baclofen causes dose-dependent neurorespiratory toxicity in rats. however, due to increased poisonings, its safety profile at high doses remains to be established in humans. none. poisoning was deliberate in % of cases. mean ingested dose was . ± mg. the majority of patients presented to the emergency room at . ± h after ingestion. digestive decontamination was performed in . % (n = ) of patients. clinical presentation was dominated by neurological symptoms; including coma (n = ), hypotonia (n = ), hyporeflexia (n = ), agitation (n = ), seizures (n = ) and delirium in case. hemodynamic manifestations included bradycardia in patients, three of them required atropine infusion. one patient presented with hypotension responding to vascular resuscitation. sixteen cases required mechanical ventilation. aspiration pneumonia was noted in cases. mean duration of ventilation was . h ± . mean hospital length of stay was h ± . complications included ventilation associated pneumonia in one case and moderate rhabdomyolysis in cases. all patients evolved favorably. there is no correlation between coma and assumed ingested dose. conclusion baclofen overdose causes mainly neurological effects and except for bradycardia cardiovascular effects were uncommon. prognosis is good if full supportive care is administered properly. none. introduction the lack of an effective treatment for the maintenance of abstinence from alcohol has led physicians to take an interest in baclofen. beyond efficacy, safety of baclofen, prescribed in high doses, is a concern, especially in case of drug overdose. indeed, patients with chronic alcohol abuse frequently develop psychiatric disorders, and are at risk of voluntary drug intoxications. thus, we set up a retrospective study to describe morbidity and mortality associated with baclofen overdose. conclusion baclofen, prescribed in high doses, may lead to severe intoxications: self-poisonings frequently require endotracheal intubation and are associated with an increased risk of death. dialysis decreases baclofen elimination half-time but clinical relevance of this difference could not be determined. none. introduction baclofen, a gaba-b receptor-agonist with muscle relaxant properties established since , has been recently used at elevated doses to treat dependence to ethanol. the number of prescriptions has exponentially increased without an exact evaluation of its toxicity. we aimed to describe acute baclofen poisoning requiring intensive care unit (icu) admission and study the relationships between the toxic encephalopathy and the plasma baclofen concentration. we conducted a single-centre retrospective study including all baclofen-poisoned patients admitted to the icu in - . when requested by the clinical situation, repeated electroencephalograms and measurements of the plasma baclofen concentrations were performed. toxic eeg encephalopathy on a scale of zero to five was graded according to the international rating system (markand, ). plasma baclofen concentration was determined using liquid chromatography coupled to mass spectrometry in tandem developed with a quantum ultra apparatus (thermo fisher scientific) and electrospray source ionization in positive mode (limit of quantification: ng/ml). linear regression and chi- or mann-whitney tests were used as requested for subgroup comparisons. baclofen pharmacokinetics and the relationships between the toxic encephalopathy and the plasma baclofen concentration were modeled using winnonlin software v. ) were closed to the observed values reported at therapeutic doses. the relationship between baclofeninduced encephalopathy as a function of the baclofen concentrations was described using a sigmoidal emax model. conclusion baclofen poisoning may be life-threatening. toxic encephalopathy is well-described with eeg and its grade correlated to the baclofen concentration. prescribers should be aware of the dangers of baclofen which benefits to treat dependence to alcohol are still lacking. none. results initial examination suggested that an illness other than bacterial meningitis was the cause of patients' complaints. first hypothesis was meningitis receiving uncomplete dosage regimen of antibiotics. thereafter owing to apparent loss of consciousness with abnormal eyes movements, non-tonico-clonic seizures were considered meanwhile. the ratio of individuals less y-o to those equal to and greater was / %. the male to female ratio was / %. the mean duration of hospitalisation was . ± . days (extremes - days). extrapyramidal syndrome predominant on the upper part of the body was noted by paediatrician neurologists who suggested considering a genetic disease. however, signs and symptoms were present in people from different families in different areas at the same time. the definitive diagnosis made on pictures and videos of children and adults and was facio-troncular dystonia resulting from drug-induced adverse effect. four urine samples were collected in children and sent to a toxicological laboratory in france. all urine samples were positive for haloperidol meanwhile the other causes of facio-troncular dystonia were excluded, including other neuroleptics, metoclopramide, antidepressants, amodiaquine, anti-histaminic drugs, anti-epileptics, and cocaine. from january to august , hospitalisations were recorded in patients. looking for the source of haloperidol showed that tablets sold as 'diazepam' and consumed by symptomatic patients contained haloperidol as the sole active pharmaceutical ingredient, suggesting that this large outbreak was due to haloperidol toxicity from falsified diazepam. initial treatment was diazepam to relieve severe facio-troncular dystonia which was efficient but resulted in long-lasting sedation more especially in children. a dosage regimen using bipéridène administered by intravenous and oral route was refined to prevent adverse effects related to this anticholinergic agent used in children. the complete reversal of the facio-troncular dystonia was the antidotal evidence supporting the toxicological diagnostic. the mortality rate was less than % meanwhile the direct causal relationship with adr is questionable. an epidemiological study, including toxicological analysis in controls in ongoing. indeed, facio-troncular dystonia induced by haloperidol does not result from a drug overdose but is an adr occurring in about % of patients treated with haloperidol. who is involved in the inquiry related to this counterfeature involving different countries. the cause of the error is presently under investigation. discussion this outbreak emphasizes the need to consider toxicity resulting from counterfeatured medicines when facing collective atypical signs and symptoms in countries with unrestricted access to medication with limited control of qualities of the medicinal drugs. conclusion counterfeatured medicinal drug may result not only in poor efficacy but also in onset of unexpected outbreak of unknown diseases that should suggest a toxic origin. in late -early , médecins sans frontières (msf) had to face an outbreak of severe facio-troncular dystonic syndrome (ftds) in north-east congo. this outbreak resulted from counterfeature of pills sold as diazepam. toxicological analysis revealed one pill contained about mg of haloperidol. ftds induced by haloperidol does not result from a drug overdose but is an adverse drug reaction (adr) occurring in about % of patients treated with haloperidol. nine-hundred and twenty-five individuals were admitted in msf structures for ftds. the ratio of individuals less than y-o and equal to or greater of age was / %, including ( . %) of children less than y-o. initial treatment was based on diazepam which relieved ftds but resulted in long-lasting sedation, preventing given any drug by the oral route. owing to the definitive diagnosis, a shift to the use of a more specific antidote was chosen. biperiden was selected as existing in the intravenous and oral form in the swiss pharmacopea. the study was approved by the ethical committee of the ministery of health of the republic democratic du congo. patients and methods as a whole, biperiden was used in cases ( % of the total). treated children presented with severe dystonia as evidenced by inability to cooperate and to swallow. verbal informed consent was obtained from relatives. the dosage regimen to treat drug-induced dystonic syndrome in the swiss pharmacopea is as follows: for parenteral use in children, intravenously or intramuscularly: . mg/kg or . mg/m bsa every , according to response and tolerance; a maximum of four doses per day should be used. the internal msf recommendations for biperiden use in children were . - . mg/kg of body weight that might be repeated four times a day. initially, biperiden administration was administered under medical supervision by the msf referent at the scene. results there was no pediatric preparation of biperiden. accordingly, the adult preparation was used in children. the preparation contained mg of biperiden in one milliter of solvent. the initial planned dose for children of y-o and less and those up to y-o were and mg, respectively. the mg ( ml) of biperiden was diluted in ml of saline resulting in a final dilution of mg/ml. six children were treated according this dosage regimen. however, the one mg dose was either of limited efficacy while being associated in others of signs suggestive of adr, including agitation, heart rate greater than b/ min, the upper limit for children aged of y-o and less. two children greater than y-o presented severe abnormal behavior resulting in an attempt at escape. owing to question about safety, the dosage regimen was changed, as follows: mg ( ml) of biperiden was diluted with ml of saline resulting in a final dilution of . mg/ml. an initial dose of . mg was administered intravenously as a bolus dose. the effects were looked for over min. in the absence of improvement in facial dystonia, a second bolus dose of . mg was administered, a third dose could be considered min later if the ftds did not resume. the cumulative initial dose should not be greater than mg. in addition to the reversal of facial dystonia, the therapeutic effect of biperiden included the return of swallowing to normal allowing to give further doses of biperiden by the oral route for three days. the first oral dose was administered no less than h after the last initial dose at a dose equal to the efficient initial cumulative dose. the following doses were halved every h. no adr related to biperiden were reported using this dosage regimen. the mean duration of hospitalisation was . ± . days. discussion the bioavailability of biperiden by the oral route is equal to %. accordingly, the corresponding intravenous dose should be divided by a factor three. dosage regimen of anticholinergic drugs in children are poorly documented. the dosage regimen recommended by the pharmacopea resulted in frequent and severe adr. titration of biperiden resulted in efficient and safe dosage. conclusion when biperiden administration is required by intravenous route in children of y-o and less, biperiden should be administered intravenously and titred using bolus dose of . mg till the therapeutic effect is obtained. introduction severe poisoning by rodenticides is frequent. it represents nearly % of patients admitted to the new intensive care unit (icu) of the region. that is why we decided to perform this study. the aim of this work was to describe the epidemiology, clinical features and management of all patients admitted to our unit for acute poisoning with rodenticides. patients and methods it was a retrospective study performed in the year from january to december. the study included all patients admitted in the icu for rodenticide poisoning. results patients were enrolled in the study. our patients were young with a mean age of ± years. poisoning was more common in females (n = ; %). the mean delay between rodenticide poisoning and first medical contact was about ± h in the cases where this information. most of our patients ( %) attended the emergency department of zaghouan with a non-medical transportation. it was a suicide attempt in most cases ( %) and an accidental poisoning in % of patients. the most frequent cause of poisoning in our study was organophosphorus pesticide (n = ; %). the second cause was alpha-chloralose poisoning with seven cases ( %). one patient ingested accidentally an anticoagulant rodenticide. most of patients had ingested (oral route) the rat poison (n = ; %). clinical examination found normal vital signs in ten cases ( %). nine patients ( %) had a shock, eight patients ( %) had an acute metabolic disorder and five patients ( %) had acute respiratory failure or were comatose. all patients enrolled in the study were admitted in the icu for a period of clinical observation of h. stomach pumping (gastric lavage) was performed in patients ( %). an antidote which was atropine was needed in twelve patients. three patients ( %) who ingested alpha-chloralose needed intubation and mechanical ventilation. all patients had a good outcome and were discharged from icu and from hospital. the mean icu length of stay was ± days. conclusion this is the first study of acute poisoning with rodenticides admitted in the new icu. the results of our study were similar to those published in recent literature. cases of acute poisoning with rodenticides reported in this work were not severe. none. introduction the systemic arterial load imposed to the left ventricle (lv) is a major determinant of normal/abnormal cardiovascular function. the lv mean ejection pressure (lvmep) is the best estimate of load faced by the lv throughout ejection. the contribution of the steady and pulsatile blood pressure (bp) component of arterial load to lvmep is debated. we studied the hemodynamic correlates of lvmep using carotid tonometry. intensive care unit patients equipped with an indwelling catheter were studied, thus allowing precise calibration of the tonometer. patients and methods carotid tonometry (complior analyse ® alam medical, france) was prospectively performed on hemodynamically stable, spontaneously breathing patients ( f, mean age ± sd = ± years). carotid waveforms were calibrated from diastolic bp and time-averaged mean bp invasively obtained at the radial (n = ) and femoral (n = ) artery. all patients were free of aortic stenosis. lvmep was the area under the systolic part of the carotid pressure waveform divided by ejection time. results lvmep ( ± mmhg) was strongly related to central systolic bp ( ± mmhg; r = . ) and was also related to mean bp (r = . ), peripheral systolic bp (r = . ), peripheral (r = . ) and central (r = . ) pulse pressure (each p < . ). the lvemp was not related to age, heart rate and stroke volume. systolic pulse wave amplification ratio from carotid to periphery was . ± . . conclusion lvmep was most strongly related to central systolic bp, which combines the influences of the steady and pulsatile components of central arterial load (r = . ). lvmep was less strongly related to peripheral systolic bp, which may be less informative given variable systolic pulse wave amplification across patients. introduction myocardial dysfunction is one of the main predictors of poor outcome in septic patients, with mortality rates next to %. many pathological findings were found in the sepsis induced cardiomyopathy including myocardial ischemia, alterations in microcirculation and proinflammatory cytokines. the aim of this study was to assess the prognostic value of a recently developed highly sensitive cardiac troponin i (hstni) assay in patients with septic shock. we performed a prospective observational study in septic shock icu patients within h of admission. exclusion criteria were age > years; pregnancy; post-cardiac arrest and braindead. hstni was measured soon after admission and , , and h after. patients were subjected to transthoracic echocardiography (tte) at study inclusion and regular biochemical and hemodynamic assessments were performed. pearson's chi square and fisher's exact tests were used. p < . was considered significant. conclusion circulating hs-ctni is present in patients with septic shock. a rise of hstni may be an indicator of poor outcome. also, right heart functional abnormalities exist in patients with septic shock. none. evolution of the right distribution width as a pronostic marker during the differents state of shock introduction right distribution width (rdw) has been recently proposed as a pronostic factor in different pathologic situations and especially to the septic patients who stay in icu. some works substantiate the relationship between an alteration of the red blood cell rheology during the septic shock and a severe state of the disease. no one has studied the rdw between the differents shocks yet. we are going to determinate the relationship between rdw and apache ii score, mortality rate in the intensive care unit (icu), at the hospital, at the day and . we investigated those parameters near patients who were admitted at the icu and needed norepinephrine between the first of march and the st of december. they were stratified in différent groups: septic shock n = , cardiogenic shock n = , hemorragic shock n = and obstructive shock n = . results we did not observe any correlation between the rdw and the icu mortality, hospital mortality and at the day and . only a poor significant correlation has been found between the cardiogenic shock and the mortality rate: at the hospital (p = . ), at day (p = . ) and at the day (p = . ) but not in the icu (p = . ). the receiver operating characteristics (roc) curves do not show significant differences between rdw, apache ii score and icu mortality rate or intra hospital. the sample of the hemorrhagic shock and obstructive shock was not usable for this calculation. compared to other studies which were focused on the septic shock where the mortality was approximately %, we determinated a mortality rate near %. conclusion the delta of the rdw d /d did not present any correlation with the mortality rate. in our study, the rdw in the different kind of shocks do not look like to be a good predictive marker of the mortality, except for the patients included in the cardiogenic shock where a poor significant correlation could be highlighted. conclusion cardiogenic shock was the most frequent complication of ami who led to icu admission, whereas mechanical complications are rare at the era of early coronary reperfusion strategies. in addition to severity score, serum creatinine and cardiogenic shock appeared as independent factors of hospital death. none. introduction pulmonary embolism (pe) in high-risk is a partial or total obliteration of the pulmonary arterial network by a fibrin-clot cruoric more than %, the management requires a rapid reduction of pulmonary arterial resistance and right ventricular post load through rapid revascularization by thrombolysis. our aim is to determine the value of thrombolysis in pulmonary embolism and describe the clinical, paraclinical and outcome pulmonary embolism at high risk. patients and methods this is a descriptive study of cases of pulmonary embolism at high risk admitted to the cardiology department to chu oran between and . signs of gravity of (pe) comprising: syncope, circulatory collapse, cardiogenic shock or acute pulmonary sonographic sign of heart. it was confirmed in chest ct. all patients received thrombolysis using the protocol accelerated by two types of molecules: streptokinase or actilyse. the sex ratio was . ; mean age years, ranging from to years; risk factors were dominated by contraception was % and the postoperative % the clinical picture was dominated by cardiogenic shock in % of cases. % cardiovascular collapse and syncope in %; doppler echo all patients had signs of dysfunction of the right ventricle represented by the dilatation of the right cavities and pulmonary hypertension. the cta found a (pe) bilateral in % right in %. thrombolysis using actilyse in patients and streptokinase in cases. the outcome was favorable in patients; with two cases that are complicated by chronic pulmonary heart and the death of patients with cancer. discussion the female predominance is explained by the increase of risk factors hormonal contraception, whose first generation combination hormonal. our patient had a high probability with clinical signs of severity based on the score wells [ ] . this diagnosis was confirmed by chest ct; which shows the vascular bed obstruction degree with a very good sensitivity and specificity. the suspect patients with severe pe and that presented signs of acute pulmonary heart ultrasound have effectively (pe). the indication of thrombolysis was chosen on hemodynamic criteria; success is found in % of patients with improved hemodynamics dice the early hours. this success is explained by the role of thrombolytic in lysis clot to obtain pulmonary arterial revascularization; and reduce pulmonary arterial resistance and the right ventricular afterload which accelerates the healing of right heart failure and improvement of pulmonary capillary volume. the cases who developed a chronic pulmonary heart; it was done immediately a right ventricular dysfunction with pulmonary arterial outset of very high pressures suggestive that the embolism occurred on an already pathological right heart. no cases of massive bleeding were noted in our series. conclusion severe pulmonary embolism is burdened with high mortality; diagnosis is based on the stratification of risk score, was facilitated by the non-invasive strategies that articlent around the doppler echocardiography and ct angiography; thrombolysis can reduce the high mortality related to severe pulmonary embolism. introduction hypertension is a frequent motif for admission to emergencies. the diabetic is increasingly exposed to this risk [ ] . the objective of this study is to evaluate the proportion of diabetic patients presenting to the emergency department with high blood pressure (bp) and to identify their epidemiological and clinical characteristics. introduction sepsis associated liver dysfunction (sld) is usually attributed to systemic and/or microcirculatory disturbance. hypoxic hepatitis, also known as shock liver or ischemic hepatitis, is a life threatening event associated with high morbidity and mortality. doppler ultrasonography is a non invasive method to measure doppler hepatic hemodynamic parameters. the primary objective of this study was to assess the accuracy of the hepatic hemodynamic parameters (portal venous blood flow pvbf and resistance index of the hepatic artery hari) in predicting sld in septic shock patients. the secondary aims were to identify factors associated with sld, investigate the effects of volume expansion (ve) on systemic and intrahepatic hemodynamics and to assess the intra-and interoperator reproducibility. we also analyzed -day mortality. in a prospective design, we included consecutive patients with septic shock ( males; median age: . years) admitted to the icu with septic shock in charles nicolle hospital of tunis from february to july . all patients were resuscitated following the surviving sepsis campaign guidelines. we measured systemic hemodynamic variables (mean arterial pressure (map), and cardiac index (ci)) and performed hepatic doppler before and after volume expansion. we measured pvbf and computed the hari. we recorded the liver function tests (alt, ast and bilirubin) for h. sld was defined as an increase in serum bilirubin ≥ µmol/l (hepatic sofa ≥ ). accuracy of the hepatic hemodynamic parameters to predict sld was measured by the area under the roc curve. p < . was taken to indicate statistical significance. the median sofa score at t was points and the median igs score was points. the sources of infection were as follows: the lungs (n = ), the abdomen (n = ) and the urinary tract (n = ). the incidence of sld in our cohort was . % (n = ). there was no significant difference between "sld group" and "no-sld group" in all hepatic hemodynamic parameters especially the pvbf and the hari. lactate levels were significantly higher in patients with sld (median . vs. . mmol/l). similarly, the platelet count was significantly lower in the "sld group" [mean (± sd) . ± . ( /l) vs. . ± . ( /l); p = . ]. there was no difference in duration of mechanical ventilation, icu length of stay and -day mortality between the groups. the pvbf was significantly lower in patients who died before d (median: vs. l/min in the survivors; p = . ). volume expansion caused a significant increase in ci, mean hepatic artery velocity and the pvbf. the intra-and interoperator reproducibility was good to excellent for the systolic and mean velocities of the hepatic artery, portal vein diameter and the pvbf. conclusion our results don't support the hypothesis that the hepatic sonography is predictive of sld in septic shock. our pilot study showed higher lactate levels and hematologic sofa in sld group. the pvbf was significantly lower in patients who died before d . more experience will be necessary to define the ultimate role of doppler ultrasonography in the evaluation of hepatic perfusion in patients with septic shock. introduction early surgery is the current trend for management of patients with valvular disease. that said many of them, particularly from developing countries, are still operated at a very advanced stage of disease. despite improvements in myocardial protection and surgical techniques, postoperative care after multiple valve surgery (mvs) for advanced rheumatic heart disease (rhd) remains to be a clinical challenge. we conducted a study to determine postoperative complications and morbidity-mortality risk factors in this subgroup of patients. results sixty-two patients were included: with out-of-hospital refractory cardiac arrest and with in-hospital refractory arrest. the initial rhythms was shockable rhythm in ( %) cases. at ecls initiation, the mean no flow was . ± . min and mean low flow (time between the time of refractory cardiac arrest and time at which an ecls flow was provided) was ± min. the mean ecls flow rate was . ± . l/min. initial blood test results were: arterial ph = . ± . and plasma lactate = . ± . mmol/l. eleven ( %) patients survived ( / ( %) acute coronary syndrome, / ( %) severe poisoning due to drug intoxication, / ( %) dilated cardiomyopathy, and / ( %) others). survival was lower for patients with out-of-hospital refractory cardiac arrest, of ( %), than for patients with in-hospital refractory cardiac arrest, of ( %), respectively, p = . . as expected, out-of-hospital refractory cardiac arrest was associated with a more prolonged low flow ( ± min vs ± min, p < . ) and a more profound acidosis (ph . ± . vs . ± . , p = . and arterial lactate . ± . vs ± , p = . ). in univariate analysis, survival was lower for patient with refractory cardiac arrest unrelated to drug intoxication, vs %, respectively, p = . . in addition, mortality was associated with arterial ph ( . ± . vs . ± . , p = . ) and low flow ( ± vs ± min, p = . conclusion in a highly selected group of critically ill patients with refractory cardiac arrest, the potential beneficial effect of ecls could be due only to its clinical impact on reversible causes of circulatory failure (i.e. severe drug intoxication in our cohort). further studies are needed to clarify whether the use of ecls could be considered as a disproportionate tool, specifically in patients with out-of-hospital refractory cardiac arrest due to acute coronary syndrome or associated with prolonged low flow or a profound acidosis. none. post-cardiac arrest shock treated with veno-arterial extracorporeal membrane oxygenation: an observational study and propensity-score analysis wulfran bougouin , nadia aissaoui , alain combes average time between introduction and removed of the ecd was h ( - ). among the esogastroduodenoscopy performed, ( %) were strictly normal. endoscopy showed minor gastric injuries in patients ( %). within these patients, ( %) also presented minor esophageal injuries. esogastric injuries characteristics were mostly similar to usual orogastric probe injuries. one patient ( %) experienced a serious ulcerous esophagitis mimicking a peptic esophagitis, not firmly related to the ecd. no patients necessitated hemostatic local procedure and no significant gastrointestinal bleeding was observed. eight patients ( %) were alive at d , including patients ( %) with a cerebral performance category score of . this compares favorably to outcomes from previous studies. conclusion ecd seems an interesting and safe semi-invasive method of cooling in ohca patients treated with °c-ttm. although it seems slower than more invasive devices to reach °c, ecd was able to strictly maintained the tt within the maintenance phase of ttm. further studies will be necessary to define the exact place of this new device within the cooling strategy in patients necessitating a precise ttm-strategy. none. fig. see text for description introduction since post-cardiac arrest care might influence the outcome, characteristics of receiving hospitals should be integrated in survival evaluation of patients transported in hospital. we aimed at assessing the influence of care level center on survival at discharge in a regional registry of out-of-hospital cardiac arrest (ohca). we prospectively collected utstein and in-hospital data for all non-traumatic ohca patients, in whom a successful return of spontaneous circulation (rosc) had been obtained, from a large metropolitan area (great paris). receiving hospitals were categorized in groups (a, b, c) depending on their respective characteristics (annual volumes, / catheterization availability and temperature management use). we compared patients' characteristics in the groups and performed a multivariable logistic regression using discharge survival at endpoint. results during the study period (may -dec ), patients were admitted in hospitals ( in group a, in group b and in group c). overall survival rate at discharge was / ( %). patients' baseline characteristics significantly differed, as hospitals from group a treated younger patients and more frequent shockable rhythms (p < . ). unadjusted survival rate differed significantly among the groups of hospitals (respectively , and . % for a, b, c, p < . ). however in multivariable analysis, the category of hospital was no longer associated with survival. conclusion in this population-based study, characteristics of receiving hospitals are not associated with survival rate at discharge. this could result from the strategy used for triage, which aims in matching patients' characteristics and resources. introduction acute kidney injury (aki) commonly occurs after cardiac arrest and is associated with an increased mortality and a delayed awaking. early recognition of aki remains challenging, given that serum creatinine increases belatedly after aggression. introduction out-of-hospital cardiac arrests (ohca) are an absolute urgency and have a very poor prognosis. pediatric guidelines differ from adult guidelines for cardiac arrest management. since , adult guidelines apply from the onset of puberty. the main objective was to describe the epidemiological characteristics and outcome of ohca victims while taking puberty into account. the secondary objective was to determine the prognostic factors for survival at d . materials and methods all patients less than years of age, victims of ohca between july , and september , care by a mobile emergency and resuscitation service (smur) participating in french national cardiac arrest registry (réac) were included. patients were split into groups: prepubescent patients (named "children": girls - years, boys - years), pubescent patients (named "adolescents": girls from to years and boys from to years) and "adults" (men and women - years). the "adolescents" group was consecutively compared to the "children" group and to the "adults" group. results children, adolescents and , adults under the age of have been included. ohca in adolescents occurred more often on public roads ( %) or in public places ( %) and were more often traumatic ( %) than those in children and adults. respiratory causes were more frequent in children ( %) than in adolescents ( %) and adults patients ( %). the proportion of shockable rhythm increased with age ( , and % for children, adolescents and adults respectively). survival at d was greater in adolescents ( %) than in children ( %) and adults ( %) (p = . and p = . respectively). in the studied groups, initial shockable rhythm was a survival factor at d (respectively or [ . - . ] for children, adolescents and adults). other risk factors are described in table . conclusion adolescents had better survival at d than the others groups. adolescents and adults had shockable rhythm more often than children. moreover, respiratory failure was less frequent in adolescent and adults patients compared to children. puberty seems to be a good limit to differentiate pediatric patients with ohca. none. introduction non-invasive ventilation (niv) is an effective alternative to endotracheal mechanical ventilation (mv) in the management of acute respiratory failure (arf) patients. nevertheless, it can be still difficult to assess its real feasibility, application and outcome in daily clinical practice. therefore, we report our clinical experience with routine use of niv since the last national recommendations ( ). our aims were to evaluate the clinical efficacy and outcome of niv, and to identify predictive factors for niv failure based on a daily use. patients and methods we conducted an observational retrospective single-center cohort study by reviewing all medical records from january to december in our -bed medical intensive care unit (icu). eligible patients were those having received niv during their icu stay. two groups were defined according to the indication of niv: niv for hypoxemic or hypercapnic arf (arf-niv), and niv used in the post-extubation period for weaning, prevention or treatment of post-extubation arf (post-extubation niv).the main evaluation criteria were the incidence of niv use, success/failure rate of niv and risk factors for niv failure in each group. niv failure was defined as the need for stopping niv whatever the reason (intubation, intolerance, death) within days after its initiation. ( ; ), and was longer in the post-extubation niv group ( days ( ; ) ) than in the arf-niv ( days ( ; ) for hypoxemic arf, ( ; ) for hypercapnic) (p < . ). the overall icu mortality was . % ( . % in hypoxemic group, . % in hypercapnic group, and . % in post-extubation niv group) (p = . ). in multivariate analysis, the main risk factors for arf-niv failure were: saps ii on admission (p < . ), absence of cardiologic history (p = . ) and the cause of arf (p = . ) with a higher failure rate for pulmonary infections than acute cardiogenic pulmonary edema (or . , p = . ). for post-extubation niv, the only independent risk factor for failure was normocapnia before niv initiation (p = . ). conclusion our large longitudinal study demonstrates the feasibility and efficacy of niv applied in daily clinical practice. provided it is performed in a suitable environment by an experienced team, niv should be considered as a first-line ventilatory treatment in various etiologies of arf and a very useful ventilatory support in the postextubation period. nevertheless, risk factors for niv failure should be known by icu clinicians, hypoxemic arf remaining the more difficult indication to manage with niv. réanimation médicale, hôpital saint-louis, paris, france; service de biostatistique et information médicale, hôpital saint-louis, paris, france; réanimation, institut paoli-calmettes, marseille, france; réanimation introduction acute respiratory failure (arf) is the leading cause for icu admission in immunocompromised patients. in these patients, oxygenation strategy is of major interest to avoid the need for mechanical ventilation (mv), which is associated with high mortality rates. in that setting, use of non-invasive ventilation (niv) and oxygen therapy with high flow nasal cannula (hfnc) could be interesting alone or in association, but data about initial ventilation strategy in immunocompromised patients are controversial. to assess how initial oxygenation strategy actually influences the risk of mv on the coming day within the three first days of icu stay. the study end-point was the need for mv on the coming day. we restricted analyses to these first three icu days given, based on our own experience, most of mv was expected to occur by then. we performed a post hoc analysis combining three prospective studies of critically ill immunocompromised patients (two randomized control trials, the ivnictus and the minimax studies and one prospective cohort, the trial-oh study). we only considered patients with arf and a delay between icu admission and study inclusion less than h. we excluded patients who required invasive mv within the first day, those with an icu stay less than day and those with acute pulmonary edema diagnosis at icu admission. in order to estimate and compare the causal effect of daily respiratory management strategy on the probability of intubation in the coming day, we computed inverse probability of treatment weights (iptw) using propensity-score, defined as the probability of actual treatment selection conditionally on observed covariates. to handle confounding in such dynamic regimens, we considered marginal structural models (msm), which have been proposed to estimate the causal effect of a time-dependent exposure when time-dependent covariates that can be affected by the previous treatment are present. two treatment exposure models were considered: niv versus oxygen therapy regardless the device (model ) and hfnc alone, niv alone versus niv + hfnc versus standard oxygen therapy alone (model ). results patients were included in the study. in model , there was no difference between niv and oxygen groups on mv whatever the landmark time. in model , while the unweighted or for intubation at day was significantly higher in the niv group (or . , %ci . - . ) and hfnc group (or . , %ci . - . ) than those in the standard oxygen alone group, these differences disappeared in the weighted samples. using msm, no effect of the oxygenation strategy on mv was found, regardless of the oxygenation devices but the landmark time was associated with a reduced occurrence of mv. conclusion we found no evidence of any significant difference from several oxygenation strategies on mechanical ventilation probability during the first days of icu in a large cohort of immunocompromised patients with arf. none. introduction the role of noninvasive ventilation (niv) is debated in the management of patients with acute hypoxemic respiratory failure. a recent study showed that patients treated with high-flow nasal cannulae oxygen therapy (hfnc) had lower intubation and mortality rates than those treated by the association of hfnc with niv ( ). high tidal volumes (vt) delivewred with niv may be associated with an increased risk of intubation ( ) . we aimed to identify risk factors associated to intubation, in hypoxemic patients with acute respiratory failure and especially the role of vt under niv. patients and methods this is an ancillary study from a multicenter, randomized, controlled trial including patients with acute hypoxemic respiratory failure (florali-study). we focused on only patients with moderate or severe hypoxemia (pao :fio ratio ≤ mmhg) and we excluded those with mild hypoxemia. the criteria for intubation were predetermined including worsened or persisted respiratory failure, impairment of neurologic status and hemodynamic instability. results after adjustment on the oxygenation strategy, the two factors independently associated with intubation were the presence of bilateral pulmonary infiltrates at admission (or . simulation conditions enables to reproduce its occurence, using different types of tools, from physiological parameters to heart rate variability and psychocognitive tests. future research is required to evaluate the impact of these parameters on teaching. none. with stratification by centre and operator experience. an only inclusion criterion was: "patients must be admitted to an icu and require mechanical ventilation through an endotracheal tube". patients were excluded if: contraindication to orotracheal intubation (e.g., unstable spinal lesion); insufficient time to include and randomize the patient (e.g., because of cardiac arrest); age < years; pregnant or breastfeeding woman; correctional facility inmate; patient under guardianship; patient without health insurance; refusal of the patient or next of kin to participate in the study; previous enrolment in a clinical randomized trial with intubation as the primary end point (including previous inclusion in the present trial). post-hoc analysis was performed to assess occurrence of spo < % during intubation procedure between groups of preoxygenation: bvm (at a minimum flow of l/min, niv ( % fio ), hfnc (at a minimum flow of l/min, with % fio ), and nrm (at a minimum flow of l/min). between-groups difference in desaturation occurrence was adjusted for baseline covariates significantly associated with the group membership (p < . ). multivariate analysis of the occurrence of a desaturation (< %) was performed using logistic regression. bag-valve mask was considered reference. results baseline characteristics were showed in table . groups were similar at baseline except for pao /fio ratio. in univariate analysis, age (p = . ), saps (p = . ), pao /fio ratio (p = . ),spo (p = . ) and method of preoxygenation (p = . ) were associated with occurrence of desaturation below %. in multivariate analysis, spo at randomization and method of preoxygenation were only predictors of desaturation below %. bvm and hrm were associated with similar risk of desaturation occurrence whereas niv (or . introduction intubation procedure is a challenging issue in intensive care unit (icu) [ ] . cardiac arrest related to intubation in critically ill adult patients has been poorly studied. the studies were not powered to conclude on this rare outcome [ ] . the main objective of our study was to establish the incidence of cardiac arrest and to assess the risk factors of cardiac arrest in a large prospective database of intubation procedures performed in icu. five prospective studies were included, with similar data collected before, during and after intubation procedures using the same methodology. the primary outcome was the incidence of cardiac arrest related to intubation. the secondary outcomes were the death (cardiac arrest without return of spontaneous circulation (rosc)), the cardiac arrests with rosc, the complications related to intubation, the length of icu stay and the -day mortality. the factors associated with cardiac arrest related to intubation procedures were assessed by univariate and multivariate analysis based on patient, provider and practice characteristics. results among the intubation procedures included, cardiac arrests ( . %) occurred, including with rosc ( . %) and without rosc ( . %). main patient, provider, procedure characteristics and outcomes according to cardiac arrest related to intubation are presented in table . in multivariate analysis, the independent predictors of cardiac arrest related to intubation were low systolic blood pressure prior to intubation, hypoxemia prior to intubation, no preoxygenation, overweight or obesity and age > years. mortality rate at day was significantly lower in patients intubated without cardiac arrest ( . %, of ) than with cardiac arrests overall ( . %, patients of , p < . ) and cardiac arrest with rosc ( %, patients of , p < . ). conclusion cardiac arrest related to intubation in adult icu is not a rare event occurring in . % of cases with high immediate mortality of . % and at day of . %. we identified five independent risk factors to cardiac arrest which of them could be modifiable. optimal preparation to intubation procedure could help to prevent those cardiac arrests. introduction naasotracheal intubation (nti) has been progressively given up in favour of the orotracheal intubation (oti) in intensive care unit (icu). this could be explained by more frequent infectious (sinusitis and ventilator associated pneumonia) and non-infectious (epistaxis, turbinates bones injury) complications the former being thought to be more frequent with nti. however, whereas infectious sinusitis is a risk factor for vap, no study has yet demonstrated that oti decreases the infectious sinusitis rate compared with nti. furthermore, nasal route could improve patient comfort and decrease auto-extubation. finally nti can be performed without laryngoscopy with less risk of lips and dental injury. in this prospective study, we aimed to compare the complication of nti and oti and to assess the comfort of the patient. we performed a prospective observational study in a -bed medical icu including patients requiring endotracheal intubation. the intubation route was let at the discretion of the physician in care of the patient, however oti was compulsory in case of cardiac arrest, severe hypoxemia (p/f < when available) and clotting perturbation. for each patient, age, sex, sapsii, mechanical ventilation duration. intubation route were recorded as well as complications during the placement of endotracheal tube. infectious and non infectious complications during invasive ventilation period were also recorded. in patients who were successfully extubated, pain, burning feeling, dryness and the wish of tube removal were assessed using visual analogic scales (vas conclusion despite its small size, and the absence of randomization, the present study suggests that nasotracheal intubation improves the comfort and the tolerance of tracheal intubation and is not associated to higher rates of vap. none. effect of mode of hydrocortisone administration in patients with septic shock: a prospective randomized trial oussama jaoued , rim gharbi , najla the baseline characteristics of patients were similar between the two groups. sepsis was secondary to community-acquired infection in % of cases. there was no difference in mortality between groups ( % in continuous groups and % in discontinuous group). sofa score was significantly higher at days , and in discontinuous group. length of stay, duration of mechanical ventilation, number of day without vasopressors, and the occurrence of adverse events were similar in the two groups. conclusion the mode of hydrocortisone administration in patients with septic shock has no influence on morbidity or mortality. the occurrence of adverse events was similar. introduction widespread activation of coagulation with platelet consumption is a pathophysiological feature of severe sepsis and septic shock. thrombocytopenia, either defined by platelet count below g/l or by a significant relative - -percent decrease in platelet count is a potent poor prognostic factor in sepsis. besides their role in hemostasis, platelets also carry various immune and inflammatory functions that are likely to impact on host defense against infections. we aimed to assess whether changes in the platelet count induced by sepsis is associated with the development of subsequent nosocomial infections. patients and methods patients were obtained from two prospective studies about immuno monitoring of dendritic cells and innate-like lymphocytes in critically ill septic patients ( , ) . adult patients with severe sepsis and septic shock were included. exclusion criteria were any immunosuppressive condition (hematological malignancy, hiv infection at any stage, bone marrow or solid organ transplantation, daily corticosteroid therapy > . mg/kg prednisone-equivalent, chemotherapy or any other immunosuppressive treatments), pregnancy, do-not-resuscitate orders on admission. in addition patients who died or who received platelet transfusion during the first week after icu admission were also excluded. platelet counts were collected on the day of sepsis diagnosis (d ) and then on d , d and d . the relative variation in platelet count at day n compared to day was calculated as follows: (count at day n − count at day ) × / (count at day between between d and d , between d and d and between d and d were also similar between patients with and without icuacquired infections (fig. ). discussion in this preliminary study from selected cohorts of nonimmunocompromised patients, sepsis resulted in mild alterations in platelet counts, making it unlikely to become associated with the development of nosocomial infections. it would be relevant to address this question in larger cohorts of non-selected patients, as well as the impact of platelet transfusions in this setting. conclusion changes in platelet counts were not associated with an increased susceptibility towards icu-acquired infections in non-immunocompromised patients with severe sepsis and septic shock. introduction sepsis is the leading cause of mortality in the intensive care unit (icu) patients despite the progress regarding their care. the immunodeficiency due to sepsis with the consequent profound lymphocyte alterations is now well proven. the objective of this work was to determine the prognostic impact of lymphocytopenia in septic patients in icu. retrospective study including all patients hospitalized for sepsis or septic shock between / / and / / . the sepsis and septic shock definitions were adjusted with the third international consensus definitions for sepsis and septic shock. were excluded from the study patients of onco-hematology. lymphocytopenia was defined as an absolute lymphocyte count less than level of /mm during the first h of hospitalization. the prognostic factors analyzed for the lymphopenic and non lymphopenic patients were in hospital mortality, the occurrence of nosocomial infections and hospital length of stay. results among the patients, aged ± years, patients were with septic shock and patients with sepsis. igsii score and sofa score were respectively ± and ± . four patients were immunocompromised due to hiv infection in one case and an immunosuppressive therapy in cases. lymphocytopenia was observed in patients ( %). twenty-eight patients ( %) died within an average of ± days. it was noted the occurrence of nosocomial infections. the median length of stay was days with extremes of one and days. the lymphopenic patients were comparable to non lymphopenic patients in terms of medical history and severity scores. mortality was comparable between the groups with a rate of % (n = ) in lymphopenic patients and % (n = ) in non-lymphopenic patients (p = . ). the earliness of death was correlated with the duration of lymphopenia (r = . , p = . ). the occurrence of nosocomial infections was not different between the two groups: % (n = ) for lymphopenic and % (n = ) for non lymphopenic patients. the hospital length of stay was not different between the two groups but was correlated with the duration of lymphocytopenia (r = . , p = . ). conclusion lymphocytopenia is frequently found in sepsis. lymphocytopenia was not associated with excess of mortality nor with the subsequent occurrence of infectious complications during the icu stay. his persistence was associated with an earlier death and a more prolonged hospitalization. none. introduction relative adrenal insufficiency (rai) is common in icu patients, particularly during septic shock ( ). it has been shown that the rai also occurs during cardiogenic shock ( ) . septic cardiomyopathy occurs in a significant proportion of septic shock patients. the aim of this study was to evaluate the role of rai on septic cardiomyopathy. patients and methods prospective observational study conducted in the intensive care in one university hospital in france. patients meeting the criteria for septic shock without prior corticosteroid therapy and without chronic heart disease were included. total blood cortisol levels were assessed immediately before (t ) a short corticotropin stimulation test ( . mg iv of tetracosactrin) and and min afterward. Δmax was defined as the difference between the maximal value after the test and t . rai was defined as an inappropriate adrenal response with Δmax < µg/dl and septic cardiomyopathy as the appearance of cardiac systolic dysfunction (left ventricle ejection fraction < %) within the first days of septic shock. we performed a multivariable analysis using backward stepwise logistic regression to identify independent predictors of septic cardiomyopathy. discussion although the definition of rai is not consensual, a threshold of Δmax at µg/dl has been widely used in septic shock, with or without the use of t ( ). the usefulness of substitutive doses of steroids in septic shock is controversial, but many authors assume this treatment has a potential in reversing overt vasoplegia. our data suggest an implication of rai in septic cardiomyopathy. conclusion we found rai to be an independent predictor of septic cardiomyopathy. these findings may suggest a new role for substitutive doses of steroids in the hemodynamic management of septic shock. introduction regional perfusion parameters, like lactate, pyruvate and glycerol, may predict outcome in septic shock patients. continuous venovenous haemofiltration (cvvh) has been considered beneficial in septic shock patients. the aim of our study was to investigate whether cvvh, in comparison to intermittent haemodialysis (ihd), is able to improve regional perfusion in septic shock patients studied by muscle microdialysis. patients and methods it was a prospective, randomized, clinical study, including septic shock patients with acute renal failure, aged over years. patients were randomized to receive either cvvh (n = ) or ihd (n = ) for renal replacement therapy. intermittent haemodialysis was carried out during the first h of the h study period. systemic haemodynamics and interstitial tissue concentrations of lactate, pyruvate, glucose and glycerol were obtained at baseline, , , and h after initiation of renal replacement by using muscle microdialysis. results regarding systemic haemodynamics parameters, cvvh caused a decrease in heart rate in contrast to ihd after h (− ± vs + ± /mn). there were no changes in vasopressor support throughout the -h study period and so systolic blood pressure remained stable in both groups. during the h of all renal replacement therapies there was no significant change in muscle pyruvate and glucose levels. during cvvh muscle lactate decreased constantly, as did muscle glycerol levels. this decrease reaches a significant levels at h for muscle lactate and at h for muscle glycerol (fig. ) . conclusion our results suggest that among septic shok patients, cvvh may improves regional perfusion in comparison with ihd. none. introduction acquired hypernatremia (h-na) is an independent risk of death among icu patients ( ). in the rct "hyper s" study, we compared normal to % hypertonic saline during the first h in patients with septic shock with normal serum na concentration (sna) at baseline. the study was prematurely stopped for potential harmful effect associated with more frequent h-na. we assessed the role of h-na on mortality. patients and methods data are a post hoc analysis of the "hyper s" study database including patients. sna was measured at h , every h for days and then daily until d . study fluids were stopped if sna > or > mmol/l increase over h. mild, moderate, and severe h-na were defined as sna > mmol/l, > mmol/l and > mmol/l, respectively. sna profiles were compared between d survivors and non-survivors. acute kidney injury (aki) was defined by doubling serum creatinine and/or need for dialysis. results patients with available data were analysed. ( %) developed h-na (mild: %, moderate: %, severe: %). no matter the absence or presence and its severity, h-na did not affect mortality ( , , , and %, respectively without, with mild, moderate, and severe h-na, p = . ). sna profiles were similar between survivors and non-survivors (table ) . a sensitivity analysis performed among survivors at d did not change the results. compared to patients without h-na, aki occurred in % of patients with h-n vs % (p = . ), atelectasis in versus % (p = . ) and icu acquired weakness in versus % (p = . ). conclusion hypernatremia occurrence is not associated with an increased risk of morbidity and mortality during hypertonic fluid resuscitation in septic shock. none. introduction guidelines about the moderate hypokalemia treatment (between . mmol/l and . mmol/l) are based on experts estimations, and non-specific ones for patients in the intensive care units (icu). the aim of this study was to evaluate the correction of the hypokalemia in an icu and the compliance of recommendations. materials and methods an observational epidemiological, retrospective and monocentric trial has been realized during a period of months (from january to february ). the study population included hospitalized patients in the icu who have shown a first moderate hypokalemia episode, all cause considered. patients who have presented an acute renal failure with a kdigo (kidney disease: improving global outcomes) score of three the day of their inclusion were excluded. the main primary study endpoint was percent correction of the serum potassium after h. the secondary study endpoints were the incidence rate of moderate hypokalemia and the efficacy about the hypokalemia correction in accordance with the achieved treatment consistent or not with recommendations. results patients had at least one episode of hypokalemia. the incidence rate of the hypokalemia first episode was . %. the study population included patients. igs score was . (± ). patients required mechanical ventilation at the inclusion. the serum potassium was greater than or equal to . mmol/l after h about patients ( . %) (corrected group). at h one patient had a serum potassium higher than mmol/l. the average total potassium was respectively . infusion of potassium and ( . %) patients have been a management compatible with the most common recommendations (input potassium chloride of mmol, use of the enteral administration and lack of continuous intravenous infusion). the percent correction of the hypokalemia after h was respectively of / ( . %) in the group in which recommendations had been respected and of / ( . %) in the other one (p = . ). discussion in our knowledge there are no previous studies that have specifically focused on the correction of the moderate hypokalemia in critically ill patients. in our study the incidence rate of the moderate hypokalemia was lower than data from the literature because we have only considered the first episode of the hypokalemia [ ] . among patients without contraindication to the enteral administration, this one was used in less than half of the cases. % of these patients received potassium with a continuous intravenous infusion and only patients received medical care conform to the guidelines. the medium potassium quantity provided was very lower to the guidelines. only % of the patients have been corrected after h without any difference in the medium potassium quantity which has been provided in relation to the uncorrected group. conclusion only . % of moderate hypokalemia in icu are corrected after h. the intravenous way is considerably used (in % of cases) with a poor return. a wide-ranging study is necessary to determine the best correction modes. none. results patients were included. mean ± sd age was ± years, % were male, mean ± sd saps ii was ± . icu length of stay was ± days and icu mortality rate was %. during the first days in the icu, % of patients received at least one nephrotoxic drug. % of patients received one, % received two, % received three and % received more than three nephrotoxic medications. diuretics, antibiotics and iodinated contrast media were the nephrotoxic drugs most frequently administered to, respectively, , and % of patients. aki (kdigo stage or higher) occurred in % of patients during the first days in icu. the proportion of patients with aki increased with the number of nephrotoxic drugs received: / ( %) of the patients not exposed to nephrotoxic drugs developed aki whereas, respectively, / ( %), / ( %), / ( %) and / ( %) of the patients receiving one, two, three, and more than three nephrotoxic drugs developed aki. the univariate association between the number of nephrotoxic medication and aki persisted in the multivariate analysis adjusted on baseline saps ii score (p < . ). conclusion the significant proportion of patients exposed to nephrotoxic drugs and the observed association with aki warrants further investigation. statistical adjustments for multiple potential confounders is needed in order to assess a potential causal relationship which would lay foundations for interventional studies. none. ( ) the minimal kidney aggression by current monomeric nonionic low-osmolar contrast media, late serum creatinine increase being explained by the occurrence of later (between the th and the nd hour) kidney injury due to critical illness or its therapy or ( ) insufficient sensitivity of early ( h) measurements of this biomarker to detect contrast-associated aki. competing interests partial financial support, no implication in data analysis and interpretation. introduction diabetic ketoacidosis, generally resulting from an absolute deficiency of insulin, is a frequent cause of hospitalization in intensive care unit. recommendations for diagnosis of diabetic ketoacidosis, care and site of admission have been published by the english society of diabetology. icu admission are recommended if one of the following criteria is present: gcs < , systolic arterial pressure (sap) < mmhg, spo < %, ketosis > mmol/l, hco < mmol/l, ph < . , potassium level < . mmol/l or anion gap > mmol/l. however, it is suspected that adhesion to recommendations remains low. in this study, we aimed at describing patients admitted for diabetic ketoacidosis in icu. we looked at adhesion to published recommendations regarding admission and care. we also described metabolic complications and looked for an association between complications and dose of initial insulin therapy. complications hypoglycemia (< . mmol/l) was observed in % of patients within the first h in which % were < . mmol/l. this was and % of patients between and h of icu stay. hypokalemia below . mmol/l happened in % of patients within the first h and in % between and h. neither hypoglycemia nor hypokalemia were correlated with initial insulin bolus or initial dosage of continuous intravenous insulin. hypophosphatemia < . mmol/l was observed in % of patients. discussion in this study, admission to icu was consistent with british recommendations since most patients presented at least one clinical or biological criterion indicating icu admission. arterial blood gas were sampled in the large majority of patients despite consistent data showing that venous blood gas might be sufficient in non-hypoxemic patients. also, initial insulin bolus and sodium bicarbonate perfusion were performed in a significant subset of patients despite absence of convincing data or recommendations supporting their use. finally, significant hypokalemia and hypoglycemia were frequent in these patients. these complications are in theory favored by insulin therapy but we did not observe a correlation between administration of an insulin bolus or the dose of continuous intravenous insulin perfusion. conclusion in this retrospective multicentre study, patients admitted in icu for diabetic ketoacidosis were correctly oriented regarding the british recommendations. metabolic complications (hypoglycemia and hypokalemia) were frequent but not correlated with initial dose of insulin. the appropriate rate for hypernatremia (h-na) correction is unknown. under-correction could be associated with worse outcome. experts recommend a rapid correction of acute (< days) and sever (> mmol/l) h-na with a rate of − mmol/l/h until na < mmol/l ( ). correction should be, therefore, obtained within h. in patients with septic shock resuscitated with iso-or hypertonic saline and who acquired acute severe h-na, we assessed if the correction rate was associated with mortality. patients and methods data are a post hoc analysis of the rct "hyper s" database comparing normal to % saline for h in septic shock. serum na (sna) was measured at h , every h for days and ) . h-na correction rate was more rapid in non-survivors, p = . (table ). over-correction occurred similarly in survivors ( %) and non-survivors ( %). the time to reach sna normalization was shorter in nonsurvivors (p = . ). after adjustment for sapsii and maccabe scores, more rapid correction rate remained significantly associated with mortality: or . ; % ci ( . - . ), p = . . conclusion in the context of acute severe h-na induced by fluid resuscitation, a rapid correction rate might be associated with even aggravated rather than improved mortality. introduction systemic capillary leak syndrome (slcs) is a rare disease characterized by recurrent life-threatening attacks of capillary hyper permeability in the presence of a monoclonal gammopathy (mg). during acute episodes, the leak of fluid and proteins from the intravascular compartment to the interstitium results in clinical signs of both acute hypovolemia and interstitial edema. biological profile is pathognomonic with marked hemoconcentration and paradoxal hypoproteinemia. hypovolemic shock is the classical feature of severe scls attacks. however, beside this typical hemodynamic profile, several case report described myocardial dysfunction during scls attacks. the objectives of this study were to assess frequency, characteristics and outcome of myocardial involvement during severe scls attacks. ( %) mechanical ventilation, ( %) renal replacement therapy, ( %) veno-arterial extracorporeal membrane oxygenation, ( %) intra-aortic balloon pump and ( %) an impella. compartment syndrome occurred in ( %) patients and ( %) died in icu. we then compared the patients with myocardial involvement to the without clinical and biological manifestations were similar in between groups. however, chest pain ( vs %, p = . ), dyspnea ( vs %, p = . ) and respiratory failure ( vs %, p = . ) were more frequent in patients with myocardial involvement than in others. there was no difference between groups regarding treatment received in icu, complication and outcome except for the use of va-ecmo ( . vs %, p = . ). conclusion myocardial involvement seems frequent in patients with severe scls attack, occurring in % of the cases. such patients exhibited classical features of scls attacks. myocardial involvement was responsible for altered lvef or transient ventricular hypertrophy. myocardial dysfunction could be severe, even requiring mechanical circulatory support. scls attacks should be known as a cause of severe reversible myocardial dysfunction and hypertrophy. none. introduction in refractory cardiorespiratory emergencies, ecmo appears a good alternative to conventional treatment. its extracorporeal circuit justifies curative anticoagulation explaining haemorrhagic and thrombotic complications. activated clotting time (act) is empirically and commonly used to assess anticoagulation but with large inter and intraindividual variabilities. in practice, antixa activity dosage is available to approach anticoagulant effect of heparin and is less expensive, but data during ecmo are missing. we sought to demonstrate the lack of correlation between antixa and act in patients under ecmo support. we prospectively include patients supported by ecmo in chu toulouse, france, between / and / for circulatory/respiratory support. anticoagulation was achieved by unfractionated heparin: initial bolus then continuous intravenous infusion ( - iu/h), for antixa target of . - . . concomitant dosing of antixa (laboratory) and act (hemocron ® ) was conducted two times a day on the same sample throughout the ecmo period. relationship between act and antixa was analyzed by spearman correlation (rho). after transformation into categorical variables (obtained target = ; outside the target = ), analyzes were completed by a concordance study (kappa). as recognized on literature act's targets were between and . results patients were included: men ( %), median age yo ( - ). indications were veno-arterial (n = ) and veno-venous ecmo (n = ). ecmo median duration was days (hours to days). spearman correlation test found low and inconsistent correlation between antixa and act (rho spearman < . ). this correlation lack present from the day one, worsens over time. analyzed kappa showed no discrepancy between the areas "targets" of act and antixa confirming the results (table ) . conclusion use of act for ecmo anticoagulation monitoring doesn't seem appropriate and high price probably justifies preferential use of antixa in clinical practice. analyzes of relationships between antixa and bleeding/thrombotic events are needed to confirm the antixa place and its target in these indications. introduction postcardiotomy cardiogenic shock (cs) has an incidence of % to % after routine adult cardiac surgery. in . - . % of cases, an venoarterial extracorporeal life support (va-ecls) is requested. the -month survival rate is . % ( ). survivors may suffer of physical and psychological impairments as well as an alteration of quality of life. this study was designed to assess the outcomes, long-term health- since icu discharge, % of patients reported physical sequelae., ecls-related limb pain occurs in % of patients while paresthesia occurs in % and chronic-tiredness in %. mean karnofsky score was % (table ) . conclusion after va-ecls for postcardiotomy cardiogenic shock longterm physical and psychological sequelae are frequent in survivor discussion interest for fluid management is growing in critical patients. nevertheless, no study has yet investigated its impact in selected patients with cardiogenic shock treated with va ecmo. our study suggested a possible association between fluid overload and mortality but lack the power to confirm these results with multivariate analysis. conclusion fluid management is a key therapy during va ecmo but fluid overload could be associated with worsen outcomes. further studies with larger population are warranted before considering fluid restriction trials. introduction extracorporeal life support (ecls) has taken an important place in the treatment of cardiogenic shock (cs) or refractory cardiac arrest (ca). however, ecls deplore a high mortality rate in the first days raising important ethic and economic consequences. in this context, continuation of support should be reassessed precociously. the aim of this study was the research of prognostic factors of -days mortality, h after ecls implantation for cs or ca. materials and methods all patients undergoing ecls in our tertiary center during a -year period were prospectively included. the ecls were managed with a multidisciplinary protocol based on consensus. clinico-biological data were collected just before and h after ecls implantation. these data were compared between survivors and deceased at month. , cpc score was respectively for patients, for , for . at months, cpc score changed only for the patients with a cpc score at (one died after another suicide attempt, one changed his cpc score to ). in the group without ca (n = ), had normal neurological status at months and at months (one patient died because of a cancer). among these patients, % returned at home and % returned to work. ( %) patients re-attempted suicide in the year. the major risk factor of mortality is the presence of a cardiac arrest on hanging site. all the other factors found to be related to mortality are well known risk factors in cardiac arrest of other origin. in univariate analysis, risk factors of neurological sequelae at months were a cardiac arrest on hanging site (p = . ) an elevated diastolic blood pressure ( vs mmhg; p = . ), a lower initial glasgow score ( vs ; p = . ), and an elevated blood glucose ( . vs . g/l p < . ) at admission in icu. discussion our cohort of self-hanging patients can be divided in two parts: a) patients with ca in the pre-hospital period with a high mortality and a good neurological recovery in / surviving patient, but with a small group; b) patients without ca with a very low mortality and a very good neurological recovery. these results seem to be better than in the most important cohort [ ] published until now in self-hanging patients without ca and not treated by hbot (mortality at . % and . % of poor neurological recovery). conclusion patients surviving a self-attempted hanging who have not presented ca and treated by hbot have mainly a good neurological outcome. randomized control study should be undertaken to confirm hbot effectiveness in that indication. introduction venoarterial extracorporeal membrane oxygenation (va-ecmo) is increasingly used to treat refractory cardiogenic shock or cardiac arrest. acute brain injury (i.e. ischemic stroke, haemorrhage and/or failure to awaken because of diffuse brain injury) may occur in up to % of patients on va-ecmo and is associated with increased mortality and poor functional outcome in survivors. however, early indicators of neurological outcome are lacking in this population. we aimed to assess the prognostic value of early electroencephalography (eeg) alterations during va-ecmo. we conducted a prospective single-center study in the medical icu of a university hospital on consecutive patients cannulated to va-ecmo. a standardized clinical neurological evaluation including the rass score, the gcs score, the full outline of unresponsiveness (four) score and brainstem reflexes was coupled to an intermittent eeg. eeg was recorded as soon as possible within the first h after va-ecmo cannulation. eeg characteristics were analyzed by a neurophysiologist who was blinded to the patient's condition. a severely altered eeg pattern was defined as a predominant delta frequency, discontinuous, unreactive and/or an isoelectric background. the primary endpoint was poor neurological outcome, defined as the composite of death or acute brain injury on neuroimaging within days. data are presented as median (interquartile range) or number (percentage). false-positive rates (fprs, corresponding to -specificity) of poor neurological outcome were calculated for each significant predictor, using an exact binomial % confidence interval (ci). results sixty-nine (age ( - ) years) patients with a sofa score of ( - ) were included. main indications for ecmo were: post cardiac surgery (n = , %), terminal dilated cardiomyopathy (n = , %), and acute myocardial infarction (n = , %). cardiac arrest before ecmo cannulation was noted in ( %) patients. eeg was recorded ( - ) days after va-ecmo cannulation and ( %) patients were sedated at time of eeg. at day , ( %) had a poor outcome (n = deaths and n = patients alive with acute brain injury). in univariate analysis, a lower rass score (p = . ), a lower four score (p = . ), a lower score on the motor component of the glasgow coma scale (p = . ), and a lack of cough reflex (p = . ) at the time of eeg were significantly associated with a poor outcome. a severely impaired eeg pattern or presence of a discontinuous background activity were also associated with a poor outcome (p = . and p = . , respectively). indicators of poor neurologic outcome are presented in the table . among all parameters, a discontinuous background activity was the only variable that constantly predicted poor outcome (false-positive poor outcome prediction rate of %, % ci - %). conclusion early intermittent eeg has a strong prognostic value for sedated patients on va-ecmo. presence of a discontinuous eeg background activity seems to be more accurate than clinical alterations to predict a bad neurologic outcome at days. none. table ). it was not found a significant association of ctp to mortality ( % in the case group and % in control group, p = . ). other factors that increased mortality were coma, seizures, shock, oedema, cellularity in csf > units/mm . otherwise, the ventilation length was prolonged with ctp group ( . vs . days, p = . ) and neurological sequels namely the epilepsy was more frequent with the group ctp: ( vs %, p = . ). conclusion the occurrence of ctp on bacterial meningitis was significantly associated with ct scan lesions which seems to be an association be in both directions. also, the positive culture predisposed more to the ctp. mortality was higher with the presence of ctp but without real significance. the ctp was a factor that extends the ventilation time and exposed to the post infectious epilepsy. introduction acute bacterial meningitis requires rapid triage and therapeutic decision-making. the aim of this study was to assess the overall ability of a point-of-care glucometer to determine bacterial infection in cerebrospinal fluid (csf). we performed a prospective, observational study. we included patients for whom an analysis of csf was indicated by the physician in charge with blood sampling performed for glucose concentration measurement within h. we simultaneously measured the glucose concentrations in csf and blood using a central laboratory and point-of-care glucometer. the diagnosis of bacterial meningitis was determined by two physicians after reviewing the complete medical chart. we compared csf and blood glucose concentrations and csf/blood glucose ratios obtained at the bed-side with a glucometer versus those obtained by the central laboratory. we determined the performance characteristics of the csf/blood glucose ratio provided by a glucometer to detect bacterial infection in the csf immediately after csf sampling. conclusion we demonstrated that the csf/blood glucose ratio measured by a glucometer can serve as a clinical decision support tool for the early detection of csf with a high probability of bacterial infection. this costless point-of-care method has the potential to expedite medical decision-making for the triage of adult patients with suspected meningitis in the emergency department immediately after lumbar puncture. none. introduction cardiac arrest remains a frequent cause of admission in intensive care unit. a majority of patients will die during their hospital stay mainly from consequences of hypoxic-ischemic brain injury after a decision of withdrawal of life sustaining therapy support by a prediction of poor outcome. the reliability of prognostication is crucial, but is still a difficult and uncertain exercise. eeg is the most widely used prognostic tool to support a clinical examination and is accessible in most hospitals. it is recommended for both prognostication and ruling out subclinical seizures. there is no high-level evidence for predicting poor prognosis using eeg because of the wide variety of classification systems used and the interrater variability. our objective is to assess the prognostic value of simple eeg features based on the recent american clinical neurophysiology society (acns) standardized classification and to study the interrater variability. we conducted a retrospective monocentric observational study in a bed medical intensive care unit of the university hospital la timone, marseille, france. all patients aged of more than year-old admitted for a resuscitated cardiac arrest between november and july who underwent therapeutic hypothermia and a full multimodal prognostic evaluation including a eeg were included in the study. outcome was classified according to the cerebral performance category score measured at day . unfavorable outcome was defined as death (cpc ), persistent vegetative state (cpc ), or severe neurological disability (cpc ). favorable outcome was defined as moderate neurological disability (cpc ), or no disability (cpc ). eeg was performed in all patients still comatose after rewarming between and h after admission and after discontinuation of sedation. eeg interpretation was made by independent senior neurophysiologists, blind to the outcome. eeg features are based on the latest acns classification. for each eeg feature, sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) for predicting an unfavorable outcome were calculated. results during the study period, cardiac arrest were admitted of which patients went through a full neurologic evaluation and were finally included in the study. according to neurological outcome, % had a favorable evolution, and % had an unfavorable outcome. the presence of burst suppression, and epileptiform activity was constantly associated with an unfavorable prognostic with a % specificity and % false positive. a non-reactive eeg is strongly associated with an unfavorable evolution with a % specificity and % false positive. other features including periodic or rhythmic patterns and low voltage were inconstantly associated with unfavorable outcome. kappa score for all eeg feature was slight or fair and always under . . discussion this study allowed us to identify a homogenous cohort of comatose patient after cardiac arrest who underwent therapeutic hypothermia. we identified simple eeg features based on the new classification of the acns constantly associated with unfavorable outcome. these features must be known by intensivists to better integrate eeg in the multimodal evaluation of neurological prognostic. there is important interrater variability that must lead to caution and to always use multimodal approach to prognostic an unfavorable outcome. conclusion bedside eeg is an excellent tool for predicting outcome of post-anoxic coma through simple eeg features. burst suppression, epileptiform activity and non-reactive eeg are strongly associated to neurological outcome after cardiac arrest. however, the interrater variability emphasize the need of being well trained for the standardized methods of evaluating eeg parameters. introduction emergent reintubation is a well-known risk of laryngotracheal trauma and of ventilatory acquired pneumonia. to precisely define its risk before extubation for each patient is a part of quality of care in intensive care units. none of these consecutive children representative of picu activity has been reintubated. the coming prospective muticentric study which aims to validate alt in childhood must precisely define this criteria of evaluation. conclusion the different methods of alt are feasible in real clinical conditions in picu. because of the increasing use of cuffed etts in a wide variation of patients with different body weight, the best alt to use at the bedside must be definitively validated in this population. introduction prolonged mechanical ventilation (pmv) and chronic mechanical ventilation (cmv) in neonates is associated with a high morbidity and mortality. the objective of the study is to identify, among the patients with pmv, those that evolved to cmv, as well as the adverse respiratory, neurological and feeding sequelae. we conducted a retrospective study of the last years at the chu sainte-justine (montreal, canada). chart review included patients with pmv (≥ days) using the paediatric definition adapted from the namdrc consensus conference ( ) . demographic and clinical data, including follow-up at and months corrected age, was collected for each included patient. the evolution of pmv neonates with cmv (≥ days) and without ( - days) was compared. we identified neonates that met criteria for pmv. patients born between and (n = , % of the cohort) were analyzed. around half of the patients ( - patients a year) are transferred from the neonatal unit to the paediatric intensive care unit. in our center, they represent around % of total admissions, but their length of stay is among the longest. among these newborns, % were preterm (n = ) with % (n = ) born before weeks gestation. of all patients with a malformation ( %, n = ), had a thoracoabdominal anomaly and had congenital heart disease. thirty-six patients had cmv with mean ventilation time of days (range - days). survival at months corrected age was % ( / ) in the pmv group and % ( / ) in the cmv group. at months corrected age, % of patients were dependent on artificial enteral feeding (nasogastric tube or gastrostomy), with % in the pmv group and % in the cmv group. nine percent of patients had oxygen supplementation ( patients in the pmv group and in the cmv group), and % were mechanically ventilated. ten percent of patients had a tracheostomy ( patients in the pmv group and in the cmv group). discussion neonates with cmv have more sequelae. their rapid identification (at days of ventilation) is essential to implement multidisciplinary development care in order to minimize neurodevelopment impairment. conclusion most newborns in our pmv cohort have a congenital malformation. survival at months corrected age appears equivalent in both pmv and cmv group. artificial enteral feeding is more frequent in the cmv group and most patients have no respiratory support at months corrected age. none. the value of pressures and volumes in assessing the fluid responsiveness depend on the systolic cardiac function in adult ( ). we have studied the relative value of static filling volume and pressure to predict the fluid responsiveness, according to systolic cardiac function in children during acute circulatory failure. patients and methods patients under years old with an acute circulatory failure of two intensive care units during a year period of inclusion were analyzed. an exhaustive cardiac echography was performed initially (indexed end-diastolic volume (edvi) and e/e' from transmitral and tissue doppler were recorded), and the stroke volume index (svi) was measured before and after a fluid challenge (a ml/ kg of crystalloid over min results twenty-five children with acute circulatory failure were included. fluid responsiveness occurred in of the fluid loading events with low lvef, and in of the fluid loading events with normal lvef. pressure approach: for low and normal lvef, the auc-roc for fluid responsiveness was respectively . (ci . - )/ . ( . - ) for a e/e' .the best thresholds of e/e' in low lvef was . with a sensitivity of (ci - ) % and a specificity of (ci - ) %. for low and normal lvef auc roc was respectively . (ci . - . )/ . (ci . - . ) for the pvc. volume approach: for low and normal lvef, the auc-roc for fluid responsiveness was respectively . (ci - ) and . ( . - ). the best thresholds in normal lvef was an edvi below ml/m wit a specificity of (ci - ) and a sensitivity of (ci - ) %. discussion our study shows a variation of the diagnostic value of e/e' and edvi according to the left ventricular systolic function. therefore, the systolic function should be taken into account to analysed the e/e' and edvi value. few preload dependency markers are validated in children and none for children in spontaneous ventilation ( ) . our study suffers from a lack of power that calls into question the validity of our results. another limitation is that both approaches with volume and pressure are not very discriminant as it is known for static value in adults. our study illustrates that, on a pressure-volume curve, when the cardiac inotropism is reduced, the filling of the left ventricle is moved to the up and right of the curvilinear diastolic function curve. therefore, pressure variations are larger than volume variations. these values should be monitored on a larger scale to define their exact diagnostic value. conclusion static pvc value is a low preload-dependency surrogate. when lvef is low a pressure evaluation based approach seems more accurate. when lvef is normal a volume evaluation based approach seems informative as predicted by the slope of the end diastolic pressure volume curve. those both static approaches remain of poor diagnosis accuracy. introduction acute viral bronchiolitis is a primary cause of respiratory distress in paediatric intensive care unit (icu). prone position (pp) is commonly used in neonates to improve respiratory mechanics and has been found beneficial to adult patients with acute respiratory distress syndrome. we aimed to evaluate the effect of pp on work of breathing as compared to supine position (sp) in children with severe bronchiolitis requiring non-invasive ventilation. the protocol was approved by our irb ( -a - ). fourteen infants ( boys) with median age days [firstthird quartiles - ] with severe bronchiolitis requiring cpap were included after written informed consent. children were investigated in pp and sp each applied for h in a random order with a washout period of min between them. level of cpap was set at cmh o in both conditions. oesophageal pressure probe was inserted orally (cto- pressure transducer, gaeltec, scotland) to measure oesophageal pressure. flow and airway pressure (pmo in fig. ) were simultanuously recorded using a neurovent data acquisition system (neurovent inc, toronto, canada). one hundred breaths were analyzed in each condition, in which work of breathing was estimated from oesophageal pressure-time product (ptpes) and oesophageal swings (fig. ). data were expressed as median (first-third quartiles) and compared by using the wilcoxon two-sample paired sign test. a p-value below . was considered significant. . the edtb contains data from ventilated patients (invasively and non-invasively) and details concerning ionotropic and sedative treatment during picu courses. discussion as far as we know, this edtb is currently the only one as exhaustive available in picu worldwide. after almost years of multidisciplinary collaboration, we are able to collect many useful physiological, therapeutic and medical data in an ongoing edtb. although many concerns remain concerning data validation, organisation and exploitation, this edtb already contribute to the development of clinical decision support systems and virtual patient validation and we create international collaborations to further develop these tools. three research protocols using the database are ongoing including: validation of a neuromonitoring clinical decision support system, validation of a cardio-respiratory simulator, developement and validation of the automatic diagnosis of pediatric acute respiratory distress syndrome and development of spo forecast using artificial neuronal network. conclusion thanks to informatics and electronic devices improvement, data gathering in intensive care units has empowered. we hope that our work in picu will encourage other teams on the way of data gathering, in order to build an international picu edtb in a close future. none. introduction severe trauma is rare in the pediatric setting ( % of all trauma in france). however, its morbidity and mortality remain high, in relation to brain injury. pediatric traumatic brain injury (tbi) prehospital care is challenging for non-pediatric retrieval teams. though, we disseminated pediatric tbi pre-hospital care regional guidelines and thereafter intended to assess severe pediatric trauma pre-hospital care and secondary cerebral insults control. we conducted a retrospective study in a single pediatric trauma center. children admitted in emergency room with severe trauma and moderate to severe tbi (glasgow coma scale ≤ ) from june to march were included. pre-hospital and hospital data regarding primary care, equipment, medications and secondary cerebral insults control (i.e. blood pressure, oxygenation, co level, temperature, glycemia) were collected from medical files. two pediatric transport team experts assessed the quality of pre-hospital care, based on two major endpoints. results twenty-nine files were analyzed. median iss was . all the children had been referred directly from the trauma scene to the pediatric trauma center. they were all intubated in the prehospital setting, ( . %) presented with spo < % before or at emergency room admission, and ( . %) presented with a pco > mmhg at admission. at least one peripheral catheter was inserted in all the children. mean total fluid bolus was . ml/kg (± ). nor-epinephrine was administered in ( %) children. mean blood pressure was below age threshold in ( %) children during transport or at admission. an intracranial hypertension treatment (apart from sedation) was delivered in ( %) children before admission. body temperature was monitored in patients and were hypothermic at emergency room admission. experts concluded on sub-optimal care in children: major endpoint was "respiratory care", "hemodynamic care" and "neurologic care" in , and patients respectively. discussion on this small series, we showed pre-hospital sub-optimal care regarding secondary cerebral insults control, especially regarding co level, blood pressure and body temperature. our results will help to design new care improvement strategies (e.g. sedation, fluid bolus and ventilation optimization, early use of vasoactive drugs, systematic body temperature monitoring…). conclusion data on pre-hospital secondary cerebral insults care are rare in the pediatric setting. based on our results, we aim to improve quality of care of children presenting with traumatic brain injury, and to reduce its morbidity and mortality. introduction unsuccessful extubation from mechanical ventilation increases mortality and morbidity. to reduce the extubation failures in our intensive care unit we used a mechanical ventilator weaning protocol, based on published data. during the first part of the study, risk factors and incidence of extubation failure were first described. afterwards in the second part, our mechanical ventilator weaning protocol was tested to determined its efficiency regarding the extubation failure. patients and methods a monocentric and observational study, was first conducted. we included children aged from birth to old, during a period of months and collected for each patient their medical history, intubation and extubation parameters, and existing events of extubation failure or extubation complication. the second part of the study was prospective, we include patients extubated by applying our mechanical ventilator weaning protocol. results average duration of mechanical ventilation was . h in the first part of the study. using a univariate analysis, duration of mechanical ventilation was a risk factor of extubation failure with an average duration of . discussion our study confirms published data about extubation failure risk factor like duration of intubation, chronic respiratory affection, history of previous intubation, and the administration of benzodiazepine. it is the first pediatric study that shows a reduction of extubation failure by using a specific mechanical ventilator weaning protocol. the mean bias of our its retrospective and prospective character. conclusion our study shows the interest of a mechanical ventilator weaning protocol to reduce the incidence of extubation failure. we currently continue the apply our protocol to include more patients in order to confirm our results. stroke of the child is formidable though it is ten times rarer than in adults, but this scarcity can have adverse consequences on the speed and quality of the management and the consequences on later psychomotor development. our goal is to describe the clinical and therapeutic aspects of these pediatric stroke while bringing our experience. patients and methods retrospective study of cases of children hospitalized in general intensive care unit to the pediatric hospital canastel oran for stroke during the period from january to january . the clinical, etiological, para clinical, and scalable were studied and transcribed on a standard electronic form.all patients had a brain ct. magnetic resonance imaging(mri) was possible in patients for lack of availability of the technical facilities during the study. results ten cases were selected. the mean age was months ( month to years), % are male, patients had a history of chd like tetralogy of fallot and complicated bronchiolitis myocarditis, one patient had a history of petechial purpura, other was a factor deficiency, headache history was noted in patients, and patients with no particular antecedent was found. all patients arrived comatose / score on the scale of glasgow, isochores reactive pupils with a motor deficit of hémicorps, patients have degraded their neurological score with onset of clinical signs of hypertension intra cranial namely anisocoria and hypertension requiring osmotherapy, sedation and mechanical ventilation with an average duration of - day. o child arrived brain dead, patients had generalized tonic-clonic seizures which yielded after taking a benzodiazepine (diazepam) and phenobarbital (like gardenal). cerebral ct was performed in all cases and could we revealed the nature of the stroke hemorrhagic in cases and ischemic stroke in cases. two patients have benefited from an mri that found a thrombosis of the artery internal carotid right sylvian. besides symptomatic treatment, treatment was initiated based on the type of stroke, patients received low molecular weight heparin (lmwh) at . ml/kg in addition to symptomatic treatment, patients received vitamin k. four patients died in an array of autonomic disorders and evolved favorably and six patients were transferred to a pediatric unit. the average length of stay in icu was . days ( - days). discussion the mortality rate is important since no specialized center for children, and difficulty especially in the diagnostic imaging field while suspected stroke should be confirmed by imaging and the diagnostic delay. which is due to a poor assessment of the initial situation in half of the cases by the parents, the other half by the swiss magazine consulté.une doctor showed that in a study in % of children with stroke, this diagnosis was not primarily discussed and that in % of cases the cause of the stroke was poorly evaluated [ ] . heart disease certainly represent the second most important risk factor. a collaboration of a team must be multidisciplinary, death has affected mostly older children whose age is between and years, who have a hemorrhagic stroke against by infants who have an ischemic stroke have evolved and oriented they exceed the acute phase to pediatric services for further investigation and monitoring. conclusion the child may also be having a stroke, which usually reaches the elderly. this justifies a good knowledge of this disease, and multiply the initial management efforts to reduce mortality and improve prognosis. anwar armel , benqqa anas , samira kalouch , khalid yaqini , aziz chlilek introduction nosocomial infections are a main problem for public health for their cost as well as for the morbidity and mortality they generate. they are particularly common in intensive care units due to patient's lower defenses and of invasive procedures proliferation. work's purpose: • determine the epidemiology of bacterial noso-comiales infections (ibn) in the medico-surgical pediatric intensive care department of children's university hospital of casablanca. • to identify factors associated with these infections. we led a retrospective study of hospitalized patients, spending more than h in medical-surgical pediatric intensive care department, at the university hospital ibn rochd of casablanca, over a period of months from january to december . results during the studied period, patients were admitted at intensive care with a stay of more than h. thirty episodes of inb were recorded. the incidence rate was . % and the incidence density was . % per hospitalization's days. the admission average age was . ± -month starting from month to years with a male predominance ( %). most of admissions ( %) was related to medical background, . % received from other hospital department. furthermore, % of the patients received prior antibiotics, usually prescribed before icu admission. invasive procedures (intubation, central catheterization) were used in . % of patients, vvp only in . %, tracheotomy in . and . % had received surgery. gram-negative bacilli (bgn) were isolated for a lot of patients, dominated by acinetobacter baumannii. these bacteria were isolated throughout the study year. risk factors analysis underlined that the presence of invasive procedures enhances in risk, that is central venous catheter and the need for mechanical ventilation. conclusion nosocomial bacterial infections are dominated by pneumonia and central catheter infections, and are mainly due to bgn. the factors associated with these infections were identified. the guillain-barré syndrome (gbs) is the most common cause of acute flaccid paralysis in children since the acute anterior poliomyelitis eradication. few studies have been held on the topic and knowledge of gbs in children, although it is recognized that the etiologic mechanisms, and clinicobiological background, are the same as in adults, prognosis remains different. our work's aim is to study this disease's mortality factors of children hospitalized in pediatric intensive care. patients and methods it is a retrospective, descriptive, mono centric study to review patients with gbs between january and december and hospitalized at pediatric intensive care department of abderrahimharouchi hospital of casablanca. the used software is spss . to compare the bivariate variables, we used the khi test, and to compare quantitative variables, the anova to factor test was used. the level of significance was fixed at % with % confidence interval. the disease was predominant in male with a sex ratio of . men/women. after a prodromal event, usually infectious ( . %) and a free interval of days on average to start motor disorders. these are of two types: either a hypo or areflectic flaccid paralysis of the lower limbs ( . %) of ascending evolution in . % of the cases. either flaccid tetraplegia or hypo areflectic, ( . %). ventilation was required in . % of the cases, and specific treatments based on immunoglobulins were administered in . % of the cases. death's rate is still high ( . %) and mainly due to hospitalization complications. in our study respiratory disease was noted in . % of the cases, also other signs of serious illness such as swallowing disorders ( . %) and autonomic disorders ( . %) also noted what led to management in intensive care for all our patients. these patients study allowed to identify some mortality prognosis factors of the disease in intensive care units (such as male gender, ig administration duration, the occurrence of autonomic disorders like blood pressure instability), the most discriminating remains the occurrence of nosocomial infections. conclusion it must be underlined, that in view of our strict inclusion criteria, focusing only on patients admitted at intensive care and of the relatively small sample size ( cases), our results must be qualified and must be enhanced by additional and more varied studies to better understand this disease in children. introduction early surgical treatment is recommended for refractory intracranial hypertension (htic) in children to improve vital and functional prognoses, whether traumatic or vascular cause. the main objective of this study was to compare the mortality and morbidity of children with severe intracranial hypertension after severe head trauma (tc) or due to vascular cause after decompressive craniectomy (dc) or medical therapy alone. the secondary objective was to identify the initial severity factors associated with higher mortality. patients and methods a retrospective study was performed with data collected from patients aged under years-old admitted to our pediatric intensive care unit for severe intracranial hypertension of traumatic or vascular cause, between january and january . they were divided into groups: patients who received medical therapy alone and those treated with decompressive craniectomy after optimal medical management. results a total of children were included. among them, were treated with dc ( htic of vascular cause and htic of traumatic cause), and were supported by medical means only ( htic of vascular cause and htic of traumatic cause). in the population "traumatic intracranial hypertension", we note that children in the "dc" subgroup are more often in mydriasis upon arrival (p = . ) than in the subgroup treated medically. in this same population, children in the "dc" subgroup received higher doses of mida-zolam (p = . ), of mannitol (p = . ) and hypertonic saline (p = . ) than in the other subgroup. in the population "vascular intracranial hypertension" the two subgroups were comparable. in the case of traumatic intracranial hypertension, mortality rate in the "dc" subgroup was . % against . % for children treated medically (p = . ); "dc" children had more metabolic complications such as hypernatremia than "not dc" children, p = . . mortality rate in the «vascular intracranial hypertension» group was % for children treated with decompressive craniectomy, and . % for children treated medically alone (p = . ). patients treated surgically in the «vascular intracranial hypertension» group had longer overall stays (p = . ) and longer icu stays (p = . ). popc score (pediatric overall performance category) upon discharge for children with intracranial hypertension of traumatic cause treated with decompressive craniectomy was . ± . against . ± . among children treated medically, p = . . in "dc" children with intracranial hypertension of vascular cause, popc upon hospital discharge was . ± . against . ± . among non-operated children, p = . . the schooling rate was higher among children treated medically for intracranial hypertension of traumatic cause, p = . . the severity factors related with higher mortality identified in the population "traumatic intracranial hypertension" were mydriasis upon admission, a pim score higher and a lower temperature (< . °); the latter being the only factor identified for htic of vascular cause. in the case of traumatic intracranial hypertension, icp monitoring in survivors was . % against . % in children died, with no significant difference. in the population "vascular intracranial hypertension", all the patients who died had not been monitoring pic. discussion the severity factors related with higher mortality identified in the population "traumatic intracranial hypertension" were mydriasis upon admission, a pim score higher and a lower temperature (< . °); the latter being the only factor identified for htic of vascular cause. other studies have related other severity factors as initial glasgow scale, tardive decompressive craniectomy. conclusion decompressive craniectomy doesn't seem to improve the mortality rate or the outcome in patients with hypertension of traumatic cause in our study but the dc traumatic subgroup was more serious than the subgroup treated medically. in children with refractory intracranial hypertension of vascular cause dc significantly improves survival and outcome. further studies are needed to clarify the role of decompressive craniectomy and its timing in the therapeutic management of refractory intracranial hypertension. introduction shortage of heart grafts is a major problem, leading to a significant mortality rate in the national waiting list, essentially for young children with low weight. the potential paediatric brain-dead donors often have myocardial dysfunction (md), which seems to be reversible. the aim of this study is to assess prevalence, causes and consequences of md when the potential paediatric donors are taken over, up to multi-organ retrieval, and the evolution after cardiac transplantation. materials and methods this observational, monocentric, retrospective study included all brain-dead children aged - years old, who had their myocardial function assessed through a cardiac ultrasound performed by a cardiologist and identified from to . all adult patients and those who didn't undergo a cardiac ultrasound were excluded. md was defined as an lvef ≤ % with or without abnormal segmented cinetic parameters. the main evaluation criteria was the prevalence of md in potential identified donors. the secondary evaluation criteria were the causes and consequences of md on heart retrieval and the origin of this md. results out of included patients, had md. prevalence of md was of %. there was no significant difference between groups regarding aetiology of brain death nor administration of catecholamines. having a cardiopulmonary arrest during intensive care unit stay was associated with a significant risk of presenting a md (p = . ). having a md had no consequences on organ retrieval in general (p = . ), but was significantly associated with a decrease in heart retrieval opportunities (p = . ). the cause of heart grafts refusal was a poor ventricular function in % of cases ( cases out of ). the cause for non-retrieval was parental refusal in one-third of cases. evolution of the cardiac grafts was favorable in cases on , one transplanted patient died (from a non-cardiac cause) and patient was lost to follow up. conclusion md in paediatric brain-dead patients has direct consequences on heart retrieval and transplantation, and otherwise, organ shortage is a major ongoing problem. a better transplant management regarding hemodynamics (with the use of a protocol) could increase the number of heart transplants, especially in small children, and reduce mortality rate in national waiting list. the prone positioning (pp) is a strategy widely used in the treatment of severe forms of acute respiratory distress syndrome (ards) in adults. its early use significantly reduces mortality ( ). however, the studies do not strongly demonstrate its prognostic impact in pediatric ards. the aim of this study was to describe the prone positioning practices in the french-speaking pediatric intensive care units (picu). patients and methods this survey was conducted by email questionnaire to pediatric intensivists belonging to the french society of intensive care medicine and the french-speaking group of pediatric intensive care and emergency medicine. it was conducted from february to may . the survey was addressed to doctors, nurses, physiotherapists practicing in picu. it included questions about indications, contraindications, techniques and medical devices used, and complications. results one hundred and three persons answered ( doctors and nurses) which work in french hospitals and canadian hospital. sixty-eight percent of interviewed persons have more than years experience and % of them treat each year more than children ards. only % of the picu have a pp medical protocol. fifty percent of interviewed persons frequently use pp for the medical care of ards and % systematically use it. thirty-six percent begin pp at the early phase of ards during conventional ventilation, while % before the introduction of unconventional ventilatory strategies (ohf); only % use it after the respiratory failure unless unconventional ventilatory strategies. seventy-three percent report that pp is used with prolonged periods (> h/day), % with short periods (< h/day) and % with very long periods (> h/day). regarding the weaning criteria, most of interviewed persons seem to use multiple and combinated criteria: % use hypoxemia severity parameters (pao /fio , pao , sao ), % use the oxygen level (fio ) and % use the mechanical ventilation parameters (peep, p max, p plate). finally, despite a low level of scientific evidence in children, % of the persons gave a strong recommendation for pp as standard care in severe pediatric ards. see fig. . the survey confirmed the widely use of pp in pediatric ards. however, no specific protocol is avalaible in most of the picu. the timing of the pp beginning can be different according to children, early and prior to use of the conventional ventilation strategy in most cases. the duration of pp seems more consensual. most of the centers use extended periods longer than h/day. these results are close to guérin et al. advocating a duration > h/day. finally, the weaning is a great issue and depends on multiple criteria. in guerin et al. ( ) pp was interrupted if one of the following criteria were present: pao / fio ≥ mmhg, with peep of ≤ cm of water and a fio of ≤ . ; decreased pao /fio than %, compared to compared to the supine position, or the occurrence of complications. no study has validated pp weaning criteria during pediatric ards. conclusion the prone positioning is a strategy commonly used in pediatric intensive care units for the severe pediatric ards. the criterias of implementation and timing are variable, as well as the weaning criterias. more pediatric multicenter randomized studies will be necessary to confirm the benefits of pp in pediatric ards and to define clear weaning criteria. introduction allogeneic hematopoietic stem cell transplantation (hsct) recipients have profound defects in every immunity compartments that can lead to severe opportunistic infections (oi). % of hsct patients require admission to the icu because of diverse infectious or non-infectious complications with dismal outcomes. oi specific course in this population has not been described previously and the management of these infections may be a concern. the aim of this study was to investigate risk factors, management and outcomes of io in hsct recipients admitted to the icu. patients and methods this was a retrospective ( - ) single center study of patients admitted to icu after an allogeneic hsct. patients provided written informed consent according to helsinki declaration. data regarding the transplant, infections and life sustaining therapy use were analyzed. oi were considered if present at the time or during icu admission. results hundred and ninety-four patients (pt) were included. median age was [ ; ] years, . % were males. reason for transplantation was acute leukemia in ( %) pt and the hematological condition was still in complete remission at icu admission in % of patients. ( %) and ( %) had received a myeloablative conditioning regimen and anti-thymoglobulin serum respectively. % had acute graft versus host disease over grade at icu admission. oi was documented in patients ( %). an invasive fungal infection (ifi) was found in pt owing to mucormucosis, trichosporon septicemia and invasive aspergillosis ( possible, probable and proven according to eortc criteria). serum galactomannane antigen was positive in ( %). median time from transplantation and icu admission to ifi diagnosis was respectively [ ; ] and − [− ; ] days. lung was involved in % and patients with aspergillosis were admitted to the icu for acute respiratory failure in % (vs. % for others p = . ). they did not required invasive ventilation more frequently ( vs. % p = . ). and % required vasopressors and renal replacement therapy with no difference as compared to others. median icu length was [ ; ] days. demographic, stem cell source, and donor type were not associated with ifi occurrence in this population. however / had received a total body irradiation ( vs. % p = . ). ifi occurrence was not associated with icu or day mortality ( vs. % p = . and vs. % p = . respectively). a viral infection was found in pt owing to cmv, adenovirus, hsv and vrs infections. analyses were focused on cmv reactivation. median time from transplantation and icu admission to cmv reactivation was respectively [ ; ] and − [− ; − ] days. reactivation was mainly positive blood pcr but pt had cmv colitis. a preemptive treatment was started on the same day in median and lasts [ ; ] days. patients with cmv reactivation had more frequently multiple organ failure ( vs. % p = . ) and higher icu admission sofa score ( [ ; ] vs. [ ] [ ] [ ] [ ] [ ] [ ] p = . ). they trend to have higher admission creatinine serum level ( [ ; ] vs. [ ; ] umol/l, p = . ) and more frequently required emergency renal replacement therapy ( vs. % p = . ) mechanical ventilation ( vs. % p = . ) and vasopressors ( vs. % p = . ). median icu length was [ ; ] days and comparable to others. demographic, stem cell source, conditioning regimen and donor type were not associated with cmv occurrence. cmv reactivation was not significantly associated with icu or day mortality ( vs. % p = . and vs. % p = . respectively). conclusion oi was found in % of allogeneic hsct recipients admitted to the icu. ifi were mainly responsible for respiratory distress and cmv associated to multiple organ failure. non-invasive diagnostic tests were positives in a majority of these patients. in this cohort, io treatment was started quickly after the diagnostic and we did not find an association with mortality. intensivists should always consider oi in their diagnostic panel in this specific population. introduction over the last two decades, targeted therapies in patients with solid tumors have both increased their length of survival and significantly altered their immune functions. however, data on opportunistic infections in this setting remain scarce. in this systematic review, we sought to identify published cases of opportunistic infections in patients with solid tumors, with a special interest on clinical findings, trends over time and outcomes. materials and methods we performed a search of medical subject headings (mesh) on pubmed using the words pneumonia pneumocystis (pcp), invasive aspergillosis (ia), histoplasma, mucor, geotrichum, cryptococcus, coccidioidomycosis combined with the mesh term neoplasms (breast, lung, ovarian, urologic gastrointestinal, digestive system, abdominal, brain, carcinoid tumor, sarcoma, testicular, seminoma). we identify published cases of opportunistic infections in non hiv patients with solid tumors between / / and / / included. results regarding pneumocystis jirovecii pneumonia, cases could be identified. there were men and women, aged of . ( - ) years. underlying tumors were chiefly brain neoplasms (n = , %), lung neoplasms (n = , %) and breast neoplasms (n = , %). at the time of pneumocystis pneumonia onset, patients ( %) had a history of chemotherapy, ( %) had received long term or high dose steroids, and ( %) had an history of biotherapy targeting the malignancy. of note, patients ( %) had received only chemotherapy, ( %) had received steroids alone, ( %) everolimus therapy alone and ( %) received none of these treatments. regarding invasive aspergillosis cases could be identified. mean age was . ( - ) and ( %) were men. solid tumors associated with invasive aspergillosis were primarily lung neoplasms (n = , %) and brain neoplasms (n = , %). at aspergillosis onset, ( %) patients had a history of chemotherapy, ( %) were receiving long term or high dose steroids and ( %) had received targeted therapy. fourteen ( %) patients had received only chemotherapy, ( %) only steroids, and ( . %) had received targeted therapy alone. for both infection, there was a trend for a higher number of reported cases throughout the studied period. conclusion this systematic review provides objective data showing that an increased proportion of patients with solid tumors present with opportunistic infections. we are convinced that it is a clinically relevant but still neglected problem. selected oncologic population may be becoming eligible for antimicrobial prophylaxis against pneumocystis or aspergillus. care unit of strasbourg in france. patients were included only if they are non-immunocompromised according to the european organisation for research and treatment of cancer (eortc). invasive aspergillosis was defined as an association of microbiological evidence, a radiological imaging and a clinical context. results eighteen patients ( males) were identified during the study period. the median of igs ii was . (interquartile range (irq), . - . ). ninety-four percent was under mechanical ventilation. fourteen ( %) patients were suffering from liver failure. among liver failure, twelve ( %) were beforehand suffering from cirrhosis. the median meld score was (interquartile range (irq), - ). sixty-four percent of aspergillosis were due to aspergillosis fumigatus. hundred percent were pulmonary aspergillosis. fifty-six percent of aspergillosis were associated with bacterial pneumonia. the mortality rate at the date of the latest news (an average of years) was seventytwo percent. discussion invasive aspergillosis is not exceptional in the non-immunocompromised patient especially in patient developing liver failure. an active research of colonization/infection with aspergillus in these patients remain to be discussed. conclusion invasive aspergillosis in icu has a poor prognosis. the liver failure seems to be the most important risk factor in non-immunocompromised patients according eorct criteria. introduction chest wall elastance (ecw) has been found to increase in prone (pp) as compared to supine position (sp) in ards patients [ ] . this makes respiratory system elastance (ers) not reflecting lung elastance (el). little is known about the changes of ecw, el and lung resistance (rl) when moving the patient from the sp to the pp via the lateral position (lp). the goal of present study was to measure ecw, el and rl in ards patients in sp, lp and pp during the proning procedure. patients and methods it was a prospective, single-center, controlled study. ards patients intubated, sedated and paralyzed with pao /fio ratio < mmhg, peep ≥ cmh and an indication of pp were included. mechanical ventilation was delivered in volume controlled mode with constant flow inflation and end-inspiratory pause . s included into the inspiratory time. ventilator settings were unaltered during the procedure. an esophageal balloon catheter (nutrivent device) was used for esophageal pressure (pes) measurement. pressure at the airway opening (pao) and airflow were measured by fleish pneumotachograph proximal to endotracheal tube and upstream heat and moisture exchanger. pao, pes and airflow were continuously measured during min in sp, then during min in lp and min in pp. the side for the lateralization was that selected by routine practice (in the opposite side from central venous line). ers and resistance of the respiratory system (rrs) were obtained by fitting flow and pao signals breath by breath to the first order equation. ecw and resistance of the chest wall (rcw) were similarly obtained by fitting flow and pes signals breath by breath to the first order equation pertaining to the chest wall. el and lung resistance (rl) were obtained by subtracting ers and rrs from ecw and rcw, respectively. our ethical committee approved the protocol. data are shown as median (first and third quartiles). comparisons between positions were made by using paired-t-test. results twenty-nine patients, males, of ( - ) years, saps ( - ) and sofa score ( - ) were included ( - ) days after ards criteria were met. the ards severity was moderate in cases ( %) and severe in ( %). tidal volume averaged . ( . - ) ml/kg predicted body weight, peep ( - ) cmh o, fio ( - ) %, pao /fio ( - ) mmhg. the cause of ards was pulmonary in cases ( %), extra pulmonary in ( %) and undetermined in ( %). lateral positioning was on the right side in ( . %) and on the left side in patients ( . %). the results are shown in the table . conclusion during prone positioning in ards patients, as compared to sp we observed a higher rl in lp and an increased ecw in pp. introduction neuromuscular blocking agents (nmba) could exert beneficial effects in acute respiratory distress syndrome (ards) through properties on respiratory mechanics and particularly in modifying transpulmonary pressures (pl). patients and methods prospective randomized control study in moderate to severe ards patients within the first h of the onset of ards. all patients were monitored by an esophageal catheter and followed during h. moderate ards patients were randomized in two groups according to the systematic administration of a h continuous infusion of cisatracurium besylate or not (control group). the severe ards patients group received a h continuous infusion of cisatracurium besylate. the evolution during the h of the study of the oxygenation and the respiratory mechanics including inspiratory and expiratory transpulmonary pressures and driving pressure were assessed and compared. delta transpulmonary pressure (∆pl) was defined as inspiratory pl minus expiratory pl. results thirty patients were included, in the moderate ards group and in the severe ards group. nmba infusion was associated with an improvement in oxygenation both the moderate and the severe ards patients group accompanied by a decrease in both the plateau pressure and the total positive end expiratory pressure. the mean inspiratory and expiratory pl were higher in the moderate ards patients group receiving nmba as compared with the control group (fig. ) . in contrast, there was no modification of both the driving pressure and the ∆pl related to nmba administration. conclusion nmba could exert beneficial effects in moderate ards patients through higher observed inspiratory and expiratory transpulmonary pressures. none. introduction prone position (pp) is a major treatment in management of acute respiratory distress syndrome (ards). the use of pp in patients with severe ards associated with brain injury is at high risk of intracranial hypertension. the aim of this study is to analyze the effect of pp on intracranial pressure (icp) and cerebral perfusion pressure (cpp) in patients with ards and acute neurological condition requiring monitoring of icp. patients and methods it is a retrospective descriptive study including sixteen patients with acute brain injury (subarachnoid hemorrhage, severe head trauma, and hemorrhagic stroke) and continuous monitoring of icp who developed a severe ards during icu stay from january to december and for which pp was performed. pp sessions were analyzed. hemodynamic and respiratory parameters, blood oxygenation, pic and ppc were studied in supine, before pp and after pp. the study was approved by fics ethic comity. results a significant increase in pao /fio ratio was observed in pp, from ± to ± (p < . ). in pp, the icp was increased ± . - ± . mmhg (p < . ) while the cpp was stable ± versus ± mmhg (ns). median duration of pp session was h ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . increasing of icp during pp required medical treatment in sessions ( %). pp session was interrupted in sessions ( %). in subgroup of patients who respond to pp in terms of oxygenation, the increase of icp was lower than in non-responders ( vs %) (p < . ). cpp was not modified whatever the nature of the response to pp ( ± - ± in non-responders and from ± to ± in responders (ns)) (fig. ). discussion our study shows an improvement of oxygenation during pp in severe ards patient with acute brain injury. we observe a constant increment of pic during pp sessions. the increment of icp is less in responders to pp. significant increased icp requiring an enhancement in the medical treatment was observed in % of the cases, and lead in most cases to a discontinuation of the session. our data underlined the absolute necessity to monitor icp during pp session in patients with acute brain injury and ards, even if icp is controlled previously in supine. only prospective ( , ) and one retrospective studies evaluate the effects of pp on icp in patients with acute brain injury and acute respiratory failure (arf). they results are similar to ours. in all these studies, the severity of arf was often not well specified. roth and al. ( ) had included only % of ards in a population of patient with icp not controlled. in others studies, monitoring of icp during pp was not systematic. despite the retrospective nature of the study and the small number of patients, it is the only work studying the effects of pp on intracranial pressure in patients with acute brain injury at risk for intracranial hypertension and severe ards according to the berlin's definition. conclusion our work suggest that pp is a quite secure technique for use for the treatment of severe ards even patients at risk of intracranial hypertension with a benefit in terms of oxygenation without major increase of icp particularly in pp responders. introduction influenza-associated acute respiratory distress syndrome (ards) requiring extracorporeal membrane oxygenation (ecmo) support is known to have a good prognosis ( ). however, the incidence and impact of co-infection in this setting remain unknown. we conducted a retrospective, observational analysis of data prospectively collected from all patients admitted to our medical icu who received ecmo support for influenza-associated ards between and . co-infection was defined as isolation of a pathogen in the lower respiratory tract at a significant level or in the blood during the h following hospital admission. when no pathogen was identified in a patient receiving antibiotics prior to bacteriological sampling, an independent adjudication committee reviewed all charts to assess if the patient had a "high probability" or "low probability" for bacterial co-infection, based on clinical, radiological and biological results available. results are presented as median [iqr] . results among the patients hospitalized for an influenzaassociated infection in our icu, had an ards requiring support by either veno-venous-(vv, n = ), venoarterial (va, n = ) or venoarterio-venous-(vav; n = ) ecmo. - . ), pre-ecmo sofa score > (or . ; % ci . - . ) as independent predictors of hospital mortality, but not co-infection (or . , % ci . - . ). in a second analysis, patients with proven co-infection and high probability of co-infection were grouped and compared to patients with no co-infection and low probability of co-infection; and results were similar. as compared to others co-infected patients, those co-infected with a pvl-positive s. aureus had same characteristics and similar mortality rate, but all received a treatment active against pvl production. conclusion co-infection is frequent in patients with influenzaassociated ards supported by ecmo, occurring in roughly % of the cases. mortality of patients with co-infection is higher than those without, but seems mainly due to the severity of the disease. s. aureus was the most frequently identified pathogen, with a high prevalence of pvl-positive s. aureus, infection with a pvl-positive strain was not associated with a poorer outcome as compared to other co-infections. whether a treatment active against pvl production should be given in those patients remains to be determined. none. the pancreaticoduodenectomy (pd) is major surgery in visceral surgery. this technique performed for the first time in by whipple has seen much progress and development over the years that have enabled a significant reduction in mortality, while the morbidity remains high. the aim of this study was to analyze postoperative morbidity pancreaticoduodenectomies. we retrospectively studied cases of cephalic duodenopancreatectomy at the department of surgical emergencies resuscitation (wing ) spanning years, between january and december . the average age of patients was . years with % of females and % of males, the frequence of pancreatic resections was years. the indications of cephalic duodenopancreatectomy were: tumors of pancreatic head ( %), ampulla vater ( %), duodenum tumors ( %). the restoration of continuity after cephalic duodenopancreatectomy was realized with a rate of % for pancreaticogastrostomy and % for pancreaticojejunostomy. the average hospital stay was , days, with extreme lengths of - days. the postoperative course was marked by the occurrence of deaths ( %), the morbidity rate was , % after pj and % after pg; the most frequent complications were the pancreatic fistula ( %), the postoperative peritonitis ( %), the digestive bleeding ( %), the gastroparesis ( %). conclusion advances in the overall care of patients by surgical teams, anesthesiologists and intensivists, the dpc mortality is currently low in experienced centers. the multidisciplinary, involving surgeons, radiologists and especially intensive care, to manage more effectively the complications of this surgery remains burdened with high morbidity. introduction severe acute pancreatitis (sap) is a common but potentially lethal pathology due to the multiplicity and severity of complications that can occur at all stages of evolution. in the last decade, mini-invasive interventional treatments of infected pancreatic necrosis (ipn) have been developed. the aim of the present study was to assess the management and outcomes of sap patients, as well as to identify the role of ipn. this was a retrospective study of prospectively collected data from all consecutive patients admitted in intensive care unit (icu) in a single french center (hospital of nantes) from to . using logistic regression, we evaluated the association between ipn and patients characteristics at baseline and the outcomes. (fig. ) , highlighting the prognostic importance of respiratory failure and acute renal failure at the time of lt, as well as complex interactions between donor and recipient features. conclusion ventilator support and/or acute renal failure at the time of lt are major predictors of mortality but complex recipients/donors relationships may moderate these associations, as demonstrated by our cart analysis. none. subtotal gastrectomy ( / ). enlarged gastrectomy was performed in patients ( %). the mean operative time was . ± min. per-operative transfusion was required in patients ( . %). the average length of stay in icu was . ± days. postoperative mortality was . %. in our series, patients ( . %) had at least one postoperative complication: an anastomotic fistula diagnosed in patients ( . %), patients ( . %) had postoperative peritonitis and patients had ventilator associated pneumonia. reoperation was necessary for patients ( . %), it was performed after . days ( - days). in univariate analysis, risk factors for postoperative morbidity after gastrectomy was hypoalbuminemia (p = . ), anemia (p = . ), bmi (p = . ) and malnutrition (p = . ). age, sex, neoadjuvant chemotherapy, extended lymphadenectomy, splenectomy or pancreatosplenectomy, total gastrectomy and operative time were not significantly associated with higher postoperative morbidity. in multivariate analysis, malnutrition (p = . ) and bmi (p = . ) were significantly associated with the occurrence of postoperative complications. conclusion the results of our study are similar to those reported in medical literature. preoperative evaluation and nutritional rehabilitation are crucial to improve patient's outcome and reduce morbidity and mortality after gastrectomy for cancer. the mesenteric ischemia is a condition relatively rarely. it is marked by high mortality. mortality is primarily related to the land on which ischemia occurs and especially the time taken to diagnose. this delay is due to the low specificity of clinical signs and the absence of diagnostic laboratory test. the mesenteric ischemia remains a diagnostic and therapeutic challenge. patients and methods twenty cases of acute mesenteric ischemia have been collected at the surgical resuscitation (resuscitation ) at the hospital center ibn rochd of casablanca from january to december . results the mean age of our patients is year old. it is about a disease that the incidence increases these last years, particularly because of the waxing number of old patients and/or suffers from advanced cardiovascular diseases. the cardiovascular risk factor has been present in % of our patients. the abdominal pain has been present in all the patients. it is a sudden, intensive pain localized the most often at the level of the epigastria, becomes diffuse in few hours or even few days. other clinical signs have been described as the bilious vomiting that becomes fecaloid after few days. the digestive hemorrhages as the moelena and the hematemeses. a stop of the matter and the gazes was noticed in % of our patients. the absence of specificity of the clinical signs forced the realization of complementary examinations. the scanner becomes the reference imaging. it permits a differential diagnosis, the search of direct signs of vascular obstruction and the emphasis of intestinal pain. four etiologies are noticed: the arterial occlusion by emboli ( %), the arterial thrombosis ( %), the venous thrombosis ( %) and the "non occlusive" form ( %). the strategy of management of the acute mesenteric ischemia is multidisciplinary, based on the equips of radiology, vascular surgery and/ or visceral surgery and resuscitation. the treatment consists in measures of general resuscitation, the techniques of endoluminal vascular disobstruction and techniques of surgical revascularization. in spite of the improvements in the diagnosis and the therapeutic procedure of the ima, the disease still know a rate of mortality between and % according the studies. in our study, we noticed cases of death ( %), cases of good recovery ( %), cases are unknown evolution ( %). conclusion it is a vital emergency that the evolution still knows great mortality. it is very important to remind the acute mesenteric ischemia in the case of any acute abdominal symptom in order to anticipate about the natural evolution and to act in a reversible stage of the ischemia. none. introduction emergency departments staff are frequently exposed to many complex stressful situations and consequently burnout syndrome. our study aimed to describe epidemiological particularities and determine the risk factors of burnout syndrome in different categories of emergency. patients and methods we studied five academics and four regional hospitals. the level of burnout was assessed using the "maslach burn out inventory" score and the degree of depression with major depression inventory (mdi) test. results one hundred and forty-three correctly completed questionnaires were collected. the mean age of study population was ± years. sex-ratio was at . . fifty-one per cent of the care staff were married. physicians represented % and paramedical %. the general frequency of burnout syndrome was % (n = ). low level burnout was present in %, moderate level in % and high level in %. the depression frequency was %. a statistically significant correlation was found between burnout and depression firstly (p = . ) and between burnout and lack of equipment (p = . ). their relative risk was . [ . , ] and . [ . , . ] respectively). main risk factors associated with high level burnout are detailed in table . conclusion burnout syndrome frequency in our emergency departments is alarming. helping to resolve social and psychological problems and improving work conditions may help to decrease it. the healthcare activity is recognized as a major polluting activity. in france, it generates , tons of waste cremated each year, and represents % of the tertiary energy consumptions. in the united states, it generates tons of waste per day and % of total co emissions in were attributed to him. ultimately, such waste production is associated with adverse environmental and health effects. nevertheless, near half of the hospital waste would be recyclable, particularly in our intensive care units (icu) [ ] . furthermore, sustainable development solutions generate profits. the aim of this study is to make an overview of waste produced in a icu and offer solutions to conserve natural resources and reduce the carbon footprint bound to the healthcare activity. materials and methods experimental study, single-center, concerning a period of months in an icu-high surveillance unit compound of beds. we have identified all waste generated. our packaging were given to the recycling company in connection with the hospital. then we have studied the impact of the implementation of sustainable development solutions. results firstly, we have studied the non-recycled waste and the quantity produced over a period of month. approximately kg of waste is produced per patient per day with % of infectious waste and % of general waste. these results were linked with a bad distribution of garbage bags in the rooms ( l of infectious waste versus l of general waste). secondly, we have improved our way to sort and consume and we have created recycling dies without compromising patient safety. all these measures have not increased workload. changing bags in the rooms ( l of infectious waste and bags of l of general waste) allowed to reach the normal goals of sectors with a net benefit estimated at euros per year. the medical broken glass containing drugs was thrown into plastic containers of l for infectious waste to prevent the risk of cuts. by creating a specific die intended to the general waste, we could quantify the production of this glass to kg per week and to spare the use and the incineration of containers of l per year (global economy of euros). plastic packaging represented an important proportion of the cremated waste. we have created sectors of recycling including the polypropylene ( - kg per month), the polyethylene colorless and colored polyethylene. this plastic is sold to be recycled without additional cost for the hospital. the linerboards was cremated. we have created a recycling die ( kg per month). this sector was subsequently extended to the entire hospital structure, particularly the pharmacy that produces containers of l per month. they are now sold without additional cost. many unnecessary plastic waste is generated daily. we have removed using mild soap plastic bottles of ml by using the same mild soap in pump of ml (economy of euros). the use of l plastic bags for the transitional deposit of linen has been deleted (economy of euros). concerning the paper: % of the impressions were made in simplex. printers were parametrized on both sides by default allowing the economy of reams per year ( , sheets), several thousand liters of water and the reduction of co emissions. discussion recycling is only one component of the sustainable development in health. other avenues that could be considered to improve icu sustainability would include examining water use (for linen), electricity use (reducing non-essential use at night…). beyond these actions, we need to encourage our suppliers to turn to sustainable and recyclable packages to reduce the use of polluting and depletable fossil fuels such as oil. but also to develop with them circular economies where waste is returned to them to be reused. conclusion we must ask the question also resuscitate our tons of waste. our icu produce large quantities of waste (over tons per year per bed). however, a significant proportion, especially plastic, is recyclable with a significant environmental and financial benefit. waste management also requires an optimal and rational use of supplies because "the best waste is that which is not produced" and that excess is not a guarantee of quality. as already said st exupéry in : "we do not inherit the earth from our parents, we borrow it from our children. " so do not expect tomorrow to reduce major adverse ecological impact paradoxically generated by a great profession whose ultimate goal is to cure people. moreover, an external consultant is rarely applied and palliative cares are insufficiently developed after «non-readmission» decisions. for providing corrective measures, this study lead to propose a «nonreadmission» process by integrating the discussion for a real «patient's care project» at the end of the icu hospitalization. this process would lead to collect patient's opinion through advance directives, to ensure a collegial discussion including an external consultant and to allow reevaluation of global patient's clinical status and one or more organ failure(s). then, «non-readmission» decisions would be integrated in a therapeutic project which would promote the initiation of a palliative care program if necessary. the purpose of this process is well to respect patient's autonomy and dignity as required by french law and medical ethics. the proportion of elderly patients is steadily increasing. due to the growth of this part of the population who suffer from multiple pathologies, the need for hospitalization in intensive care increases. according to the simulations, the proportion of octogenarian patients in icu will increase reaching the third of icu patients. while chronological age is not a significant factor of poor prognosis in the icu ( ), many factors should be taken into account to evaluate the relevance of icu admission in the senior population and withholding such intensification should be consensually discussed between clinicians and obviously as often as possible with the patient himself ( ) . the aim of the study was to assess the role of stakeholders (ward physicians, intensivists, family doctor and patient himself ) in the decision of withholding icu admission for elderly patients in our internal medicine department. we made a prospective observational monocentric study, including all the elderly patients (defined as older than ) admitted in the internal medicine department from january to june . the only non-inclusion criterion was patient's refusal to participate to the survey. collected data involve physiological (cognitive, autonomy, nutritional status), morbidities (acute and chronic diseases) and social parameters (marital status, relatives). and evaluation of quality of life by the patient himself using an analog visual scale was also obtained. internal medicine physicians were asked to report any icu withholds decision for their patients. in absence of notification, every physician was questioned again the day of the concerned patient's discharge. results one hundred ninety-one patients were included between january and june . factors associated with a significant reduction of in hospital mortality were higher age (p = . ), higher lactate level (p = . ), chronic obstructive pulmonary disease (p = . ), diabetes mellitus (p = . ), immunodepression (p = . ) and respiratory failure (p = . ). conclusion in patients hospitalized for vs high body mass index, low left ventricular systolic function, high white blood cell count, low creatinine clearance, high lactate level and st-segment depression are the variables correlating significantly with high-sensitivity troponin-t concentrations. peak of hstnt was not significantly associated with in-hospital mortality in this setting. introduction mitochondria are evolutionary endosymbionts that are derived from ancestral aerobic bacteria and so might bear and release bacterial molecular motifs supporting the role of mitochondria in danger signal regulations. free circulating mitochondrial dna (mtdna) is elevated in a wild range of critical illness observed in intensive care units, and is associated with bad outcomes and mortality. the mtdna is a molecular pattern that belongs to mitochondrial damage associated molecular patterns (mtdamps), and can interact with pattern recognition receptors (prr) to induce self defense reaction. free mtdna activates inflammatory signaling pathways through toll-like endosomal receptor (tlr ) interactions. nevertheless, new evidence advocates a role of the receptor for advanced glycation end-products (rage) in mtdna signaling. experimental data suggest a role of mtdna-prr interaction in systemic inflammation and organ dysfunctions as septic acute kidney injury or pulmonary inflammation. impact of free circulating mtdna on endothelial cell is not known. the main purpose of this study was to test whether mtdamps and mtdna can induce endothelial dysfunction. we also evaluated the role of mtdna-rage axis in mtdamps induced endothelial dysfunction. mitochondria were isolated from livers of wild type c b mice. isolated mitochondria were sonicated on ice to obtain mtdamp preparations. semi quantitative evaluation of mtdamp content was tested by qpcr, with specific markers of mtdna (cytochrome b (cytb), nadph oxidase (nd )). intraperitoneal injection of mg of mtdamps was used as experimental model in wild type and rage ko mice, as previously described [ ] . the mtdamps were also administrated after ex vivo dnase preparation. endothelial function was assessed with a mulvany-halpern style myograph, h after mtdamp administrations on aorta (conductive vessel) and on d division of mesenteric artery (resistive vessel). endothelial-dependent relaxation was studied by cumulative expositions of the vessels to acetylcholine ( . - - . - m). endothelial-independent relaxation was studied by sodium nitroprussiate exposition. results the mtdamps preparation contains a high quantity of mtdna with a /cycle threshold (ct) ratio of . for cytb expression. intraperitoneal administrations of mtdamps induced a decrease of endothelial-dependent relaxation mainly on conductive vessel (p = . , n = per group) and to a lesser extent on resistive vessel (p = . , n = per group). rage-ko mice were protected from mtdamps-induced aorta dysfunction (p = . , n = per group). the ex vivo exposition of mtdamps to a dnase preparation decreased mtdna content in mtdamps solution with a /ct ratio of . for cytb expression. eventually, the pretreatment of mtdamps with a dnase preparation prevented the mtdamps-induced aorta dysfunction (p = . , n = ). discussion more than prognostic markers, mtdamps particularly mtdna seems implicated in endothelial dysfunction in critically ill patient. new evidence suggest rage interaction in endosomal tlr pro-inflammatory and pro-oxidant response to mtdna [ ] . also in sepsis, physiological clearance of circulating dna might be impaired, this results comfort the possibility of therapeutic regulation of free circulating mtdna to prevent septic organ dysfunction related to mtdamps accumulations. conclusion exogenous mtdamps can induce endothelial dysfunction in mice. the mtdna-rage axis is a key component of the signaling pathway involved in this dysfunction. the use of dynamic parameters to assess fluid responsiveness was supported by cyclic changes in stroke volume induced by mechanical ventilation. however, these parameters have several limits. venous to arterial carbon dioxide difference inversely related to cardiac index. consequently, fluid administration would be beneficial if carbon dioxide gap increases. objective to investigate whether carbon dioxide gap predicts fluid responsiveness in patients with acute circulatory failure. patients and methods we conducted a prospective study in the medical intensive care unit of hospital taher sfar at mahdia, between march and april . patients with circulatory failure and who required mechanical ventilation were included. we measured the variation of cardiac index between baseline and after volume expansion of ml of saline fluid. the picco was used to measure cardiac index. response to fluid challenge was defined as a % increase in cardiac index. before and after fluid administration, we recorded carbon dioxide difference and hemodynamic parameters. results among included patients, ( %) were responders. the causes of acute circulatory failure were septic shock (n = ), cardiogenic shock (n = ), and hypovolemia (n = ). carbone dioxide gap was significantly higher in responders group ( ± vs ± mmhg, p = . ). the area under the roc curve for carbon dioxide gap was . ( % ci . - . ). the best cutoff value was mmhg (sensibility = %, specificity = %, positive predictive value = % and negative predictive value = %). the area under the roc curve for delta carbon dioxide was . ( % ci . - . ). conclusion in this study, baseline carbon dioxide gap was not universal indicator to predict the fluid responsiveness in patient with circulatory failure. introduction supraventricular arrhythmia (sva) is commun in intensive care unit (icu). its incidence seems to be higher in patients with sepstic shock. sepsis-associated myocardial dysfunction promote the occurrence of sva by constituting an arrythmogenic substrate or under the effect of inotropic drugs. the aim of this study is to assess the incidence and prognostic impact of sva in patients with septic shock. patients and methods we retrospectively studied all patients with new onset sva suffering from septic shock in non cardiac surgical icu. myocardial dysfunction was evaluated by transthoracic echography (tte) after an adequate cardiac resuscitation using intravenous fluids expansion and adjunctive vasoactive agents. sva was detected by the electrocardiogram scope. during the study period clinical and biologic characteristics, hemodynamic tolerance (vasopressors doses, arterial pressure changes), current treatment (such as corticoid), duration of mechanical ventilation, duration of vasopressor requirement and hospital mortality were collected. results sixty patients were included in the study. the sva occurred in patients, with an incidence of %. the median time to onset was days. cardioversion was performed for patients with an effectiveness of %. clinical and biological characteristics were similar between the groups with and without sva: saps and sofa score at the beginning of septic shock, the existence of ards and cardiac biomarkers (nt-probnp, troponin). however, renal failure and the use of corticoid in septic shock were more frequent in the group with sva. the maximum doses of vasopressor agent were not significantly different between the groups with or without sva. myocardial dysfunction in sepsis defined by the left ventricle ejection fraction (lvef) less than % (or the need for inotropic drug for lvef > %) was not associated with the occurrence of sva (+sva group: n = ; −sva group: n = ; p: . ). sva was poorly-tolerated, observed by a significant decrease in mean arterial pressure and a significant increase in norepinephrine doses within h of the start of sva. the occurrence of sva was associated with longer duration of use of vasopressor agent and a longer duration stay in icu (+sva group: days, −sva group: days; p = . ). there was no difference in duration of mechanical ventilation and hospital mortality between the two groups. conclusion the occurrence of sva is common in septic shock, poorly tolerated hemodynamically and associated with longer duration stay in the icu and vasopressor need. sepsis myocardial dysfunction isn't necessarily associated to the occurrence of sva. introduction a short term beneficial effect of prone position on cardiac index has been shown in % of ards patients, and was related to an increase in cardiac preload in preload responsive patients ( ) . the aim of this study was to evaluate the long term hemodynamic response to prone position in a larger series of ards patients. patients and methods single center retrospective observational study performed on ards patients hospitalized in a medical icu between july and march . patients included were adults fulfilling the berlin definition for ards, undergoing at least one prone position session, under hemodynamic monitoring by the picco ® device, with availability of hemodynamic measurements performed before (t ), at the end (t ), and after the prone position session (t ). prone position sessions were excluded if they were performed > days after ards onset. the following variables were recorded: demographic, sapsii, ards severity and risk factor, sofa score and cumulative fluid balance at pp onset, delay between ards session and pp session, hemodynamic, arterial blood gas, ventilatory settings, plateau pressure, catecholamine dose and additional treatments. statistical analyses were performed using prone position session as statistical unit and mixed models taking into account both multiple prone position sessions by patient and multiple measurements during a prone position session. p < . was chosen for statistical significance. data are expressed as mean ± standard deviation. results patients fulfilled the inclusion criteria over the study period, totalizing prone position sessions ( ± sessions per patient). patients' age was ± y, % were male, % fulfilled the criteria for severe ards, and sapsii at icu admission was ± . ards risk factors were pneumonia in ( %), aspiration pneumonia in ( %), and sepsis in ( %) patients. duration of prone position sessions was ± h. hemodynamic measurements were performed in pp ± h after pp session onset. at session onset, sofa score was ± , and cumulated fluid balance was . ± . l. vasopressor were used in %, inhaled nitric oxide in %, and neuromuscular blocking agents in % of the sessions. hemodynamic and respiratory parameters before, during and after the prone position sessions are reported in table . cardiac index increased by at least %, decreased by at least % or remained stable in ( %), ( %), and ( %) of the sessions, respectively. as compared to both other groups, pp sessions with significant increase in cardiac index had the following significant differences at t by univariate analysis: lower cardiac index, lower global end-diastolic volume, lower cardiac function index, and lower vasopressor dose. multivariate analysis is under investigation. conclusion prone position is associated with an increase in global end-diastolic volume, reversible after return in supine position that may explain the positive effect of pp on cardiac index observed in ¼ of the pp sessions. introduction make sure that our patient have a good circulatory condition is a daily challenge for the intensivist. one of the therapeutics is fluid and one of his purpose is to increase venous return and then cardiac output. in order to examine that, there are several tools as the transthoracic echocardiogram wich allows the visualisation and the study of the respiratory variability from the inferior vena cava (ivc). unfortunately there are some situations where the ivc visualisation is difficult (obesity, gut surgery, emphysema). the ivc is easily seen by a transhepatic ultrasound in her retrohepatic section. we make the hypothesis that the shape of the ivc could be predictive of fluid responsiveness. we have performed fluid challenge in patients under mechanical ventilation. the need for fluid therapy is the intensivist in charge decision. we performed a echocardiogram and we take two measures of the icv: major axis and minor axis, the icv is measured avec the sus hepatic vena. a elastometry index (ei) is determined which is the ratio of minor axis to minor axis. the fluid challenge is ml of isotonic saline then we perform a new echocardiogram. a tag is written on the patient to take the same ultrasound slice. we retain one increase of % of the cardiac index (ic) as a success of the filling. we exclude the presenting patients a right cardiac insufficiency, an arrhythmia and/or a htap. the statistical analysis is realized with the software r. results between august, and january, we included patients. the average age is of years ( - ), igs of ( - ), ejectionnal fraction of % - ) and the s wave tricuspid is ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the causes of the filling were an oliguria ( %), a low blood pressure ( %), a low cardiac output ( %), a hyperlactatémia ( %) and an other cause in % of the cases. we find a positive correlation between the ei and the increase of the ic, also for the area of the vci and the respiratory variations of the vci (p . ) the other variables are not predictive (bp, e/e' , e/a). the data are summarized in the picture . roc curves has been established ( only % of the journals studied required authors to use stard. a high impact factor and the year of the study were the items associated with a better sqs the presence of a conflict of interest was associated with a lower sqs in univariate analysis. a higher impact factor (> ), was the only independent factors statistically significantly (p = . ) associated with higher sqs in a multivariate regression model. discussion our study showed that the sqs were very low. assessment of a study depends on quality of reporting. blindness and participant sampling are the cornerstone to evaluate such bias as spectrum, verification, review and selection bias of a study, and were unfortunately scarcely reported compared to existing data in diagnosis accuracy reporting. one of the limitation is the years sample of the study. we have planned to continue the analysis for a -year review starting just after the stard publication. conclusion our study showed that several items remain poorly reported. we recommend systematic use of stard criteria in the elaboration and reporting of future studies that evaluates the preload dependence. introduction neurological impairment, i.e. encephalopathy, is commonly observed in patients with decompensated cirrhosis and/or portosystemic shunts admitted in icu. often ascribed to high plasmatic levels of ammonia, encephalopathy could also be induced by drugs or infection, due to altered blood-brain barrier (bbb) permeability. this latter setting is often underdiagnosed and encephalopathy related to hyperammonemia (so called hepatic encephalopathy-he) being pointed out as the culpit of all neurological symptoms in cirrhotic patients. quinolones and betalactamins were recently found in the cerebrospinal fluid of he patients and it has been shown that the expression of efflux pumps, responsible for drugs passing through the bbb, was altered in animal models of he. the purpose of this study was to assess the incidence of neurological impairment, i.e. encephalopathy, in cirrhotic patients hospitalized in discussion overall, we reported a higher rate of lumbar puncture than those reporting in others studies concerning status epilepticus. furthermore the rate of % of pleocytosis directly linked to status epilepticus is slightly higher than in most studies. unfortunately we didn't realize a second lumbar puncture to assess the pleocytosis normalization during the days following the first lumbar puncture. the pathophysiological hypothesis of this phenomenon may be that prolonged/repeated seizures during status epilepticus would induce a blood-brain barrier dysfunction thereby favoring a cerebrospinal pleocytosis. conclusion in our study, % of status epilepticus without infectious or neoplastic origin had a cerebrospinal pleocytosis directly linked to status epilepticus. this pleocytosis was significantly associated with myoclonic seizures and blood leukocytosis. these data may help to interpretation of cerebrospinal fluid pleocytosis during status epilepticus. introduction neurological prognostication from cardiac arrest survivor is a current concern. eeg patterns and nse dosage are two important prognostic factors. nse threshold for prediction of poor outcome appear controversial, in part, because of variability in dosage timing and measurement techniques. synek score is routinely used in our center to classify comatose patients in post cardiac arrest. the aim of this study was to assess the prognostic value of nse and synek classification to predict poor neurological outcome. introduction traumatic brain injury (tbi) is a major public health problem. it is the leading cause of death and disability in young subjects. one of the principles of the tbi management is prevention of secondary cerebral insults including maintaining perfusion and cerebral oxygenation, control of intracranial pressure (icp). an increase in icp above mmhg is associated with poor outcome. cerebral hypoxia can occur with normal level of icp and cerebral perfusion pressure (cpp).monitoring of regional partial pressure of brain tissue oxygen (pbto ) is a safe and reliable method for measuring cerebral oxygenation. a retrospective single-center observational study was conducted between january and december , aimed to study the influence of pbto with severe tbi patients outcome at months through glasgow outcome scale (gos). the hourly values of icp, pbto and cpp were recovered on daily monitoring sheets. we compared two groups according to their gos. during the study period, patients underwent a monitoring icp and pbto . results the mean age was . ± . years. . % were men. the initial glasgow score was . ± . . the mean simplified acute physiology score (saps ii) was . ± . and injury severity score (iss) . ± . . at months, patients had died (gos ). forty patients had a good outcome: gos - (group ). sixteen patients had poor outcome: gos - (group ). in group , there are significantly more pbto hourly values below mmhg at day ( . ± . vs . ± , in group , p = . ); and more pbto hourly values greater than mmhg at day ( . ± . vs . ± . , p = . ). conclusion pbto less than mmhg or greater than mmhg at day is associated with poor outcome at months in the severe tbi. the pbto allows a more individual approach of monitored tbi. none. introduction organ donation in patients after a decision to withdraw life-supportive therapies (wlst) (maastricht condition: m ) have been performed in our hospital since may . we report here main characteristics of donors, data on m procedure and results on renal transplant recipients. patients and methods all potential donors were included in a survey from may to june , according to the french national m protocol defined by the french organ procurement agency (agence de la biomédecine:abm) [ ] .the demographical, clinical and biological characteristics of the donors, the different deadlines and times of the protocol and data of renal transplantation were collected and analyzed. results patients had inclusion criteria. patients were admitted in intensive care unit for cardiac arrest ( %), strokes ( %), traumatic brain injury ( %), ards ( %). of them, procedures ( %) were stopped ( refusals of organ donation, medical contra-indications discovered with additional exams, failure of vessel cannulation, deaths more than h after extubation). kidneys were harvested and transplantations performed ( renal cancer discovered during procurement surgery).the characteristics of the donors, deadlines of the protocol and transplant recipients are reported in the table . conclusion the french programm maastricht offered a new possibility of organ donation in our hospital. thanks to these donors, the number of renal grafts increases and the preliminary results on transplant recipients are encouraging in line with the preliminary report of the abm. nevertheless, it is necessary to follow the transplant recipients and extend the procedure to new centres. in this study, we found some relevant risk factors for microaspiration (age, low score at gcs) consistent with literature on the subject. patients with paralytic agents had less gam which may be due to higher peep, higher cuff pressure and less enteral nutrition because of the severity of the underlying diseases. conclusion this study did not show any increased risk of microaspiration in intubated copd patients, whatever stage of copd. introduction protected specimen brush (psb) is considered to be one of the standard methods for the diagnosis of ventilator-associated pneumonia (vap). to our knowledge, there is no study assessing effect of prior antibiotherapy on direct examination, bacteriological culture and concordance of direct microscopy and culture. patients and methods all consecutive episodes of suspected vap were retrospectively evaluated between january and december in a -bed intensive care unit. patient's characteristics and preexisting conditions were abstracted from the medical charts. after assessment of vap probability using the clinical pulmonary infection score (cpis), psb were performed in patients with a cpis of or more. based on antibiotic treatment in patients when bacteriological specimens were obtained, two groups were defined: no antibiotic group and antibiotic treatment started before psb group. two independent bacteriologists retrospectively reviewed direct examination and culture of psb to assess bacteriological concordance, defined as non-concordant when direct examination and culture were different, concordant when direct examination and culture were similar and partially concordant when either direct examination or culture were comparable but with other microorganisms lacking in one or the other method. results during this -months period, among mechanically ventilated patients, episodes of suspected vap with psb were evaluated. we found % of psb (n = ) performed without antibiotic treatment and % of psb (n = ) performed under antibiotherapy. we found no significant differences in patient's demographics, characteristics, and severity between both groups. patients received antibiotics for the following reasons: aspiration pneumonia (n = ), peritonitis (n = ), vap (n = ), community-acquired pneumonia (n = ), septic shock of unknown origin (n = ), pyelonephritis (n = ), meningitis (n = ), acute pancreatitis (n = ) and others (n = ). the median duration of mechanical ventilation in the antibiotic receiving group and in the group without antibiotics was . days (iqr; - days) and days (iqr: - ), respectively. when psb was performed under antibiotic treatment, direct examination was positive in % (n = ), culture was positive in % (n = ) and those methods were concordant, non concordant and partially concordant in % (n = ), % (n = ) and % (n = ), respectively. on the other hand, when psb was performed without antibiotics, direct examination was positive in % (n = ), culture was positive in % (n = ) and those methods were concordant, non concordant and partially concordant in % (n = ), % (n = ) and % (n = ), respectively. in univariate analysis, we found a significantly higher proportion of negative direct examination and negative culture in the antibiotic group (p > . ). moreover, these methods were significantly more frequently concordant (p = . ), with a higher rate of both negative microscopic exam and culture when compared to the no antibiotic group ( %, n = vs %, n = ). surprisingly, among the patients previously treated with antibiotics with positive culture, % (n = ) of the microorganisms showed antibiotics sensitivity. discussion whether prior antibiotic treatment may induce false negative of false positive treatment is a well-recognized phenomenon, the precise effect of antibiotics on direct examination and quantitative culture is not well assessed in vap. moreover, despite recent development of clinico-radiological score, diagnosis of vap remains difficult, with no gold-standard. therefore, bacteriological guided therapy is of particular importance. we found psb realization under antibiotic treatment is associated with a lower rate of positive direct examination and culture and suggest performing these bacteriological samples without antibiotherapy. some authors have suggested lowering the diagnostic threshold point of this bacteriological technique in order to preserve its accuracy. however, we can postulate that microorganisms responsible of superinfection in mechanically ventilated patients treated with antibiotics may be resistant and therefore the psb could be positive. conclusion in patients with a high pre-test probability of ventilatoracquired pneumonia, recent introduction of antibiotics significantly reduced the diagnostic accuracy of protected brush specimen by reducing rates of positive direct examination and culture. further studies should evaluate if antibiotic discontinuation may revert this effect. ann. intensive care , (suppl ): we have had non conflict of interest in this study. results we included patients in the phase and patients in the phase . baseline characteristics of patients were similar in both groups. compliance with all the measures has been improved between the two period from to . %. the incidence density decreased from . to . vap per ventilator days between observational and interventional period, but the all-cause mortality was almost equal in the groups ( . vs. %). discussion with the implementation of our bundle, observance of the team were improved in the second group, compared to the first and the incidence density decreased from . to . vap per ventilator days between both period. this result is consistent with the littérature. sure enough, many studies show the same effect of vap prevention with a decrease of nearly % of the incidence density of vap, after implementation of a «ventilator -bundle [ ] . conclusion the implementation of a "ventilator bundle, " has significantly reduced the incidence of vap in our service. in the contrary, our study failed to demonstrate a reduction in mortality. introduction with an increasing incidence and high mortality rates, sepsis is a public health issue. there is growing evidence that sepsis induces long lasting alterations of transcriptional programs through epigenetic mechanisms that may lead to protracted inflammation, organ failure, sepsis-induced immune suppression (siis), secondary infections and death. we hypothesized that epigenetic changes contribute to the pathophysiology of siis. to test this hypothesis, we studied the effects of histone deacetylases (hdac) inhibition with trichostatin a (tsa) in a double-hit murine model of siis and secondary pneumonia. materials and methods c bl/ mice were treated with tsa ( mg/ kg ip) or saline serum (ctl) min before induction of sepsis by cecal ligation and puncture (clp). surviving mice underwent intratracheal instillation of . × cfu of pseudomonas aeruginosa days after clp. we evaluated the effect of tsa on survival and cellular responses to the primary and secondary infections. cellular responses in the blood, spleen and bal were assessed by flow cytometry after clp (days , & ) and after pneumonia ( & h). we also studied lymphocyte apoptosis and dendritic cells (dc) expression of cd , cd , and mhcii. bacterial clearance was assessed in the bal and in the blood and h after pneumonia. continuous variables represented as mean ± sd were compared using student t test. kaplan-meier curves were compared by the log rank test. p < . indicated statistically significant differences. results whereas treatment with tsa did not change survival after clp, tsa improved survival after tracheal instillation of p. aeruginosa (p = . , fig. ). tsa-treated mice had significantly higher absolute dc, t and b-lymphocytes counts with reduced lymphocyte apoptosis after clp. four hours after secondary pneumonia, tsa-treated mice had significantly higher dc counts and improved bacterial clearance in the bal, with reduced systemic dissemination of p. aeruginosa. conclusion hdac inhibition with tsa improves survival in our murine model of secondary pneumonia, improves bacterial clearance and attenuate cellular features of siis. these results suggest that sepsisinduced epigenetic changes contribute to the advent of siis. comprehensive characterization of epigenetic changes associated with siis might allow us to identify new therapeutic targets to reprogram immune cells in sepsis and avoid siis. length of icu stay was ± days. patients acquired nis ( . % bsi, . % pneumonia, . % cri and . % uti. there was no bacteriological documentation of ni in . % of cases. nis occured days post burns. the most three isolated pathogens were: acinetobacter spp. ( %), p. aeruginosa ( . %) and extended spectrum betalactamase-producing enterobacteriaceae ( %). the most frequently administered antibiotics were polymyxin/carbapenem/teicoplanin combination ( %), polymyxin/carbapenem combination ( %) and carbapenem/tigecycline combination ( %). in our study, mortality rate was %. conclusion nosocomial infection occured in . % of cases in burn patients, caused by acinetobacter spp, p. aeruginosa and enterobacteriaceae blse. so, eradication of infection in burn patients require effective surveillance and infection control in order to reduce mortality rates, length of hospitalization and associated costs. introduction infection of the lower respiratory tract is the most common cause of infection in intensive care unit (icu) ( ) . although the attributable mortality of ventilator associated pneumonia remains debated, the recurrence of these infections is always associated with a significant morbidity ( ) . staphylococcus aureus methicillin-sensitive (sams) is one of the most frequently germs involved in icu pneumonia especially in trauma patients. the aim of the study was to establish the risk factors associated with microbiological treatment failure of pneumonia, caused by sams. materials and methods we retrospectively identified patients who developed a first episode of ventilator associated pneumonia caused by sams during a years-period ( - ). the primary end point was the microbiological treatment failure defined as a second episode of pneumonia caused by sams corresponding to either a persistent or a recurrence of the pneumonia (fig. ) . the primary aim of the study was to identify factors associated with a treatment failure, the secondary objective was to identify factors associated with the occurrence of second episode (i.e. persistent, recurrence, superinfection and/or relapse of pneumonia caused by any bacteria) during or after treatment of the first episode caused by sams. definition of outcomes was based after analysis of current concepts available in the literature. factors associated with primary and secondary objectives in univariate analysis (p-value < . ), or clinically relevant ones, were entered in a multivariate logistic regression. the final selection was performed using the stepwise selection based on the akaike criterion. results fifty-nine patients ( . %) developed a second episode of pneumonia and among them, ( . %) were considered as a microbiological failure. in a multivariate analysis, the association of oropharyngeal flora (fop) with the sams (or, . ; % ci, . - . ; p = . ) and the need of emergency surgery (or, . ; % ci, . - . ; p = . ) were predictive of a microbiological failure. empirical antibiotic therapy with amoxicillin-clavulanic acid (or, . ; % ci, . - . ; p = . ) and performing emergency surgery (or, . ; % ci, . - . ; p = . ) were predictors of a second episode of pneumonia caused by any bacteria. conclusion in this retrospective, monocentric study, the co presence of orophryngeal flora and the need of emergency surgery were associated with microbiological failure of pneumonia caused by sams in icu. introduction ventilator-associated pneumonia is a major iatrogenic problem since it is a cause of hospital morbidity, mortality and increase of health care costs. it has been studied many times, but data's revision is always necessary. our study aimed to describe epidemiology of ventilator-associated pneumonia and identify local causative pathogens. we carried out a prospective study in an intensive care unit. were included patients intubated for more than h, from april to may , and presenting signs of ventilator-associated pneumonia (fever, abundant and purulent secretion, increase of fio greater than . , signs on chest-x ray) with positive culture of endotracheal aspirate. were excluded patients with germ colonization. results a total of patients were ventilated for more than h. among them thirty-four patients aged of ± . years presented episodes of ventilator-associated pneumonia (that is . ± . episodes per patient). the mean sofa score was . ± . . the main reasons of mechanical ventilation were loss of consciousness secondary to poisoning ( %), respiratory distress ( %) and status epilepticus ( %). the mean duration of stay was . days with extremes at and days. the average time between hospitalization and suspicion of ventilator-associated pneumonia was . ± . days. the average value of the clinical pulmonary infection score at suspicion was ± . . the average time between recurrences was . days with extremes at and days. the culture of endotracheal aspirate identified two pathogens in %. it reveled acinetobacter baumanii in % in which % were imipenem resistant, pseudomonas aeroginosa in %, klebsielle pneumoniae in %, staphylococcus fig. see text for description aureus methicillin resistant in %. extended spectrum β-lactamases bacteria were found in % and carbapenemases producers in %. empirical antibiotherapy was always association of imipenem and colistin. it was necessary to adapt it to antibiograms in / . ventilator-associated pneumonia was complicated by septic shock in % and acute respiratory distress syndrome in %. patients evolved to healing in % of episodes (n = ), to superinfection in % (n = ) and to death in % (n = ). pseudomonas aeruginosa was the most frequent germ in superinfection ( / ) , acinétobacter baumanii was the most pathogen associated to death ( / ). conclusion ventilator-associated pneumonia is an iatrogenic disease that threatens lives. it's in part avoidable. preventive measures have to be implemented to reduce its frequency, consequences and costs. introduction during mechanical ventilation, mismatch between respiratory muscles activity and the assistance delivered by the ventilator results in dyspnea and asynchrony and is commonly observed in intensive care unit (icu) patients. proportional assisted ventilation (pav) is a ventilatory mode that adjusts the level of ventilator assistance to the activity of respiratory muscles estimated by an algorithm. to date, pav has been mostly studied in patients without severe dyspnea or asynchrony. we hypothesized that, compared to pressure support ventilation (psv), pav will prevent severe dyspnea or asynchrony. patients and methods were included icu mechanically ventilated patient exhibiting severe dyspnea or asynchrony with psv. three conditions were successively studied: ) psv on inclusion (baseline), ) psv after optimisation of ventilator settings in order to minimize dyspnoea and asynchrony (optimisation), and ) pav. ten-minutes recording were performed with each condition. the intensity of dyspnea was assessed by the visual analogic state (vas, only in patients able to communicate) and by the intensive care respiratory distress operating scale (ic-rdos) for all the patients. the electrical activity (emg) of extradiaphragmatic inspiratory muscles was measured. the fig. bayesian nma with random effect prevalence of asynchrony was quantified by the visual inspection of the airway flow and pressure traces. results patients were included, % male, aged [ - ] years, saps [ - ], mechanically ventilated for [ ] [ ] [ ] [ ] [ ] [ ] days. the tidal volume (tv) was higher in the optimisation and pav than in the basal condition (table ). the respiratory rate(rr) was lower with pav than in the other conditions. the dyspnea-vas was lower with optimisation and pav than with the basal conditions. the ic-rdos was lower with pav than with the two other conditions. the asynchrony index was lower with pav than with the two other conditions. parasternal emg activity was lower with pav and optimisation (fig. ) . conclusion in icu patients receiving mechanical ventilation with psv and exhibiting severe dyspnea or asynchrony, the optimisation of ventilator settings with psv and the pav mode decrease in the simiar way the severity of dyspnea and the prevalence of patient-ventilator asynchrony. introduction in spite of recent research and progress in weaning protocols, extubation failure still occurs in - % of patients and is associated with poor outcomes, with a mortality rate of - %. many risk factors for planned extubation failure have been suggested, including hypercapnia at end of spontaneous breathing trial (sbt). however, performing arterial blood gases at the end of sbt is not routinely recommended whereas etco may be routinely monitored during a low pressure support sbt. the aim of this prospective observational study was to determine the clinical usefulness of etco to predict extubation failure. patients and methods we recorded clinical data and etco during a successful h low level pressure support sbt (at the beginning, after min and at the end of the trial). patients ventilated through tracheostomy and unplanned extubations were excluded. extubation failure was defined as death or the need for reintubation within h ( ) after extubation; this delay was prolonged to days ( ) in case of noninvasive ventilation after extubation, which was systematic in older patients or those with cardiorespiratory disease, as per our weaning protocol. multivariable logistic regression analysis was performed to identify independent variables associated with extubation failure. results one hundred and fifteen ventilated patients were enrolled in our study from july to june . the median age of these patients was [ - ] years, their median simplified acute physiology score (saps) ii was [ - ] points and . % (n = ) were female. seventeen ( %) patients had chronic obstructive pulmonary disease. reintubation rate was % (n = ). etco at other time points as well as its changes during the sbt were also similar between groups. the three variables predicting extubation failure in the multivariable logistic regression model were a past medical history of cirrhosis, acute respiratory distress syndrome before weaning and lower minute ventilation at the end of sbt. conclusion etco during a successful sbt seems useless to predict outcome of extubation. introduction airway management in intensive care unit (icu) patients is challenging [ ] . "airway failure", defined as the inability to breathe without endotracheal tube, differs from "weaning failure", defined as the inability to breathe without an invasive mechanical ventilation. however, most of the studies assessing predictive factors of extubation failure did not separate airway from weaning failure. we aimed to describe incidence of extubation failure in critically ill patients, separating for the first time airway from weaning failure, in a prospective multicenter observational study. patients and methods a prospective, observational, multicenter study was conducted in french icus. all adult patients consecutively extubated in icu were included. an ethics committee approved the study design (code uf: , register: -a - ). the study was registered on clinicaltrials.gov (identifier no.nct ). clinical parameters were prospectively assessed before, during and after extubation procedure. extubation failure was defined as the need to reintubate less than h after extubation. extubation failure could be due to airway failure, weaning failure or mixed airway and weaning failure. results from december to may , intubation-procedures were studied in patients from centers. patients ( . %) were intubated twice. the median number of intubation-procedures included by center was . the flow chart of the study is shown in fig. . incidence of extubation failure was . % ( of intubation-procedures). incidence of airway failure, weaning failure and mixed failure were respectively . % ( of ), . % ( of ) and . % ( of ). conclusion extubation failure at h occurred in . % of the extubation procedures recorded, % due to airway failure, % to weaning failure and % to mixed airway and weaning failure. specific risk factors will be determined using this multicenter database. introduction acute on chronic liver failure (aclf) have been recently defined by an acute decompensation of a chronic liver disease associated to organ failure and a high mortality rate. few authors reported on the use of total plasma exchange (tpe) in patients with the current definition of aclf. the aim of this pilot study was to evaluate the efficiency and safety of tpe in critically ill cirrhotic patients admitted with aclf in the icu. patients and methods a prospective cohort of cirrhotic patients admitted to the icu between february and february . tpe was performed using a plasma filter (tpe , hospal ® ) on a cvvhdf machine (prismaflex ® , baxter ® ) connected to the patient with a femoral double lumen f catheter. the plasma volume exchanged per session was . - . of the total plasma volume. ratio and type of fluid replacement were % with % albumin solution followed by % with fresh frozen plasma. clinical and biological parameters, and the following scores meld, sofa, clif-sofa, clif-of and child pugh were evaluated prior, after tpe session and days distant of treatment. results seven male patients with a mean age of . ± . years comprised the study and had a total of tpe sessions. the etiology of cirrhosis was alcoholic (n = ) or post-hcv (n = ). the reasons of aclf were acute alcoholic hepatitis (n = ), variceal bleeding (n = ) and sepsis (n = ). prior to tpe, the mean scores of sofa, clif-sofa, clif-of, meld and child-pugh were respectively . , , . , . and c . . mean total bilirubin prior and after tpe sessions was reduced from . ± . µmol/l to . ± . µmol/l (reduction of . %; p = . e− ); at day , mean total bilirubin was still lower at ± µmol/l (p = . ). mean inr prior and after tpe improved from . ± . to . ± . (reduction of inr of . %, p = . e− ) and at day of treatment at ± . (reduction of %, p = . ). mean ggt levels reduced by . % (p = . ). mean platelet counts ( . ± . g/l) reduced by . % (p = ns). the probability of survival at , and days was . , . and . %. one patient was transplanted and still alive. tolerance during sessions was good similar to cvvhdf. two side effects related to the femoral catheter were observed (bacteremia and hemorrhagic shock post catheter ablation). conclusion this preliminary study of tpe in aclf showed a marked reduction of liver enzymes and improvement in coagulation parameters with a relative good safety. a specific caution should be undertaken regarding catheter related complications. tpe worth to be fig. flow chart of the free-rea study introduction extubation is a key moment for the patient on his way to recovery. extubation failure concerns - % of icu patients and is closely linked to nosocomial pneumonia. the practice concerning enteral feeding interruption at time of extubation has not been investigated. fasting before extubation may prevent aspiration and development of nosocomial pneumonia. thus, fasting and gastric content suctioning before extubation may be reasonably considered as a mean to reduce this burden. fasting before extubation may prevent aspiration and development of nosocomial pneumonia. thus, fasting and gastric content suctioning before extubation may be reasonably considered as a mean to reduce this burden. however, fasting, as recommended before elective general anesthesia is likely to be ineffective in the setting of extubation in the icu, due to patients' gastroparesis and prolonged gastric stasis. beyond the potentially unnecessary burden in terms of paramedical workload, fasting may have some side effects such as caloric deficit, hypoglycemia, or delayed extubation. given the current lack of objective data concerning the clinical practice of feeding/fasting and gastric tube suctioning before extubation in the icu, we undertook this descriptive study to assess current practice. materials and methods we conducted a retrospective, multicenter study in eleven intensive care units in the west of france over a month timespan. all patients extubated were included and data about enteral feeding during the peri-extubation period as well as extubation failure and nosocomial that pneumonia occured within days were recorded. data observed in the eleven participating centers were completed with a short email survey concerning declarative practice performed among intensive care units. results during the study period, patients were included. overall, patients ( %) failed extubation and needed reintubation within the days following planned extubation. pneumonia was significantly more frequent reintubated patients than the other ( vs. %, p < . ). hundred patients ( %) received enteral feeding at the time of extubation. compared to patients who did not receive enteral feeding, those patients had a higher disease severity (sapsii score , [ ; ] vs. [ ; ], p < . ; longer duration of mechanical ventilation [ ; ] vs. . [ ; ] days, p < . ). accordingly, those patients had a higher rate of extubation failure ( vs. %, p = . ) and pneumonia ( vs. %, p = . ). among the patients receiving enteral feeding, fasting was implemented before extubation for patients ( %). similarly, the incidence of pneumonia was not different between groups (n = ( %) vs. n = ( %), p = . ). after extubation, the fasting patients experienced a longer delay until feeding resumption as compared to non-fasting patients ( h [ ; ] vs. [ ; ] ), but this difference did not reach statistical significance. overall gastric content suctioning before extubation was not commonly performed; before extubation: % of the fasting patients and % of the non fasting patients. among the participating centers, while some centers imposed a fasting period before extubation to all their patients, some did it infrequently. however, no center never imposed fasting, illustrating between and within center heterogeneity. this heterogeneity was confirmed on the larger scale declarative email survey ( % response rate amont units) which showed that only % of the units had a written standardized operational procedure for extubation. survey respondents reported to practice fasting before extubation "always", "frequently" and "never or rarely" in respectively , and % of cases. conclusion both practices, fasting as well as pursued nutrition until extubation are commonly performed in icus, with little standardization of practice. safety seems equivalent, as no clinically significant difference in terms of reintubation rate and pneumonia were observed. thus, the equipoise condition appears met to undertake a trial evaluating feeding strategies in the peri-extubation period. introduction noninvasive ventilation (niv) has become a cornerstone for the supportive therapy of acute respiratory failure (arf). survival benefits in chronic obstructive pulmonary disease (copd) and cardiac patients have been demonstrated. although arf and copd patients are at risk of malnutrition that adversely affects patient outcomes, few data are available regarding the management of nutritional support in non-invasively ventilated patients. we sought to describe nutritional management in patients receiving niv as the first line therapy for arf. secondary objectives were to assess the impact of early nutrition use on the need for invasive mechanical ventilation, occurrence of icuacquired pneumonia, length of stay, and death. patients and methods we conducted an observational study from the multicenter french database fed by french icus. our institutional review board approved this study. adult medical patients admitted to the icu and receiving niv for more than days were included. exclusion criteria were patients admitted after surgery, readmitted in icu, patients with neuromuscular disease and treatment-limitation decisions on admission. four groups of patients were defined according to nutrition received during the first days of niv: ( ) no nutrition; ( ) enteral nutrition: patients who received enteral nutrition with or without parenteral nutrition; ( ) parenteral nutrition only ( ) oral nutrition only. the impact of nutrition on day- mortality was assessed through the use of a cox model adjusted on clinically relevant covariates. the impact of nutrition on other secondary end-point i.e. icu-acquired pneumonia occurrence, need for invasive mechanical ventilation were assessed using a fine & gray models. patients were censored after days of follow-up. choice among collinear variables was performed considering clinical relevance, rate of missing variables and reproducibility of definitions. results were given as hazard ratio (hr) for cox models and subdistribution hazard ratios (shr) and % confidence intervals (ci). the impact on duration of stay was estimated by a multivariate poisson regression. p values less than . were considered as significant. statistical analysis was performed using sas . (cary, nc). results during the study period, , patients were included in the database and met inclusion criteria. among them, received no nutrition; received enteral nutrition, received parenteral nutrition only, and received oral nutrition only. overall, patients developed icu-acquired pneumonia ( %), required invasive mechanical ventilation ( . %) and died before day- ( %). median length of stay was days [ ; ]. after adjustment for confounders, type of nutrition support was associated with an increase day- mortality (p = . ). compared to oral nutrition, enteral nutrition was associated with an increase day- mortality [shr . , % ci . - . ; p = . ] whereas parenteral nutrition and no nutrition did not influence this outcome. the type of nutrition was not associated with the occurrence of icu-acquired pneumonia (p = . ). however, patients who received enteral nutrition experienced more frequently icu-acquired pneumonia [shr = . , % ci . - . ; p = . ] as compared to oral nutrition patients. ventilator free days within the days were negatively associated with the type of nutrition (p < . ). compared to oral nutrition, parenteral and enteral nutrition were negatively associated with ventilator free days within the days [rr per day = . , % ci . - . ; p < . and rr per day = . , % ci . - . ; p < . ]. delta paco measured between the first days was not associated with any type of nutrition. conclusion more than half the patients receiving niv were fasting within the first two niv days. oral nutrition was prescribed for onethird of them and was well tolerated. lack of feeding or underfeeding had no impact on mortality and ventilator free days within the days. however, enteral nutrition was associated with an increased occurrence of icu-aquired pneumonia and a higher mortality rate. was high, caloric debt during temporary ecls was low in comparison with previous results [ ] . overnutrition was frequent in the nec group and would justify implementation of nutrition protocol. incidence of gi intolerance remains frequent and could justify systematic used of motility agents with introduction of en. conclusion enteral nutrition in patients treated with temporary extracorporeal life support is feasible and may be improve with systematic motility agents and implementation of nutritional protocol. introduction cardiac surgery with cardiopulmonary bypass (cpb) is associated with a generalized inflammatory response with concomitant immune paresis which predisposes to the development of postoperative infections and sepsis ( ) . lymphocytes are essential agents of innate and adaptive immune responses during infections or inflammation processes. lymphopenia has been associated with immune dysfunction during septic shock, and it has been shown that low absolute lymphocyte count was predictive of postoperative sepsis ( ) . furthermore, impaired lymphocyte function probably occurs after cpb. thus, we investigated mechanisms involved in postoperative lymphopenia and impaired lymphocyte function after cpb. the aims of this study were: ) to describe a potential relationship between lymphopenia and occurrence of postoperative infections. ) to demonstrate that cpb induces lymphocytes apoptosis. ) to demonstrate that cpb impaired lymphocyte function (ability to proliferate). ) to demonstrate that il- , pd-l (programmed cell death ligand ) and indoleamine , -dioxygenase (ido) could be interesting targets to restore lymphocyte ability to proliferate after cpb. patients and methods blood cell counts with differentials obtained within the first postoperative week were analyzed in patients undergoing cardiac surgery in . postoperative lymphopenia was defined as a lymphocyte count < . × cells l − . postoperative infections were defined following cdc criteria. study procedures: the following analysis were performed before (t ) and h after (t ) cardiac surgery with cpb: lymphocyte apoptosis; t-cell proliferation ability following polyclonal stimulation; hla-dr and pd-l expression on monocytes; plasma ido activity and il- levels; and the ability of lymphocytes to undergo a clonal proliferation when stimulated using specific inhibitors of il- and ido. the study was approved by our local ethics committee. patients were informed of the observational nature of the study and gave their consent. . early lymphopenia after cpb was associated with the occurrence of postoperative infection: postoperative infections occured with a median delay of days. patients who developed postoperative infections had a significantly lower lymphocyte count at day , day and day than patients without postoperative infections. . cpb induced lymphocyte apoptosis and decreased t-cell proliferation ability. . cpb during cardiac surgery decreased mhla-dr expression. . cpb increased ido activity, pd-l expression and il- plasma levels. . il- or pd-l inhibition of inhibition could restore ability of lymphocytes to proliferate, although ido inhibitors did not show any effect. we provided new evidences that cpb induces immunosuppression. we also demonstrated that il- and pd-l could be interesting targets to restore ability of lymphocytes to proliferate. as maintaining mv during cpb decreased plasmatic levels of il- , our study brings new evidences that ventilator strategies could be of interest to decrease postoperative infections. respectively . % (n = ), . % (n = ) and . % (n = ) of the included patients. mortality was of . % in the overall population (n = ) and was higher in neutropenic patients ( . vs. . % in non-neutropenic patients; p < . ). neutropenia was independently associated with poor outcome when adjusted for underlying malignancy, allogeneic stem cell transplantation and severity as assessed by organ support (or . ; % ci . - . ). mortality decreased progressively over time in both non-neutropenic (from to %; p < . ) and in neutropenic patients (from to %; p < . ). when adjusted for confounders, admission during a more recent period was independently associated with favourable outcome and did not change the final model. conclusion this preliminary analysis suggests a meaningful survival in neutropenic critically ill cancer patients despite an independent association between neutropenia and mortality. additional analyses are on-going in order to adjust for study weight, heterogeneity across studies, assess the influence of neutropenia duration or g-csf use, and confirm the influence of neutropenia in a predefined subgroup of patients. introduction candida bloodstream infections (cbi) are frequent and increasing in hospitalized patients, especially in intensive care units. considering the results of some experimental in vitro and animal studies, it seems that yeasts belonging to candida genus are able, so as to survive, to modulate the immune response of the host by guiding t cells polarization to th profile. th and th cytokines are known to be involved in host defense against cbi. however, these data are mainly experimental or collected after candidemia. the aim of this study is to precise kinetic of cytokines network during human cbi. this was an ancillary study of an institutional project dedicated to pathophysiology of candidiasis. we have included patients with candidemia and controls ( matched hospitalized controls and healthy subjects). the sera of cases were gathered before (almost days before), during and after the isolation of yeasts from blood culture, defined as day (d ). quantitative analysis of cytokines by luminex ® technology and of ( , )-β-d-glucans by fungitell ® test were performed on samples. the amplitude of th profile response was expressed by summing the amount of the most relevant cytokines for th , th and th profiles, in pg/ ml. for each patient, the highest level of response was considered as %. results are expressed for the population by means of the results. we then performed univariate analysis (fischer exact test for qualitative variables, mann-whitney and wilcoxon test for quantitative variables, spearman for correlation; graphpad prism v software) and a multidimensional analysis by principal component analysis (pca; igorpro software). results patients with candidemia exhibited an increase in proinflammatory cytokines (ifnγ, tnfα and il- ), in comparison with the anti-inflammatory cytokines (il- and il- ) before d (p = . ) in univariate analysis. the ratio between mean values reverses at d and d (p = . ) and the increase of th response level from d to d is correlated to the decrease of th response (r = − . ; p = . ) in univariate analysis and pca. a pro-inflammatory response (th ) is associated with a reduced mortality (rr = . [ . ; . ]) and with a lower β-d-glucans levels (p < . ). discussion we describe here a dynamic cytokine profiles in response to candidemia. pro-inflammatory response predominates before d and reverses after. this is contradictory to the postulate that an antiinflammatory background could predispose to invasive candidiasis in icu patients and exhibiting a "post-infectious immune suppression conditions". but the relative deficiency in th response compared to simultaneous anti-inflammatory cytokines secretion observed after cbi is in accordance with experimental data, suggesting the modulation of the immune response by candida. the link between cytokinic profile and mortality can also raise the hypothesis of an influence by genetic factors on the regulation and direction of the immune response and so, the existence of a high-risk population. conclusion these data suggest a relation between candida and the orientation of the immune response towards a pattern deleterious for the infected host. this could allow to determine the most relevant cytokines varying during cbi. they could be used as biomarkers to identify the patients who could benefit from an early treatment in a preemptive targeted therapeutic strategy. these data will be paralleled to genetic background and to circulating candida derived molecules to precise the relative part of the host and the pathogen in this complex interaction. introduction lung ultrasound is widely used in intensive care, ermergency and pneumology medicine, for assessing acute respiratory pathologies. it is noninvasive, radiation free and rapidly available at the patient's bedside and provides an excellent accuracy. so, lung ultrasound may be an interesting tool for the physiotherapist as it allows to assess with more accuracy the patient improving the chest physiotherapy indication and monitoring ( ) . as far as we are aware, no study has evaluated the impact of lung ultrasound on clinical-decision making by physiotherapists in the use of chest physiotherapy. this case report highlights the lung ultrasound interest in chest physiotherapy in patient with lung consolidation. patients and methods this was a case report written following the recommendations of the care guideline ( ). the case was a -years-old female patient, non intubated, hospitalized in a respiratory icu. she was hypoxemic (pao = mmhg and sao = %), with dyspnoea at rest and an increasing radiological opacity at the right lung base. hypoxemia was the indication for physiotherapist referral. at the clinical examination, the physiotherapist's findings were: decreased mobility, dullness and abolished vesicular sound at the base of right hemithorax. this clinical examination and chest x-rays analysis allowed the physiotherapist to propose several clinical hypotheses: pleural effusion, obstructive atelectasis or pneumonia. the chest physiotherapy treatment differs according to the type of lung deficiencies. for example, the physiotherapist must to refer the patient to the medical staff in case of pleural effusion or may implement hyperinflation technique in case of obstructive atelectasis. determining the nature of lung deficiencies is essential to provide the more suitable therapeutic strategy. so, the physiotherapist decided to perform a lung ultrasound examination to retain the more likely hypothesis. results ultrasound examination performed by the physiotherapist highlighted the presence of a lung consolidation at the infero-lateral and posterior parts of the right lung with a pneumonia pattern: presence of tissue-like sign, shred sign, dynamic air bronchogram and fluid bronchogram. the medical staff implemented antibiotic treatment. the ultrasound findings guided the physiotherapist to choose chest physiotherapy technique improving the alveolar recruitment: nearly prone position (left side down) and continuous positive airway pressure during min. the patient response to the treatment was monitored by ultrasound and showed a decrease of the lung consolidation size and apparition of b lines, meaning a gain of lung aeration. these findings were associated with spo improvement but without decrease of dyspnoea. discussion lung ultrasound allowed the physiotherapist to precise the nature of the radiological lung opacity. as it is more accurate than clinical examination or chest x-ray, this suggests a more suitable choice of chest physiotherapy techniques than conventional clinical decision-making process. ultrasound findings suggested a positive response to the chest physiotherapy treatment. the apparition of re-aeration signs (b lines, decreased consolidation size) showed a short-term efficacy of the chest physiotherapy treatment. this allowed the physiotherapist to continue the treatment during week and obtain a substantial clinical improvement. conclusion the use of lung ultrasound in the clinical decision-making process may help the physiotherapist to choose with more accuracy the therapeutic strategy. moreover, it allows to monitor the treatment in real-time and assess the patient's response. the use of this tool may allow the physiotherapist to determine the optimal indications for chest physiotherapy and thus avoid unnecessary or inappropriate treatments. introduction critical illness together with immobilization have deleterious effects on patients outcome, especially in the presence of sepsis. increased muscle catabolism and membrane inexcitability reduce muscular mass and impair function within the first days after sepsis onset ( ). early mobilization could potentially limit muscle wasting and functional impairment in this population. the purpose of this study was to test whether exercise during the early phase of sepsis is safe and beneficial and to which extent it can limit skeletal muscle protein catabolism and preserve function. patients and methods adult patients admitted with the diagnosis of severe sepsis were included and randomly allocated to two groups; ) control group (ctrl-g): manual passive/active manual mobilization twice a day or ) experimental group (exp-g): additional two times min of passive/active cycling exercise. both groups benefited from a reduced sedation, adjusted nutritional intake and bed to chair transfer as soon as possible. skeletal muscle biopsy and electrophysiological testing were realized at day- and day- . muscle histology, biochemical and molecular analyses of anabolic/catabolic and inflammatory signalling pathways were performed. a group of four healthy subjects was used to obtain non pathological values. hemodynamic parameters and patients perception were collected during each session. results twenty-one patients were included, however died before the second muscle biopsy. ten patients in ctrl-g and nine in exp-g were finally analysed. muscle fibre cross sectional area (µm ) was significantly preserved by exercise (relative changes were ctrl-g: − ± % vs exp-g: ± %, p = . ). markers of catabolic systems were highly increased during sepsis compared to healthy subjects and reduced in both groups days after admission. however the reduction in mrna (relative change) tended to be more important in exp-g: murf- (ctrl-g: − ± % vs exp-g: − ± %, p = . ), mafbx (ctrl-g: − ± % vs exp-g: − ± %, p = . ), lc b (ctrl-g: ± % vs exp-g: − ± %, p = . ) and bnip (ctrl-g: ± % vs exp-g: − ± %, p = . ). anabolic and inflammatory markers were not affected by exercise. electrophysiological testing, including direct muscular stimulation, was abnormal on day- in of evaluated patients. since only a limited number of patients could be reassessed a second time, comparison between groups was not possible. in general, all activities were well tolerated by patients with no adverse events. the pulmonary auscultation is used by respiratory therapist (rt) to evaluate the efficiency of a treatment. listen to the noises coming from the primary bronchi (pb) is important because it is the place where secretions can be accumulated. therefore, it is crucial to know exactly where to place the stethoscope's chestpiece on the chest. few studies have analyzed the chest area where the pb were located. our hypothesis is that pb are localized on a line that joins axillary fossa (bi-axillary line: bal). the aim of our study is to evaluate the probability to find the primary bronchi by analysis of chest radiography. patients and methods a retrospective study was performed by analysis of chest x-ray using the software: tm reception ® , which allows precise measures to the tenth of millimeter. all the x-rays were made on confined to bed patients hospitalized within intensive care unit, internal medicine and abdominal surgery rooms. the following measures (in mm) were made between: the exclusion criteria were: bmi < . kg/m and bmi > kg/m , scoliosis, minor patient, lack of visibility of one of the axillary fossa, lack of visibility of pb, clavicular asymmetry, kyphosis, lack of symmetry in the shot, atelectasis and pneumothorax. statistics: normality test: ks. mean values are expressed with their sd and % ci. discussion in this study, we performed analysis of chest x-rays of bedridden patients and we demonstrated that it is possible to localize easily, on either side of the bs, the right and left pb at ± mm distance (lp) above a line joining axillary fossa. this study constitutes a new tool for the rt who, by using stethoscope with a chestpiece of cm surface area, will be able to listen to noise coming from pb. conclusion the data presented herein (fig. ) show that right and left pb are located at a mean distance of (± ) mm and (± ) mm above the bal, on both sides of the bs. the bal represents thus an easy and precise mode to detect right and left pb by bedridden. finally, the distance between the hyoid bone and the sc is about cm. as the pb are located after the bifurcation, this information constitutes another useful way for the localization the right and left pb by bedridden patient. introduction critically ill patients frequently develop muscle weakness, which is associated with prolonged intensive care unit and hospital stay ( ). this randomized controlled trial (clinical trials nct ) was designed to investigate whether a daily training session using a tilt table, started early in stable critically ill patients with an expected prolonged icu stay, could improve strength at icu and hospital discharge compared to a standard physiotherapy program. the study protocol was approved by an ethics committee and informed consent was obtained from all patients. patients admitted in adult icu of marie lannelongue hospital, france, who were mechanically ventilated for at least days were included. exclusion criteria were cerebral or spinal injury, pelvic or lower limb fracture. patients were assessed each day for temporary contraindications for mobilization out of bed (rass score <− or > ; hemodynamic instability; a continuous intravenous dose of epinephrine/ . no significant difference was observed in terms of mrc score or in terms of pts with or without weakness (mrc > ) at icu or hospital discharge. however, the number of pts with weakness was significantly higher in the group before tilt mobilization, suggesting a more rapid improvement in the tilt group. the icu and hospital lengths of stay were not different between groups. discussion the prevalence of muscle weakness in our population is high before mobilization ( . %, % ci . - . ), is still . % at icu discharge but represents only ~ % at hospital discharge. this low hospital discharge prevalence is probably related to the early and intense physiotherapy in both groups, which may explain our inability to demonstrate superiority of the addition of tilt table positioning, although a faster recovery is suggested. conclusion training sessions using a tilt table, in addition to early and intense physiotherapy did not improve muscle strength evaluated using mrc score in surgical icu patients with muscle weakness. proposed modifications to the duke criteria for the diagnosis of infective endocarditis quantifying the relationship between staphylococcus aureus bacteremia and s. aureus bacteriuria: a retrospective analysis in a tertiary care hospital recommandations formalisées d'experts «prise en charge des infections intra-abdominales strategies to reduce curative antibiotic therapy in intensive care units (adult and paediatric) risk factors for developing esbl e. coli: can clinicians predict infection in patients with prior colonization? kdigo clinical practice guideline for acute kidney injury citrate anticoagulation for continuous venovenous hemofiltration benefits and risks of furosemide in acute kidney injury national nosocomial infections surveillance (nnis) system report, data summary from prospective evaluation of the risk factors, etiology and the antimicrobial susceptibilities of the isolates in nosocomial bacteriemic patients unrecognized endobronchial intubation of emergency patients endobronchial intubation detected by insertion depth of endotracheal tube, bilateral auscultation, or observation of chest movements: randomised trial nosocomial infections in a burn intensive care unit outcomes of critically ill cancer patients in a university hospital setting outcome and prognostic factors of lung cancer patients admitted to the medical intensive care unit intensive care of the cancer patient: recent achievements and remaining challenges chronic critical illness the epidemiology of chronic critical illness in the united states designing and conducting a cluster-randomized trial of icu admission for the elderly patients: the ice-cub study estimation de la mortalité maternelle en france: une nouvelle méthode emergency postpartum hys-terectomy for uncontrolled postpartum bleeding music interventions for mechanically ventilated patients effets de la musicothérapie en réanimation hors sédation chez des patients en cours de sevrage ventilatoire versus des patients non ventilés. annales françaises d' anesthésie et de réanimation evaluation of a low-flow oxygen-conserving nasal cannula performance of six types of oxygen delivery devices at varying respiratory rates high-flow oxygen through nasal cannula in acute hypoxemic respiratory failure international study of the prevalence and outcomes of infection in intensive care units medical intensive care urinary and serum biomarkers for the diagnosis of acute kidney injury: an in-depth review of the literature diagnostic strategies for renal impairment in the intensive care unit recommendations formalized expert acute renal failure in the perioperative and intensive care (excluding extrarenal purification techniques) frutos-vivar f. comparison of the berlin definition for acute respiratory distress syndrome with autopsy the presence of diffuse alveolar damage on open lung biopsy is associated with mortality in patients with acute respiratory distress syndrome: a systematic review and meta-analysis ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome alveolar macrophages contribute to alveolar barrier dysfunction in ventilator-induced lung injury references . akoumianaki e. mechanical ventilation-induced reverse-triggered breaths: a frequently unrecognized form of neuromechanical coupling neuromuscular blockers in early acute respiratory distress syndrome driving pressure and survival in the acute respiratory distress syndrome mortality and pulmonary mechanics in relation to respiratory system and transpulmonary driving pressures in ards organ donation, reasons for family refusal: a retrospective study in a french organ harvesting center - du avril relative aux droits des malades et à la fin de vie comité consultatif national d'ethique c. fin de vie, autonomie de la personne, volonté de mourir reliability, validity, and health issues arising from questionnaires used to measure psychosocial and organizational work factors (powfs) among hospital nurses: a critical review aging and death signalling in mature red cells: from basic science to transfusion practice most reliable indices in differentiation between thalassemia trait and iron deficiency anemia stratification du risque dans l'embolie pulmonaire aiguë.realites cardiologiques • n° -cahier • septembre european resuscitation council guidelines for resuscitation : section . executive summary breakthrough in cardiac arrest: reports from the th paris international conference changing use of noninvasive ventilation in critically ill patients: trends over years in francophone countries noninvasive mechanical ventilation in acute respiratory failure: trends in use and outcomes high-flow oxygen through nasal cannula in acute hypoxemic respiratory failure failure of noninvasive ventilation for de novo acute hypoxemic respiratory failure: role of tidal volume réanimation médicale polyvalente, hôpital de la source early identification of patients at risk for difficult intubation in the intensive care unit: development and validation of the macocha score in a multicenter cohort study clinical practice and risk factors for immediate complications of endotracheal intubation in the intensive care unit: a prospective, multiple-center study profound and persistent decrease of circulating dendritic cells is associated with icu-acquired infection in patients with septic shock intensive care med specific mait cell behaviour among innate-like t lymphocytes in critically ill patients with severe infections a -level prognostic classification in septic shock based on cortisol levels and cortisol response to corticotropin prognostic value of relative adrenal insufficiency during cardiogenic shock: a prospective cohort study with long-term follow-up. shock calcium supplementation during sepsis exacerbates organ failure and mortality via calcium/calmodulin-dependent protein kinase signaling hypocalcemia in critically ill patients diabetic ketoacidosis traps joint british diabetes societies guideline for the management of diabetic ketoacidosis peter radermacher , for the hyper s investigators and reva research network réanimation médicale, hôpital henri mondor réanimation medico-chirurgicale, hopital avicenne institut für anästhesiologische pathophysiologie und verfahrensentwicklung département de réanimation médicale et de médecine hyperbare, c.h.u. d' angers, angers, france; service de réanimation médico-chirurgicale réanimation médico-chirurgicale, assistance publique -hôpitaux de paris, hôpital ambroise paré, boulogne-billancourt, france; service de réanimation adulte quantitative measurement of heparin in comparison with conventional anticoagulation monitoring and the risk of thrombotic events in adults on extracorporeal membrane oxygenation elso guidelines for cardiopulmonary extracorporeal life support, version . . ann arbor: extracorporeal life support organization early and late outcomes of consecutive adult patients treated with extracorporeal membrane oxygenation for refractory postcardiotomy cardiogenic shock the french sf- health survey: translation, cultural adaptation and preliminary psychometric evaluation weaning of extracorporeal membrane oxygenation using continuous hemodynamic transesophageal echocardiography predictors of successful extracorporeal membrane oxygenation weaning after assistance for refractory cardiogenic shock extracorporeal membrane oxygenation in the adult: a review of anticoagulation monitoring and transfusion back from irreversibility: extracorporeal life support for prolonged cardiac arrest microcirculatory perfusion is preserved during off-pump but not on-pump cardiac surgery extracorporeal membrane oxygenation causes loss of intestinal epithelial barrier in the newborn piglet the pulsatile perfusion debate in cardiac surgery: answers from the microcirculation? a meta-analysis of complications and mortality of extracorporeal membrane oxygenation impact of fluid balance on outcome of adult patients treated with extracorporeal membrane oxygenation cumulative fluid balance and mortality in septic patients with or without acute kidney injury and chronic kidney disease comparison of two fluid-management strategies in acute lung injury renal function and survival in patients undergoing ecmo therapy extracorporeal membrane oxygenation and the kidney plasma concentrations of inflammatory cytokines rise rapidly during ecmo-related sirs due to the release of preformed stores in the intestine in-hospital mortality and successful weaning from venoarterial extracorporeal membrane oxygenation: analysis of patients using a national inpatient database in japan is the . % rate of in-hospital mortality in patients receiving venoarterial extracorporeal membrane oxygenation really that high? predicting survival after ecmo for refractory cardiogenic shock: the survival after veno-arterial-ecmo (save)-score outcomes and long-term quality-of-life of patients supported by extracorporeal membrane oxygenation for refractory cardiogenic shock usefulness of cardiac biomarkers to predict cardiac recovery in patients on extracorporeal membrane oxygenation support for refractory cardiogenic shock etiology of troponin elevation in critically ill patients esc guidelines for the diagnosis and treatment of acute and chronic heart failure: the task force for the diagnosis and treatment of acute and chronic heart failure of the european society of cardiology (esc) accf/aha guideline for the management of heart failure: a report of the american college of cardiology foundation/american heart association task force on practice guidelines nearhanging injuries: a -year experience unité de réanimation neurologique s rapid diagnosis of bacterial meningitis using a point-of-care glucometer geoffroy rousseau service d'information médicale, epidémiologie et economie de la santé guillain-barré syndrome outbreak associated with zika virus infection in french polynesia: a case-control study early predictors of mechanical ventilation in guillain-barré syndrome paediatric prolonged mechanical ventilation: considerations for definitional criteria cardiac filling volumes versus pressures for predicting fluid responsiveness after cardiovascular surgery: the role of systolic cardiac function respiratory variation in aortic blood flow peak velocity to predict fluid responsiveness in mechanically ventilated children: a systematic review and meta-analysis. pediatr anesth effect of prone and supine positions on functional residual capacity, oxygenation, and respiratory mechanics in ventilated infants and children positioning effects on lung function and breathing pattern in premature newborns l'accident vasculaire ischémique en pédiatrie. quand y penser -quoi faire outcomes of early decompressive craniectomy versus conventional medical management after severe traumatic brain injury: a systematic review and meta-analysis myocardial dysfunction evaluation in pediatric brain death donor service de régulation et d'appui, agence biomedecine prone position for acute respiratory failure in adults prone positioning in severe acute respiratory distress syndrome effects of the prone position on respiratory mechanics and gas exchange during acute lung injury does prone positioning increase intracranial pressure? a retrospective analysis of patients with acute brain injury and acute respiratory failure prone position in mechanically ventilated patients with reduced intracranial compliance maud loiselle -maud.loiselle@outlook.fr annals of intensive care boulogne-billancourt, france; réanimation polyvalente adulte, centre hospitalier intercommunal andré grégoire, montreuil, france; réanimation médicale an audit of intensive care unit recyclable waste s point of care ultrasonography: is there a place for pocket size ultrasonography devices? gabriel preda , vincent dubée , naïke bigé eric maury -ejhmaury@gmail.com annals of intensive care outcome, functional autonomy, and quality of life of elderly patients with a long-term intensive care unit stay berlin: springler; . s icu nurses' perception of end-of-life decision making: a french multicenter survey akli chermak , alexandre lautrette ap-hp) anesthésie réanimation et traitement chirurgical des grands brûlés réanimation médicale hôpital d'instruction des armées percy correspondence: fanny.ardisson@gmail.com (fanny ardisson) annals of intensive care circulating mitochondrial damps cause inflammatory responses to injury rage is a nucleic acid receptor that promotes inflammatory responses to dna beneficial hemodynamic effects of prone positioning in patients with acute respiratory distress syndrome stard : an updated list of essential items for reporting diagnostic accuracy studies towards complete and accurate reporting of studies of diagnostic accuracy: the stard initiative s neurological impairment in cirrhotic patients admitted to icu: hepatic versus drug-induced encephalopathy unité de soins intensifs d'hépatogastroentérologie réanimation polyvalente, hôpital de mercy, ars laquenexy, france; réanimation médicale, centre hospitalier universitaire d' angers, angers, france; réanimation médicale cerebrospinal fluid findings after epileptic seizures effect of epileptic seizures on the cerebrospinal fluid-a systematic retrospective analysis s synek score and nse to predict poor neurological outcome after cerebral anoxia and therapeutic hypothermia réanimation médicale correspondence: dimitri titeca beauport -titeca.dimitri@chu-amiens.fr annals of intensive care guidelines for the management of severe traumatic brain injury. vi. indications for intracranial pressure monitoring reduced mortality rate in patients with severe traumatic brain injury treated with brain tissue oxygen monitoring s organ procurement and kidney transplantation under maastricht condition (m ): update on year of activity coordination prélèvements organes s reference . conditions à respecter pour réaliser des prélèvements d'organes sur les donneurs décédés après arrêt circulatoire de la catégorie iii de maastricht dans un établissement de santé. agence de la biomédecine. version n° mai crcl by cockroft-gault, mean (ml/mn delayed graft function, n (%) ( . %) réanimation médico-chirurgicale infectious diseases society of america. guidelines for the management of adults with hospital acquired, ventilator-associated, and healthcare-associated pneumonia reducing ventilator-associated pneumonia in intensive care: impact of implementing a care bundle chiche@aphp.fr annals of intensive care national nosocomial infections surveillance system. national nosocomial infections surveillance (nnis) system report, data summary from critères d'infection chez les brulés unité d'épidémiologie et recherche clinique international study of the prevalence and outcomes of infection in intensive care units risk and prognostic factors of ventilator-associated pneumonia in trauma patients ventilator-associated pneumonia: never enough, never give up! sahar habacha , bassem chatbri , aymen m'rad , youssef blel , nozha brahmi sahar habacha -sahar.habacha@gmail.com annals of intensive care weaning patients from the ventilator automated versus non-automated weaning for reducing the duration of mechanical ventilation for critically ill adults and children: a cochrane systematic review and meta-analysis unité de réanimation et de surveillance continue, service de pneumologie et réanimation médicale noninvasive ventilation and weaning in patients with chronic hypercapnic respiratory failure: a randomized multicenter trial risk factors for extubation failure in patients following a successful spontaneous breathing trial s a multicenter prospective observational study of extubation procedures in intensive care units: the free-rea study audrey de jong -audreydejong@hotmail.fr annals of intensive care early identification of patients at risk for difficult intubation in the intensive care unit: development and validation of the macocha score in a multicenter cohort study faouzi saliba -faouzi.saliba@pbr.aphp.fr annals of intensive care réanimation médicale polyvalente, hôpital de la source mickael landais -mickaelandais@gmail.com annals of intensive care perioperative fasting in adults and children: guidelines from the european society of anaesthesiology the decision to extubate in the intensive care unit service de réanimation médicale s refeeding hypophosphoremia in a medical critical care unit: -month observational study gioia gastaldi -gioia.gastaldi@chu-rouen.fr annals of intensive care refeeding hypophosphatemia in critically ill patients in an intensive care unit. a prospective study refeeding syndrome: problems with definition and management biosit and inserm u , faculte de medecine, université rennes immune dysfunction after cardiac surgery with cardiopulmonary bypass: beneficial effects of maintaining mechanical ventilation s influence of neutropenia on mortality of critically ill cancer patients: results of a systematic review on individual data quentin georges brazil; department of critical care medicine and division of pulmonary and critical care medicine united kingdom; department of intensive care centre d'infection et d'immunité de lille equipe -basic and clinical immunity of parasitic di delta nlr") were calculated. statistical analysis used appropriate non parametric tests and cox regression for survival analysis. the ability of the variables to discriminate survivors from non-survivors was determined using roc curves results during the study period, cirrhotic patients were admitted in icu. the etiologies of liver cirrhosis were alcoholic in % of cases with severe score: median child-pugh score = %) deaths after icu discharge during the same hospitalization. nlr decreased for survivors between d and d univariate analysis, for predicting survival, higher values of nlrd , delta nlr, meld score at admission, sofa score at admission and at day and delta sofad -d were significant factors. predictors of death in multivariate analysis are shown in fig. . area under delta nlr roc conclusion the blood nlr is a novel inflammation index that has been shown to independently predict poor clinical outcomes. we have demonstrated that delta nlr is an independent predictor of mortality in critically ill cirrhotic patients the association between the neutrophil-to-lymphocyte ratio and mortality in critical illness: an observational cohort study gene-and exon-expression profiling reveals an extensive lps-induced response in immune cells in patients with cirrhosis celine dupre -duprecece@gmail.com annals of intensive care diagnostic accuracy of procalcitonin in critically ill immunocompromised patients the role of pattern-recognition receptors in innate immunity: update on toll-like receptors esm- is a novel human endothelial cell specific molecule expressed in lung and regulated by cytokines thoracic ultrasound: potential new tool for physiotherapists in respiratory management. a narrative review the care guidelines: consensus-based clinical case reporting guideline development department of physical medicine and rehabilitation icu-acquired weakness and recovery from critical illness o where should we place the stethoscope's chestpiece to hear the noise of the primary bronchi? frédéric duprez , bastien dupuis , grégory cuvelier , thierry bonus frédéric duprez -dtamedical@hotmail.com annals of intensive care o aerosol delivery using two nebulizers through high flow nasal cannula: a randomized cross-over spect-ct study correspondence: jonathan dugernier -jonathan.dugernier@uclouvain.be annals of intensive care introduction in , an international consensus conference took stock of the various measures to be implemented for the prevention of ventilator acquired pneumonia (vap) [ ]. these measures are often gathered in groups of or under the term of "ventilator-bundle. " the effectiveness of these "bundles" was poorly evaluated in african environment. objective to establish a vap prevention program and assess its impact on morbidity and mortality of patients under mechanical ventilation in our service. patients and methods prospective, mono centric, quasi-experimental before-after study. it took place in the intensive care unit of the university clinics of kinshasa in the democratic republic of congo (drc). this service is equipped with beds and a respirator for two beds. the observational period (phase ) was carried out from february st to december st, and the intervention period (phase ) from february st, to february st, . all consecutive patients intubated and mechanically ventilated for more than h were included. five preventive measures were held: hand hygiene, the elevation of the head of the bed at °- °, the daily lifting of sedation, oral decontamination with chlorhexidine and control cuff pressure of the endotracheal tube. compliance with this bundle was assessed by direct observation without the knowledge of caregivers. the diagnosis of "vap" was held before a clinically modified sore (m cpis) > . the main outcomes were the incidence of vap and mortality. the protocol for this study was approved by the ethics committee of the school of public health of the university of kinshasa, under the approval number: esp/ec/ / .introduction nosocomial infections (ni) are common in burn patients due to the loss of the first line of defense against microbial invasion, immunocompromising effects of burn injury, and invasive diagnostic and therapeutic procedures. the objective of this study was to identify the incidence of nosocomial infection (ni), the pathogens and their antibacterial patterns, and prognosis of these burn patients. patients and methods a retrospective study was conducted in a bed intensive burn care unit during months. patients were eligible for the study, if they met the following criteria: total burn surface area (tbsa) > %, length of icu stay ≥ h, and infected in accordance with the criteria of the national nosocomial infections surveillance (nnis) and the criteria of the sfetb [ ][ ]. in this study, nis were classified into four main groups: pneumonias, bloodstream infections (bsi), catheter related infections (cri), and urinary tract infections (uti). for included patients, skin levy, blood cultures, urine and sputum cultures were drawn during fever or clinical features of sepsis. results during the -month study period, patients were admitted to the icu, patients were included ( . %). were male and female. the mean age was ± yr. the mean tbsa was ± %. % were admitted from another hospital. burn injuries were due to domestic accidents in % and self immolation in %. the mean none. none. none. none. none. none. none. none. none. none. ann. intensive care , (suppl ): none. none. none. none. none. none. none. none. none. none. none. none. none. none. none. none. none. none. none. consulting activities with fisher & paykel. none. none. none. none. none. none. none. none. none. none. none. none. none. none. none. none. none. none. none. failure extubation in intensive care unit: risk factors, incidence and evaluation of a mechanical ventilator weaning protocol lucie petitdemange , anne sophie guilbert none. none. none. none. none. none. opportunistic infections in patients with solid tumors: a systematic review julien poujade , elie azoulay none. invasive aspergillosis in non-immunocompromised patients hospitalized intensive care unit guillaume trumpff , max guillot , thierry braun , ralf janssen-langenstein , marie-line harlay , jean-etienne herbrecht introduction characteristics and outcomes of adult patients with invasive aspergillosis in intensive care unit have rarely been described. we performed a retrospective study on consecutive adult patients with invasive aspergillosis who were admitted form january through january to the intensive none. noorah zaid , nawel ait-ammar , christine bonnal , jean-claude merle , francoise botterel , eric levesque anesthesia and intensive care medicine, chu henri mondor, créteil, france; unité de parasitologie-mycologie, département de virologie, bactériologie-hygiène, parasitologie, hopital henri mondor, créteil, france correspondence: eric levesque -eric.levesque@aphp.fr annals of intensive care , (suppl ):s introduction liver transplant recipients have high rate of invasive fungal disease (ifd) with high morbidity and mortality, in part due to its delayed diagnosis. the fungal cell wall component ( , )-betad-glucan (bg) is a biomarker for fungal infection but its utility remains uncertain. this prospective study was designed to review our experience in ifd and to evaluate the impact of bg in the diagnosis of ifd. patients and methods from january to may , liver transplantation were performed in our institution. serum samples were tested for bg (fungitell; cape cod inc., usa) least weekly between liver transplantation and their discharge from hospital. ifd was defined as proposed by the european organization for research and treatment of cancer/mycoses study group. results nineteen patients ( %) were diagnosed with ifd including cases of candidiasis infection (ci) in eleven out of patients, invasive pulmonary aspergillosis (including one who had previously ci) and one case of septic arthritis of the hip caused by scedosporium spp. ifd was associated with significantly high mortality (log-rank p = . ). the area under the roc curves, for bg to predict ifd, was . ( % ci . - . ). using a cutoff of pg/ml, the most discriminative cut-off point from the roc curve, the sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) values of bg for overall ifd was % ( % ci, - ), % ( % ci, - ), % ( % ci, - ) and % ( % ci, - ). conclusion based on its high npv, bg value appears to be a good biomarker to rule out the diagnosis of ifd when the value is below pg/ml. a single point bg may guide the investigation and the decision to start antifungal therapy in patients at risk for ifd. none. monitoring of changes in lung and chest wall mechanics in the supine, lateral and prone positions during the prone positioning maneuver in ards patients zakaria riad , mehdi mezidi , hodane yonis , mylène aublanc, , sophie perinel-ragey, , floriane lissonde , aurore louf-durier, , romain tapponnier , jean-christophe richard , bruno louis, , claude guérin , plug working group réanimation médicale, hôpital de la croix-rousse, lyon, france; inserm, u , equipe , équipe biomécanique cellulaire et respiratoire, université paris-est créteil -faculté de médecine, créteil, france correspondence: zakaria riad -zakaria.riad@icloud.com annals of intensive care , (suppl ):s none. introduction systemic rheumatic diseases (srd) are autoimmune diseases that are rare but cause substantial morbidity and mortality. srds chiefly affect the lungs, however, data on critically ill patients with srd admitted for arf are scarce. patients and methods retrospective cohort conducted in french icus ( . the major comorbidities were cardiovascular ( %), tobacco exposure ( %), chronic kidney disease ( %) and neoplasia ( %). two-thirds of patients were on systemic corticosteroids at admission, the median dose of (iqr) mg per day. srd diagnosis was made in the icu in . % of patients. clinically or microbiologically documented bacterial pneumonia was the leading arf etiology ( . %). in % of cases, arf was related to an opportunistic infection (mainly aspergillus (n = ) and pneumocystis (n = )). others arf etiologies included specific lung involvement ( . %) and cardiac pulmonary edema ( . %). sofa on day one was [ ] [ ] [ ] [ ] [ ] [ ] [ ] . associated organ dysfunctions were mainly hemodynamic ( %) and renal ( %). mechanical ventilation was needed in % of patients (non invasive only in . % or invasive in . %), % needed vasopressors, and % renal replacement therapy. systemic corticosteroids were started in % of patients and % of patients received pulse steroids. cyclophosphamide and plasma exchange were required in and % of patients, respectively. length of icu stay was [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] days. icu-acquired infection occurs in % of cases. in total, patients ( . %) died throughout the icu stay. arf etiology was not associated with mortality. by multivariate analysis, shock on admission (or . [ . - . ], p < . ) and the use of invasive mechanical ventilation (or . [ . - . ], p = . ) were independently associated with mortality, whereas non-invasive ventilation was associated with decreased mortality (or . [ . - . ], p = . ). by considering among the connective tissue diseases, the groups of myositis and scleroderma (n = ), these diseases were associated with a trend for a higher mortality (or . [ . - . ], p = . ). conclusion in patients with srd, arf is associated with a high case fatality, primarily when mechanical ventilation is needed. particular attention must be given to specific srd-sub groups for which pulmonary flare may require intensive immunosuppression. none. none. none. severe acute pancreatitis in icu: management and outcomes of infected pancreatic necrosis charlotte garret , matthieu peron , emmanuel coron , cédric bretonnière , jean reignier , christophe guitton réanimation médicale, chu hôtel-dieu nantes, nantes, france; the acute pancreatitis appears as a pathology that we can define with difficulty because of its clinical presentation or prognosis. patients and methods in our study, we analysed cases of acute necrotic and hemorrhagic acute pancreatitis, hospitalized at the department of resuscitation of the surgical emergencies (p ) of the uhc ibn rochd casablanca during the period ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the purpose of this study is to do a descriptive analysis of the epidemiologic, clinic, radiological, therapeutic and evolutive data of the acute necrotic pancreatitits, we included in our study patients with epidemiologic, clinic, radiologic, biologic criteria of acute necrotic pancreatitits diagnosis whatever is the biliary or alcoholic etiology. the valuation gravity of the pancreatitis has been based on:• ranson bioclinical score > /apache ii > ; • visceral failure.• spreading of the necrosis. the analysis of the results shows that: about the epidemiologic aspect: mean age ( year old), the biliary etiology predominates ( %). about the clinical aspect: pain ( %) vomiting ( %), stop of the transit ( %), the visceral distresses are: the shock ( %), respiratory distress ( %), and neurological distress ( %). about the radiological aspect: pleural effusion ( %), abdominal echography: vesicular lithiasis ( %), dilated principal biliary duct ( %), abdominal computerized tomography: stage e ( %). about the biological aspect: hyperglycemia ( %), hyper-amylasemia ( %). the indexes of gravity that have been appreciated in this study are: ranson score > ( %), imrie score > ( %), igs score ≥ ( %), osf score ≥ ( %). the treatment of the anhp has been symptomatic in particular and the evolution has been characterized by mortality about %, the cause was particularly infectious. the prognostic factors predetermined in this study are:• female type (p = . ).• hemodynamic distress (p = . ).• respiratory distress (p = . ).• scores of gravity:• ranson > (p = . ).• imrie > (p = . ).• osf ≥ (p = ).• infection (p = . ).• duration of the hospitalization (p = . ).• rate of c-reagent protein (p = . ). in conclusion, the mortality is still high in the anhp, considerable effort of search is necessary to prevent the infectious complications of mortality. none. predicting -day mortality following liver transplantation in patients with acute-on-chronic liver failure: a decision-tree model from the french national liver transplantation system, the optimatch study, - none. none. none. none. none. none. the french law and recent expert opinions have emphasized the need for a multidisciplinary approach in decisions to forgo life sustaining therapies for the critically ill. we sought to assess how icu nurses actually rank their involvement and perceive this process. materials and methods we conducted a cross sectional survey using a web-based questionnaire between june and september . results of the icus invited to participate, ( %) agreed. a total of icu participants completed the survey of whom % were nurses and % assistant nurses. median age was (inter quartile range - ) years and % were female. median work experience was ( - ) years and time in the icu was ( - ) years. eighty-five percent of the participants have been involved at least once in a multidisciplinary end-of-life discussion. less than half of the participants reported a good ( %) or partial ( %) knowledge of the current end-of-life legal framework. the decision to start a discussion about withdraw life-sustaining therapy (wlst) was initiated by a senior intensivist in % of the cases, by a nurse in % and an assistant nurse in . %. this decision was approved by % of the participants. the decision-making process was considered to be initiated at the right time for % of the participants, too late for %, and too early for %. the discussion occurred mostly in the afternoon ( %) or during the medical staff ( %), in a dedicated place in % of the cases. a median of ( - ) health-care professionals attended the wlst discussion. half the respondents reported being reluctant to talk during the discussions and % never expressed their own opinion. indeed, although the length of the discussion was ( - ) minutes, participants estimated to talk during only ( - ) minutes. the following reasons were mentioned by the participants to explain these facts: having cared for the patient for too short time ( %), lack of medical knowledge ( %), decision of wlst already taken by the medical staff ( %), their opinion not really taken into account ( %), reluctant to talk during meetings in general ( %), consider that the discussion is limited to a medical expertise ( %), limited professional experience ( %), and fear to express a different opinion ( %). nevertheless, % of the participants were partially ( %) or totally ( %) satisfied by the way the decision making process was conducted, % considered that collegiality was applied, and % agreed with the final decisions.conclusion icu nurses rank favorably multidisciplinary wlst discussions. nevertheless their involvement in the discussion remains limited. beyond factors related to work organization and professional experience, efforts should be made to recognize their role and value, and to encourage them to share their own opinions with the other members of the icu team. none. determinants and prognosis of elevation of high-sensitivity cardiac troponin t in patients hospitalized with vasodilatatory shock marie caujolle , jérôme allyn , dorothée valance , caroline brulliard , none. free plasmatic mitochondrial dna-receptor for advanced glycation end-products: a new signaling pathway of critical illness-induced endothelial dysfunction arthur durand , rémi nevière , florian delguste , eric boulanger, none. quality of reporting of fluid responsiveness evaluation studies: a five year systematic review izaute guillame , matthias jacquet-lagrèze , jean-luc fellahi none. none. none. none. none. introduction microaspiration of gastric and oropharyngeal contaminated secretions occurs frequently in intubated critically-ill patients, and plays a major role in the pathogenesis of ventilator-associated pneumonia (vap). at basic state, patients with chronic obstructive pulmonary disease (copd) have an increased risk of microaspiration (due to gastro-esophageal reflux disease, pharyngo-laryngeal dys-function…), this risk may even be more important under mechanical ventilation. the main purpose of this study is to determine if copd is a risk factor for global abundant microaspiration (gam) in intubated critically-ill patients. we gathered data about two prospective multicentric randomized trials focused on microaspiration in intubated patients. data about copd were retrospectively collected in order to complete previous data. microaspiration of gastric and oropharyngeal secretions was respectively determined by quantitative measurements of pepsin and salivary amylase in all tracheal aspirates during the first h after intubation. gam was defined as the presence at significant level of pepsin (> ng/ml) and/or salivary amylase (> ui/l) in at least % of the tracheal aspirates. in order to find gam independent risk factors, we realized an univariate and multivariate analysis of the variables collected. results out of patients included in the studies, were analyzed among which patients with copd. patients ( %) had gam. neither copd diagnosis, nor spirometric severity nor specific therapeutics were associated with gam. risk factors for gam in univariate analysis were the age, diabetes, low score in glasgow coma scale (gcs), and no recourse to paralytic agents or vasopressors. after none. none. implementation and impact assessment of a "ventilator-bundle" at the university clinics of kinshasa: before and after study josé mavinga , joseph nsiala makunza , m e mafuta , yves yanga , amisi eric , jp ilunga , ma kilembe none. none. amel mokline , achraf laajili , helmi amri , imene rahmani , nidhal mensi , lazheri gharsallah , sofiene tlaili , bahija gasri , rym hammouda , amen allah messadi burn care department, trauma and burn center, tunis, tunisia correspondence: amel mokline -dr.amelmokline@gmail.com annals of intensive care , (suppl ):s none. none. none. introduction mechanical ventilation (mv) weaning is a crucial step in critically ill patients. mv duration is associated with an increased risk of ventilator associated events, even though its specific impact on mortality has never been clearly demonstrated ( ). automated closed loop systems might help the weaning process. a recently published meta-analysis has reported a reduction in mv duration when using an automated weaning mode as compared to non-automated mode ( ) . however, the different automated modes have not been compared to each other. the objective of this network meta-analysis was to compare the performance of the three major automated weaning modes, i.e. the automode°, the smartcare° and the adaptative support ventilation (asv°) for mv weaning in critically ill and postoperative adult patients. we included all randomised control trials that compared automated closed loop weaning applications either to another automated application or standard care, including weaning according to a written weaning protocol or nurse driven protocols. the three modes of automated modes included in the study were asv°, smartcare° and automode°. the primary outcome was the duration of mv weaning, defined as the time between randomization and a successful extubation. we also planned subgroup analyses in the icu and the post-operative populations. the quality of the studies was assessed independently by two blinded investigators, using the evaluation recommended by the cochrane collaboration. a network bayesian meta-analysis using random effect models and based on aggregate data from the included studies was performed using the gemtc package (r project, vienna). this trial was declared in pros-pero in august (crd ). results search of databased identified articles; were screened for eligibility after removal of duplicates. abstract analysis led to the exclusion of articles with a final full text analysis of randomised control trials. ultimately, trials were included in the analysis, representing ventilated patients. nine studies included patients in the post-operative period while six were conducted in icu. the automated mode was asv° (a) in studies, smartcare° (c) in studies and auto-mode° (b) in studies. all studies reported the duration of mv weaning as defined in our protocol. in all studies, the control group was standard care with a weaning process driven either by nurses or physicians. in studies ( %) a written weaning protocol was used in the control group. all icu studies used sedation protocols based on sedation scores, none of them including systematic daily sedation interruption. each one of the automated application was associated with a significant reduction in the duration of mv as compared to the control. when comparing all different modes using the network meta-analysis framework, asv° appeared to be the best automated mode when it pertains to reducing the duration of mechanical ventilation weaning (fig. ) . subgroup analysis showed similar results in the post-operative and the icu populations. conclusion compared to standard weaning practice, the major automated weaning modes significantly reduced the duration of mv weaning in critically ill and post-operative adult patients. asv° was associated with the most significant effect when compared to the two other automated modes (smartcare°, automode°). further physiological respiratory studies would help to understand the underlying mechanisms accounting for the superiority of asv. none. none. introduction in intensive care unit (icu) patients, diaphragm dysfunction is associated with adverse clinical outcomes. ultrasound measurements of diaphragm thickness (tdi), excursion (exdi) and thickening fraction (tfdi) have been proposed as estimators of diaphragm function, but have never been compared to phrenic nerve stimulation. our aim was to describe the relationship between tdi, exdi, tfdi and diaphragm function evaluated using the change in endotracheal pressure after phrenic nerve stimulation (ptr,stim), and to compare their prognostic value. patients and methods ptr,stim and ultrasound variables were measured in mechanically ventilated (mv) patients < h after intubation ("initiation of mv", under assist-control ventilation, acv) and at the time of switch to pressure-support ventilation ("switch to psv"). diaphragm dysfunction was defined as ptr,stim < cmh o. results patients were included. at initiation of mv, ptr,stim was not correlated to tdi (rho = − · , p = · ), exdi (rho = · , p = · ) or tfdi (rho = − · , p = · ). at switch to psv, tfdi and exdi were correlated to ptr,stim, (rho = · , p < . and · , p = · , respectively), but tdi was not (rho = − · , p = · ). at switch to psv, a tfdi < % could reliably identify diaphragm dysfunction (sensitivity and specificity of and %, respectively), but tdi and exdi could not. this value was associated with increased duration of icu stay and mv, and mortality. conclusion under acv, neither tdi, exdi nor tfdi were related to ptr,stim. under psv, tfdi was strongly correlated to diaphragm strength and, when decreased, was associated with poorer outcome. alexandre demoule has signed research contracts with covidien, maquet and philips; he has also received personal fees from covidien and msd. none. none. none. management of enteral feeding during extubation in the intensive care unit: a multi-center retrospective study in french intensive care units mickael landais , noemie hubert , mai-anh nay , johann auchabie , bruno giraudeau , reignier jean , arnaud w thille , stephan ehrmann none. none. nutritional support in patients receiving temporary extracorporeal life support: a retrospective cohort study arthur bailly , laurent brisard , philippe bizouarn , thierry lepoivre , johanna nicolet , jean christophe rigal , jean christian roussel , bertrand rozec réanimation ctcv transplantation thoracique, chu de nantes -hôpital nord laennec, saint-herblain, france; chirurgie ctcv transplantation thoracique, chu de nantes -hôpital nord laennec, saint-herblain, france correspondence: laurent brisard -laurent.brisard@chu-nantes.fr annals of intensive care , (suppl ):s introduction the optimal nutritional intake in patients receiving temporary extracorporeal life support (ecls), including extracorporeal membrane oxygenation (ecmo) venovenous (vv) or venoarterial (va), remains controversial. enteral nutrition (en) is suspect to increase risk of gastrointestinal (gi) intolerance and intestinal ischemia. so, total parenteral nutrition (tpn) is often preferred. the purpose of this study is to describe the nutrition practices for critically ill patients receiving ecls and identify opportunities for improving nutrition therapy in this population. patients and methods retrospective analysis of patients requiring ecmo-va or ecmo-vv between and in the cardiac surgery intensive care unit of the university hospital of nantes. nutritional support was daily monitored with parenteral intake (glucose, lipid and propofol, protein and albumin, parenteral nutrition) and enteral nutrition until ecls weaning. two groups were compared during ecls period: no enteral nutrition delivered (none or tpn) (anec, n = ) and at least once enteral nutrition delivered (nec, n = ) including en alone and supplemental parenteral nutrition (spn). primary outcome was incidence of gi intolerance and risk factors. secondary outcomes were nutritional adequacy (calculated as overall of calories and protein delivered divided by the theoretical amount requirements: kcal/kg/d and . g/kg/d) and clinical outcome. data are reported as median ( th and th percentiles) or number (%), and analyzed with student's t test for continuous variables and χ test for categorical variables. p < . was considered as significant. none.introduction refeeding syndrome (rs) is a potentially lethal condition that remains underdiagnosed. it is characterized by severe electrolyte and fluid shifts associated with metabolic abnormalities in malnourished patients undergoing refeeding orally, enterally, or parenterally. clinical criteria have been proposed for determination of its risk and reported in the national institute for clinical excellence (nice) clinical guidelines. hypophosphoremia (hp) is a prominent feature of the rs and seems to be the earliest abnormality. phosphorus is a vital component of nucleic acids, enzyme systems, and various metabolic pathways. objective to determine the incidence of refeeding hypophosphoremia (rh) < . mmol/l, and severe rh < . mmol/l in a medical critical care unit. patients and methods monocentric, retrospective and observational study with patients from french-speaking icu nutritional survey study frans. critically ill adults (more than yo) were enrolled if they were hospitalized for more than days during a -month period and had an artificial nutritional support. refeeding hypophosphoremia is defined by the occurrence of hypophosphoremia after refeeding. we studied the incidence of hr, risk factors, and prognosis. results patients were enrolled between / / and / / . rh appears in . % and severe rh < . mmol/l in . % (fig. ) . there is no correlation between rs risk factors and rh in our study. logistic regression did not permit to identify neither risk factor nor prognostic modification. there is a lack in phosphoremia measuring ( . %), and overfeeding during the first days occurs in . %. discussion we define that an hypophosphoremia appearing after refeeding is a refeeding hypophosphoremia, and we do not consider others etiologies, such as mechanical ventilation, alkalosis, sepsis, alcoholism, malabsorptive states, poor intake, some medication. our cohort is too small to find some possible correlations with risk factors or prognosis. conclusion refeeding hypophosphoremia is common in our population. hypophosphoremia is not an independent predictor of icu or in-hospital mortality in critically ill patients. the knowledge of the sri requires the follow-up of the phosphoremia during nutrition after critical illness in particular in the undernourished patients. none. introduction to determine the possible relationship between days cumulated proteins ( days cpd) and energy deficits ( days ced) observed in ventilated patients and icu length of stay, duration of ventilator support, incidence of infections and days mortality. patients and methods mixed medical or surgical ventilated for at least days adult patients from icus from chu liège belgium were enrolled into the study. they were fed by enteral route with a target of kcal and . g of proteins by corrected kg of bodyweight and by day. if % of the target was not reached on day seven, parenteral nutrition was added with the same target. ced and cpd were calculated for days, taking into account all the sources of nutrition, and was defined as the difference between the amount of energy or protein intake and the target. results from / / till / / , patients were followed. data from patients could be cumulated on the first days. there were males, mean bmi was . ± . ; saps ii score on day was . ± . , sofa score at day was . ± . . they were ventilated for a median of days (iqr - ), median icu length of stay was days (iqr - ). mean sofa max calculated for the first days was . ± . and the day mortality was . %. on day , only % reached the target of kcal/kg and % the target of . g of protein/kg. mean days ced was − . ± . kcal and mean days cpd was − . ± . g. there was a significant negative relationship between both deficits and the sofa max (p = . for ced and p = . for cpd). however, there were no correlations between any of the deficits and icu length of stay, duration of mechanical ventilation, occurrence of infections and days mortality. discussion saps ii level, sofa max level, icu length of stay, all these parameters emphasize the high severity of this cohort of patients. it could indeed been thought that it is in this group of critically ill patients that the impact of nutrition could be easily demonstrated. clear relationships between sofamax on day and the days ced and cpd could be seen. however, both the deficit and the level of organ dysfunctions could be cause or consequence. unlike previous studies, usually performed in less severely ill patients, we did not find any relationship between ced or cpd and patient's outcome. conclusion contrary to some recent studies, we found no relationship between ced and cpe and outcome of patients. future studies are needed. none. cardiopulmonary bypass induces lymphopenia and decreases lymphocyte proliferation ability: il- and pd-l as potential therapeutic targets to reduce postoperative infection fabrice uhel , mathieu lesouhaitier , murielle grégoire , baptiste gaudriot , arnaud gacouin , yves le tulzo , erwan flecher , karin tarte , jean-marc tadié fig. incidence of hypophosphoremia at admission, the first day, and refeeding hypophosphoremia none. the prognostic impact of neutropenia in criticallyill cancer patients remains controversial. hence, several studies in critically ill cancer patients failed to demonstrate the impact of neutropenia on outcome [ ] . this lack of statistical association might however, reflect a lack of statistical power. a previous meta-analysis of aggregated data suggested % ( % ci - %) raw increase in mortality in neutropenic patients. the available data were, however insufficient to allow adjustment with confounders [ ] . the aim of this study was to assess the influence of neutropenia on mortality of critically ill cancer patients using individual data obtained from studies identified by our systematic review. secondary objectives were to assess the influence of neutropenia on mortality of critically ill patients while taking into account underlying malignancy, use of g-csf or changes related to period of admission. patients and methods this systematic review and meta-analysis was performed according to the prisma statements. public-domain databases including pubmed and the cochrane database were searched by using predefined keywords. the research was restricted to articles published in english and studies focusing on critically ill adult patients from may to may . the methods and objectives of this systematic review were reported in the prospero database (crd ). selected manuscripts' authors were then contacted to obtained part of their dataset. mortality was defined as either hospital or day- mortality. this preliminary analysis reports results from the whole dataset before and after adjustment using logistic regression. period of admission and use of g-csf were then assessed and were a pre-planned analysis. results our initial search yielded citations and studies were retained for further analysis. overall, studies were excluded for redundancy with other included studies, as containing only neutropenic patients, and two as containing only palliative patients. finally datasets ( %) containing sufficient data to allow comparison were obtained from authors. overall, patients were included in this study, including patients with neutropenia at icu admission. median age was of years (iqr - ). median sapsii score at icu admission was (iqr - ). respectively and patients had underlying haematological malignancy and solid tumours, and patients underwent allogeneic stem cell transplantation. mechanical ventilation, vasopressors, and renal replacement therapy were required in none. none. ( ) . in icus, cirrhotic patients are widely admitted and revalued after receiving optimal treatments for days. however, little is known about how manage these patients after day according to their prognosis. the blood neutrophil-to-lymphocyte ratio (nlr) as a novel inflammation index biomarker has been reported to be a predictor of clinical outcomes in various malignancies and in unselected critically ill patients ( ) . nlr has also been identified as a predictor of mortality in patients with stable liver cirrhosis. to our knowledge, the ability of nlr to predict outcome in critically ill cirrhotic patients has never been studied. the aim of this study was to evaluate the usefulness of inflammatory marker such as nlr for diagnosis of infection and predicting the outcome in hospitalized critically ill cirrhotic patients. we performed a retrospective monocentric study including consecutively cirrhotic patients hospitalized in a medical icu from to . for each patient, clinical and biological data at admission and day were collected. nlr at admission ("nlrd "), at day ("nlrd ") and the variation of nlr between admission and d none.introduction diagnosis of infection in immunocompromised patients can be difficult. however, diagnosing infection is very important, particularly in critically ill. this study aims to evaluate the benefit of procalcitonin (pct) blood level as a diagnostic marker for bacterial infection in patients with hematological malignancies admitted to the intensive care unit (icu). this retrospective single-center study included all consecutive patients with acute myeloid leukemia or high grade lymphoid malignancy admitted to the icu. patients were sorted into three subgroups, according to clinical and microbiological data: «infectious disease», «no infectious disease» and «unknown». initial serum pct and when available at day and day were recorded. receiver operating characteristic (roc) curve, sensitivity and specificity were calculated. serum pct was considered as decreasing when the decrease was ≥ % at day and/or ≥ % at day . mortality rates in the icu and at day- were also studied. results fifty-four patients were included in the study. at diagnosis, pct levels were significantly different between the "infection disease" group and the "no infection disease" group (p = . ). there was no difference between the "infection disease" group and the "unknown" group (p = . ). for the diagnosis of bacterial infection, best initial serum pct threshold was . ng per milliliter. for that threshold, sensitivity was . % and specificity was . %. pct area under the roc curve was . [ci % = . - ]. youden's j statistic was . . pct levels weren't different between groups according to the presence of neutropenia or in case of inaugural disease. there was a significant difference in pct values between groups according to the sofa score (p = . ), but not the saps score. mortality rate in the icu and at day- were significantly lower for the patients with decreasing pct (p < . and p < . , respectively). when comparing serum pct and crp predictive values, pct was significantly a better marker of bacterial infection (fig. ). discussion we found that serum pct, with a threshold of . ng/ ml, is a reliable marker of bacterial infection disease in patients with aggressive hematological malignancy admitted to the icu. our study confirms the results of a previous study in unselected immunocompromised patients admitted to the icu, showing a % sensitivity, a % specificity and an area under roc curve of . [ . - . ] for a threshold of . ng/ml ( ). the main limitations of our study are its retrospective design and the small number of included patients. conclusion pct is a reliable marker of bacterial infection in patients with hematological malignancies admitted to the icu. pct kinetic seems to be an interesting prognostic marker in this population. none. in this study, we have found that kinetics of secretion and expression of endocan is faster with huvecs stimlated by tlr agonist than tlr agonist. this results could suggest that endocan may be not only a marker of septic shock but could be also a specific marker to recognize the nature of pathogenic microorganisms in septic shock. furthermore, other studies with more tlr agonists could be useful to confirm these results. conclusion studying the effects of diverse tlrs agonists could make the plasmatic dosage of endocan more specific and helpful to recognize the nature of pathogenic microorganisms in septic shock. none. lung ultrasound: help to the diagnostic and the monitoring of response to physiotherapy. a case report of pneumonia aymeric le neindre introduction chronic critical illness (cci) syndrome is a new condition affecting an increasing number of patients, who survived an acute critical illness but have persistent severe organ dysfunction, requiring prolonged specialized care. cci is a iatrogenic process, reflecting the efficacy of modern life support technologies( ), and encompasses multiple organ failure, need for prolonged mechanical ventilation (mv), organ support, and palsy due to polineuromyopathy. the transition from acute to cci is gradual: definitions are based on duration of mv, with cut-offs of , or consecutive days of mv for ≥ h/day. cci patients may come from either medical or surgical icu; their health status fluctuates between improvements and deteriorations implying recurrent transitions between different levels of care ( ) .the risk of death is reported to be as high as %. despite a relatively young age ( years on average), functional status of cci patients discharged is seriously impaired, thus cci patients require long-term rehabilitation. aim: to estimate the frequency of cci syndrome in careggi, a large academic, tertiary care hospital; to describe the clinical course of cci patients through discharge, and their functional status at discharge. patients and methods administrative data on admission, transfer, death and discharge of all cci patients, consecutively admitted in one of the icu beds at careggi hospital from january to december , , were collected. cci was defined with the cut off of ≥ days of icu stay, representing the index event (ie) without contribution of previous or subsequent hospitalization in other hospitals. reasons for admission were grouped into the broad categories of medical causes, surgery, major trauma and cardio-respiratory arrest. patients discharged were evaluated in daily living, cognitive status, and mobility using barthel index. results we identified subjects who developed cci ( males; age . ± . years, mean ± sem); of them came from an external icu, began their cci course within careggi hospital ( from the emergency room, from a regular ward). average duration of the ie was . ± . days. these sample developed accumulative length of icu stay of days, corresponding to a % icu bed occupation over the theoretical total of , . when days of subintensive care and regular ward were separately added, days of highly specialized care and days of total acute hospital stay were reached. surgical patients had longer hospitalizations (p = . ).cci patients confirmed to be highly erratic: a total of transitions across different services were recorded in the patients, with a maximum of in of them. mean age was comparable between the patients who died ( %) and the remaining who were discharged alive ( . ± . vs. . ± . years; p = . ).fourteen subjects continued their icu stay out of hospital. only , whose age was lower ( . ± . years), were discharged home; half of the participants (n = , . %) were admitted to a residential rehabilitation facility. younger subjects scored better in the domains of self care (p = . ) and cognitive status (p = . ) but not in the domain of mobility, including walking ability: patients required maximal assistance in performing activities of daily living and transfers, other required medium/maximal assistance, with no statistical difference between dg group. conclusion cci is a relevant clinical condition that need to be assessed and possibly prevented, as it causes severe morbidity, long-term functional impairment and exceeding healthcare costs. none.conclusion early mobilization during the first week of the sepsis onset was safe and preserved muscle fibre cross sectional area. none. none. study of efficacy on icu acquired weakness of early standing with the assistance of a tilt table in critically ill patients none.introduction patients with high flow nasal cannula may benefit from combined aerosol therapy. clinical efficacy depends on pulmonary deposition which is related to the type of nebulizer. all new nebulizers or delivery methods require rigorous evaluation. the aim of this study was to compare lung deposition between two nebulizers (jet nebulizer vs vibrating-mesh nebulizer) through high flow nasal cannula in healthy subjects. patients and methods aerosol delivery of diethylenetriaminepentaacetic acid labelled with technetium- m ( mtc-dtpa, mci/ ml) to the lungs using a vibrating-mesh nebulizer (aerogen solo ® , aerogen ltd., galway, ireland) and a constant-output jet nebulizer (opti-mist plus nebulizer ® , convatec, bridgewater, nj) through high flow nasal cannula (optiflow ® , fisher & paykel, new zealand) was compared in healthy subjects. flow rate was set at l/min through the heated humidified circuit. pulmonary and extrapulmonary deposition were measured by single photon emission computed tomography combined with a low dose ct-scan (spect-ct) and by planar scintigraphy. results lung deposition was only . ± . and . ± . % of the nominal dose with the vibrating-mesh nebulizer and the jet nebulizer, respectively (p < . ). dose lost in the high flow circuit, humidification chamber and nasal cannula was higher with the vibrating-mesh nebulizer as compared to the jet nebulizer ( . ± . vs . ± . % of the nominal dose, p = . ). expressed as percentage of emitted dose, lung deposition was similar with both nebulizers. conclusion this study demonstrated that aerosol delivery through hfnc is poor in the specific conditions of the study despite the higher efficiency of the vibrating-mesh nebulizer as compared to the jet nebulizer. placing the nebulizer on the hfnc circuit at l/min induces high aerosol loss on the circuit and the oropharynx. key: cord- -h p kmss authors: follet, jérôme; guyot, karine; leruste, hélène; follet-dumoulin, anne; hammouma-ghelboun, ourida; certad, gabriela; dei-cas, eduardo; halama, patrice title: cryptosporidium infection in a veal calf cohort in france: molecular characterization of species in a longitudinal study date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: h p kmss feces from animals were collected on farms in the region of brittany, france. each sample was directly collected from the rectum of the animal and identified with the ear tag number. animals were sampled three times, at , and weeks of age. after dna extraction from stool samples, nested pcr was performed to amplify partial s-rdna and kda glycoprotein genes of cryptosporidium. the parasite was detected on all farms. one hundred out of calves ( . %) were found to be parasitized by cryptosporidium. amplified fragments were sequenced for cryptosporidium species identification and revealed the presence of c. parvum ( . %), c. ryanae ( . %), and c. bovis ( %). one animal was infected with cryptosporidium ubiquitum. the prevalence of these species was related to the age of the animal. c. parvum caused . % of cryptosporidium infections in -week-old calves but only . % in -week-old animals. the analysis of the results showed that animals could be infected successively by c. parvum, c. ryanae, and c. bovis for the study period. c. parvum gp genotyping identifies iia subtypes of which . % were represented by iiaa g r . this work confirms previous studies in other countries showing that zoonotic c. parvum is the dominant species seen in young calves. cryptosporidium is a genus of protozoan parasites infecting a wide range of hosts [ ] . all groups of vertebrates are susceptible to cryptosporidium infection worldwide. this parasite is the etiological agent of cryptosporidiosis, which is mainly characterized by diarrhea in humans and livestock. massive outbreaks of enteritis in people such as in milwaukee, wisconsin (usa) have increased public awareness of this parasite [ ] . in humans, the prevalence and severity of infection increase in immunodeficient individuals such as aids patients. in immunocompetent patients, the disease is self-limited [ ] . no drug therapy is yet available and the high resistance of oocysts to environmental conditions and chemical treatment make cryptosporidiosis difficult to control [ ] . cattle have been considered to be a primary reservoir for cryptosporidium oocysts for zoonotic c. parvum [ ] . these animals could be a risk factor via environmental contamination from their manure being spread on farmland or their grazing on watersheds [ ] . on farms, transmission of cryptosporidium spp. can result from ingestion of contaminated food or water, by direct transmission from host to host, or through insect vectors [ ] . in cattle, infection by cryptosporidium spp. was first reported in [ ] . since vaccines have become commercially available against escherichia coli k , rotavirus, and coronavirus, cryptosporidium has emerged as the main neonatal diarrheic agent in calves [ ] . in farm animals, the economic impact is related to morbidity, mortality and growth retardation [ ] . among the species previously described (if the three fish species are accepted without complete genetic characterization) [ , [ ] [ ] [ ] , c. parvum, c. bovis, c. ryanae and c. andersoni usually infect cattle. c. parvum has zoonotic potential and is a frequent cause of human cryptosporidiosis [ ] . c. bovis and c. ryanae have not been found in humans and there is only one description of c. andersoni in a patient [ ] . recent reports have described an age-related distribution of these aforementioned species in dairy cattle on the east coast of the united states [ ] [ ] [ ] , india, china, georgia [ ] , malaysia [ ] , and denmark [ ] . the most prevalent species were c. parvum in preweaned calves, c. ryanae and c. bovis in postweaned calves and c. andersoni in adult cows [ , ] . in france, previous studies on the prevalence of cryptosporidium in cattle were based on microscopic determination [ ] or coproantigen detection using elisa [ ] . these studies on dairy calves reported a within herd prevalence of cryptosporidium without identifying species or the relation to the host's age. the present study was conducted in farms in brittany, france to determine the prevalence of cryptosporidium in veal calves. we used genotyping and subtyping for the molecular study of cryptosporidium isolates. follow-up of the same animal allowed us to determine the outcome of the infection and the age distribution of cryptosporidium species. fifteen fattening units in brittany (france) were included in this work. they belonged to three industrial veal producers representative of integrators in france and did not present any known history of cryptosporidium infection. these farms were located in four administrative regions ( figure ): côtes d'armor (ca -ca ), morbihan (mo ), ile-et-vilaine (iv -iv ), and mayenne (ma -ma ). during the summer and autumn of , all farms were visited three times and fecal samples were taken from animals exhibiting diarrhea at the age of weeks old. calves arrived in fattening units at the age of weeks old and were confined in small groups from four to six animals per pen. because of a concomitant welfare study [ ] , calves had to stay to weeks without any external stress despite the farmer's presence. at the age of weeks old, calves were finally sent to the slaughterhouse. consequently, sampling was done at the ages of weeks, weeks, and weeks (table ). these points of sampling corresponded to the beginning, the middle and the end of the fattening period. fecal samples were collected and shipped by a veterinarian. collectors respected the following criteria: use of a single pair of latex gloves per animal, a single plastic sterile cup per animal, and collection of at least g of feces per sample. feces were collected directly from the rectum of each animal and stored at °c in potassium dichromate ( . % wt/vol) until processed. cups were capped, labeled with the animal's ear tag number, and accompanied by a form recording the date of sampling, the animal's sex, breed, identification number, and the mean age of the herd. after washing steps in water to eliminate potassium dichromate from the samples, dna was extracted according to the method previously described [ ] without the cetyl trimethylammonium bromide (ctab) and polyvi-nylpyrrolidone (pvp) treatment steps. an s rna gene fragment was amplified by nested pcr according to xiao et al. [ ] . the partial gp gene was amplified according to gatei et al., [ ] . pcr products were analyzed on % agarose gel and visualized by ethidium bromide staining. to ensure purity and limit the presence of pcr inhibitors, all pcr-negative samples were reprocessed. samples were treated for oocyst purification by immunomagnetic separation (dynabeads ® anti-cryptosporidium, invitrogen ™, norway) according to the manufacturer's instructions. these samples were finally processed as previously for dna extraction and pcr amplification. cryptosporidium species identification pcr products were purified on an ultracel ym membrane (microcon, millipore, bedford, ma, usa) according to the manufacturer's instructions. dna sequencing reactions were performed using internal primers of the nested pcr with the abi prism big dye terminator cycle sequencing kit (applied biosystem, foster city, ca, usa). capillary electrophoresis was performed by genoscreen (lille, france). sequences were analyzed using blast at ncbi [ ] . the prevalence of cryptosporidium infection on farms from four administrative regions in brittany (france) was studied ( figure ). all cryptosporidium-positive specimens generated the expected ssu-rna products in nested pcr and revealed that no farm was free of cryptosporidium. the molecular analysis of fecal samples revealed that ( . %) were positive for cryptosporidium. as shown in table , the overall prevalence of infected animals was . % ( / ) and ranged from % on a farm in morbihan (mo ) to % on farms in ile-et-vilaine (iv , iv ) and in mayenne (ma ). amongst the specimens sampled from -week-old and -week-old animals, cryptosporidium prevalence was . % and . %, respectively (range, %- . %). in -week-old calves, the prevalence decreased to . % (range, %- . %). the prevalence of infection decreased as the age of the calves increased. for species identification, the positive nested pcr products were sequenced. sequence analysis from readable electrophoregrams revealed the presence of c. parvum, c. bovis, and c. ryanae. one additional cryptosporidium genotype showing % identity with cryptosporidium ubiquitum (eu ) (previously identified as * a calf is considered to be positive if at least one out of the three samples is positive. **the number of animals is because one calf died between the age of and weeks. cryptosporidium cervine genotype [ ] ) was detected in one calf. this sequence was deposited in genbank under the accession number gu . sixty ( . %) samples were identified as c. parvum as follows: forty-six sequences had % identity with the genbank af nucleotide sequence, had % identity with the af nucleotide sequence and three had % identity compared to both references. these sequences were deposited in genbank under the accession numbers gu to gu . for the other positive specimens, ( . %) were identified as c. ryanae (previously described as cryptosporidium deer-like genotype). thirtyone of these had % identity with the ay sequence [ ] and eight were % identical to this reference. these nucleotide sequences were deposited in gen-bank under the accession numbers gu to gu . for the last positive samples, ( %) had an identical nucleotide sequence with c. bovis (genbank accession number, ay ) formerly known as the cryptosporidium bovine b genotype. within these sequences, had % identity to the reference deposited in genbank, three sequences had % identity. these last sequences were deposited in genbank under the accession numbers gu to gu . prevalence of c. parvum, c. ryanae, and c. bovis in relation to calf age the distribution of cryptosporidium species identified in animals at the age of , , and weeks is shown in figure . the prevalence of each species changed with the age of the calves. c. parvum prevalence was . % in the -week-old calves and decreased to . % in -week-old animals. this species was not identified in -week-old calves. c. ryanae and c. bovis were identified in -weekold calves in . % and . % of the specimens, respectively. the prevalence of these species in -week-old animals increased to . % and . %, respectively. this prevalence evolved to % and % in -week-old animals. the presence of one, two, or three species of cryptosporidium was determined in each animal (n = ) for which the sequences were readable in all positive samples. three calves positive for c. parvum at the age of weeks were excluded because cryptosporidium species could not be identified in all of the following samples collected in these animals. as shown in in the time lapse of this study, % of the animals ( / ) were found to excrete two different species of cryptosporidium successively. indeed, . % ( / ) produced c. parvum and c. ryanae, . % ( / ) excreted c. parvum and c. bovis, and . % ( / ) excreted c. ryanae and c. bovis. finally, . % ( / ) of the animals studied were detected to produce c. parvum, c. ryanae, and c. bovis. the subtyping analysis was performed on c. parvum positive specimens. from targeted samples, could be used for sequence analysis. as shown in table , all alleles identified belong to the iia family. the most common subtype iiaa g r ( % identity with reference strain ab ) was found in out of samples ( . %). six samples ( . %) were typed as subtype iiaa g r ( % identity with reference strain gq ), three samples ( . %) as subtype iiaa g r ( % identity with reference strain dq ) and two samples ( . %) as subtype iiaa g r ( % identity with reference strain dq ). finally one sample ( . %) was subtyped as iiaa g r ( % identity with reference strain dq ) and another one ( . %) as subtype iiaa g r ( % identity with reference strain dq ). discussion calves under month of age are frequently infected with cryptosporidium sp [ ] which results in economic loss [ ] . in france, up to date, the prevalence of cryptosporidium in diarrheic calves has been studied only by elisa and microscopic strategies [ , , ] . no data are available on a molecular basis to study cryptosporidium species in calf herds in that country. the present study based on s rdna and gp gene analysis is the first in france to include molecular characterization to describe the prevalence and the host age related susceptibility to different cryptosporidium species after a follow up of the same animal. our results showed that all fifteen farms were contaminated with cryptosporidium. the parasite prevalence on farms ranged from % to % of the sampled animals. this observation was in accordance with results in michigan (usa) where this parameter ranged from % to % [ ] . the prevalence of . % obtained in this work tended toward the upper end of the scale compared to other investigations done in france which ranged from . % in beef herds [ ] to % in suckling calves [ ] and in other european countries where prevalence ranged from . % to % [ , ] . however, the sampling program did not allow the study of animals under weeks of age. indeed, the animals arrived in these structures at the age of to weeks and farmers did not allow sampling before two complete resting weeks for each animal. therefore, our results could underestimate the real prevalence as huetink et al. showed that the percentage of parasite excreting animal declines after the third week of age [ ] and that the first peak of prevalence is at the age of days [ ] . in our study, the higher prevalence of cryptosporidiosis was observed in calves weeks old ( . %) and the lowest ( . %) in the -week-old animals. this observation shows that prevalence of cryptosporidium infection decreases with increasing age of the cattle in france as in many other countries [ , , [ ] [ ] [ ] [ ] [ ] [ ] . additionally, our data confirmed the presence in france of a host age-related susceptibility to the infection with different cryptosporidium species. c. parvum was predominantly detected in -week-old calves ( . %) compared to c. ryanae or c. bovis detected in . % and . % of the positive samples respectively. it is noteworthy that these results are very similar to data obtained in ireland on calves under days of age with %, . %, and . % of prevalence of the same species, respectively [ ] and in the uk on animals over weeks old with % c. parvum, % c. bovis, and % c. ryanae [ ] . in contrast to previous studies [ , ] , c. ryanae and c. bovis were found with similar prevalence predominantly in week and week old calves. this association between the age of the cattle and the cryptosporidium species identification has been supported by several studies [ , , , , ] but different reports suggest that cryptosporidium species repartition regarding the age of the host could be due to a technical artifact. despite the fact that the methodological strategy based on pcr using genus specific primers and partial direct sequencing of the s rdna is commonly used to identify cryptosporidium species [ ] , this molecular tool is limited in the case of mixed infections. feng et al., [ ] suggested that the important shedding of c. parvum in preweaned calves had probably masked the concurrent infection of these animals by c. bovis or c. ryanae. furthermore, previous reports suggested that a dominant cryptosporidium species in a sample can be preferentially amplified by pcr [ , ] . it is noteworthy that this situation of mixed cryptosporidium species infection in farm animals would be more prevalent than originally believed [ ] [ ] [ ] . mixed cryptosporidium species could also explain sequencing difficulties encountered in this work. the simultaneous presence of several species in the same sample could lead to amplification and sequencing of different genetic fragments leading to unreadable superimposition of electrophoregrams. consequently, in our work based on the utilization of cryptosporidium generic primers, the amplification of a single fragment with a single sequence is not conclusive evidence that the sample contains only a single species. however, based on our results, it is possible to confirm the predominance of different species of cryptosporidium by group of age among the calves. particularly, our data showed that animals can be sequentially infected with c. parvum, c. ryanae and c. bovis as well as c. parvum, c. bovis and c. ryanae. this observation provides evidence that a previous infection with c. parvum did not protect calves against an infection with other cryptosporidium species. fayer et al. suggested that the peak of cryptosporidiosis prevalence in young calves could reflect the immaturity of the immune status [ ] . it was also suggested that the low excretion of c. parvum oocysts in older calves might be related to the development of immunity that also protected the animal against a secondary challenge [ ] . it has been reported that immunity arises in the first two weeks after infection [ ] . interestingly, fayer et al. [ ] described that calves previously challenged with c. parvum were able to excrete oocysts after a second challenge with c. bovis but not with c. parvum. the authors concluded that immunity to c. parvum was not extended to c. bovis. consistently, in our study, the presence in the same animal during sequential sampling of c. parvum, c. bovis and c. ryanae suggests that immunity against c. parvum and against c. bovis did not extend to c. ryanae. furthermore, the observation that one animal excreted sequentially c. parvum, c. ryanae and c. bovis suggests that immunity against c. ryanae did not extend to c. bovis as well. finally, the risk to human health posed by cryptosporidium infected cattle in france was assessed. the detection of c. ubiquitum (a rare infectious agent detected in humans [ ] ), c. ryanae and c. bovis (which are mainly specific for cattle) led to consider that the -week-old calves are not likely a public health concern. however, the major detection of c. parvum, a prevalent zoonotic species, in -week-old calves was in agreement with the report of atwill et al., who considered that the contribution of cattle to human cryptosporidiosis is limited to calves under months of age [ ] . to determine c. parvum subtypes, the sequence analysis of a fragment of the gp gene was done. our results show that in the region of brittany, all identified c. parvum gp subtypes belonged to the iia family which was previously found in both animals and humans [ ] . particularly, human infections with the iia subtype are commonly seen in areas with intensive animal production [ ] . among the gp subtypes formerly described in cattle [ ] , only six were identified in this work, being iiaa g r the most commonly found. this subtype has been widely reported in calves and humans in different countries such as in portugal [ ] , slovenia [ ] and the netherlands [ ] . this observation confirms previous works and suggests a zoonotic transmission of the parasite also in this region. it is noteworthy that the three predominant subtypes (iiaa g r , iiaa g r , and iiaa g r ) found in this work were also described in cattle with an equivalent distribution in the netherlands [ ] and england [ ] . thus, the subtype iiaa g r was found in . % of the samples in this work, . % in the netherlands and . % in england. the iiaa g r was identified in . % of the samples in this report, . % in the netherlands and . % in england. the iiaa g r determined in . % of our samples, was characterized in . % in the netherlands and . % in england. it is remarkable that subtypes, iiaa g r , iiaa g r and iiaa g r were equivalently underrepresented in these three countries. this observation could suggest that the proportion of a gp subtype would not be randomly represented in a population. finally, the zoonotic transmission assessment of c. parvum in france would require a comparative investigation of variable genetic loci both in human and animal samples. this is the first report on the molecular identification of cryptosporidium species or genotypes in veal calves in france. according to data reported previously in many countries, a sequential distribution of species is observed in cattle according to age. c. parvum was mainly observed in the youngest calves, while c. ryanae and c. bovis became predominant in stool specimens collected in older animals. in some cases, several cryptosporidium species were successively detected in the same calf, suggesting that the immune defense against c. parvum is not efficient against c. ryanae or c. bovis. finally, the major identification of the iiaa g r subtype in france suggests that -week old calves could be a reservoir for zoonotic parasites transmissible to humans. fayer r: taxonomy and species delimitation in cryptosporidium a massive outbreak in milwaukee of cryptosporidium infection transmitted through the public water supply the cell biology of cryptosporidium infection advances in the epidemiology, diagnosis and 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classification of cryptosporidium isolates from humans and calves in slovenia molecular epidemiology of cryptosporidium in humans and cattle in the netherlands submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this study was supported by the catholic university of lille through the "projet grande campagne ensemble innovons" genotyping program. we would like to thank the veal unit managers who participated in this study. authors' contributions jf and kg participated in the conception and design of the study, carried out the experiments and drafted the manuscript. hl designed the sampling strategy and collected samples on farms. jf, kg and afd designed the protocol for molecular assay and participated in the analysis result. ohg carried out molecular assays. edc, gc and ph participated in the coordination of the study and helped draft the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -x r w authors: guan, l.; prieur, c.; zhang, l.; georges, d.; bellemain, p. title: transport effect of covid- pandemic in france date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: x r w an extension of the classical pandemic sird model is considered for the regional spread of covid- in france under lockdown strategies. this compartment model divides the infected and the recovered individuals into undetected and detected compartments respectively. by fitting the extended model to the real detected data during the lockdown, an optimization algorithm is used to derive the optimal parameters, the initial condition and the epidemics start date of regions in france. considering all the age classes together, a network model of the pandemic transport between regions in france is presented on the basis of the regional extended model and is simulated to reveal the transport effect of covid- pandemic after lockdown. using the the measured values of displacement of people mobilizing between each city, the pandemic network of all cities in france is simulated by using the same model and method as the pandemic network of regions. finally, a discussion on an integro-differential equation is given and a new model for the network pandemic model of each age class is provided. up to now, covid- has widely spread over the world and is much more contagious than expected. the outbreak of covid- has resulted in a huge pressure of hospital capacity and a massive death of population in the world. quarantine and lockdown measures have been taken in many countries to con- trol the spread of the infection, and has proved the amazingly effectiveness of these measures for the outbreak of covid- , in particular in china (see [ ] ). quarantine is a rather old technique to prevent the spread of diseases. it is used at the individual level to constrain the movement of all the population and encourage them stay at home. lockdown measures reduce the pandemic trans- mission by increasing social distance and limiting the contacts and mobility of people, e.g. with cancellation of public gatherings, the closure of public transportation, the closure of borders. covid- may yield a very large number of asymptomatic infected individuals, as mentioned in [ ] and [ ] . therefore, most countries have implemented indiscriminate lockdown. but the long time of duration of lockdown can cause inestimable financial costs, many job losses, and particularly psychological panic of people and social instability of some countries. as declared by some governments (see [ ] ), testing is crucial to exit lockdown, mitigate the health harm and decrease the economic expensation. in this paper, we consider two classes of active detection. the first one is the short range test: molecular or polymerase chain reaction (pcr) test, that is used to detect whether one person has been infected in the past. the second test is the long range test: serology or immunity test, that allows to determine whether one person is immune to covid- now. this test is used to identify the individuals that cannot be infected again. for our research on covid- , we aim to evaluate the effect of lockdown all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint within a given geographical scale in france, such as the largest cities, or urban agglomerations, or french departments, or one of the metropolitan regions (to go from the finest geographical scale to the largest one). the estimations of effect are also considered on different age-classes, such as early childhood, scholar childhood, working class groups, or the elderly. besides, we propose to understand the effect of partial lockdown or other confinement strategies depending on some geographical perimeters or some age groups (as the one that lyon experienced very recently, see [ ] ) in the context of covid- , there have been many papers that focus on estimating the effect of lockdown strategies on the spread of the pandemic (e.g. [ ] and [ ] ). in [ ] , the lockdown effect is estimated using stochastic approximation, expectation maximization and an estimation of basic reproductive numbers. in this work, we aim at evaluating the dynamics of the pandemic after the lockdown by looking on the transport effect. in this paper, one contribution is that an extension of the typical sird pandemic model is presented for characterizing the regional spread of covid- in france before and after the lockdown strategies. taking into account the detection ratios of infected and immune persons, this extended compartment model integrates all the related features of the transmission of covid- in the regional level. in order to estimate the effect of lockdown strategies and understand the evolution of the undetected compartments for each region in france, an optimization algorithm is used to derive the optimal parameters for regions by fitting the extended model to real reported data during the lockdown. based on regional model analysis before and after the lockdown, we present a network model to characterize the pandemic transmission between regions in france after lockdown and evaluate the transport effect of covid- pandemic, when considering all age classes together. the most interesting point is the chosen exponential transmission rate function β(t), in order to incorpo- rate the complex effect of lockdown and unlockdown strategies and the delay of incubation. this paper is organized as follows. in section , the extended model is de-pandemic network of all cities in france. in the 'discussion' section, considering the age classification, an integro-differential model is presented for the pandemic network transmission, at any geographical scale, and for any set of age classes. in this paper, the scenario we consider is a large safe population into which a low level of infectious agent is introduced and a closed population with neither birth, nor natural death, nor migration. there is one basic model of modelling pandemic transmission which is well known as susceptible-infected-recovereddead (sird) model in [ ] . this mathematical compartmental model is described where s(t) is the number of susceptible people at time t, i(t) is the number of infected people at time t, r(t) is the number of recovered people at time t, d(t) is the number of deaths due to pandemic until time t, with constant parameters: β is transmission rate per infected, δ is the removal or recovery rate, α is the disease mortality rate. the compartment variables s(t), i(t), r(t), d(t) satisfy at any time instant t, here n is the total number of population of the considered area. from the differential equations ( )-( ), it is obvious that at any time instant t, the total rate βi(t) of transmission from entire susceptible compartment to infected compartment is proportional to the infected i; the infected individuals recover at a constant rate δ; the infected go to death compartment at a constant rate α. in fact, with the exception of the detected well-known data, there are some undetected data that cannot be measured but are significantly important for the analysis of the evolution of covid- in france under lockdown policy. moreover they are useful to provide efficient social policies, such as optimal management of limited healthcare resources, the ideal decision of the duration and level of lockdown or re-lockdown, and so on. inspired by [ ] , the basic sird model is extended to a more sophisticated has recovered from the pandemic and is immune. the flow diagram of this model is sketched out in figure . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint the evolution of each compartments is modelled by the following equations, di with all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint the other parameters in equation ( )-( ) are defined as follows: • γ ir is the daily individual transition rate from i to r, and γ ir = ( − all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint • γ ih is the daily individual transition rate from i to h, and γ ih = ( − • γ iu is the daily individual transition rate from i to u , and γ iu = ( − • γ hr is the daily individual transition rate from h to r, and γ hr = (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . the infection transmission rate β(t) is the rate of the pandemic transmission from an undetected infected person to susceptible individuals at time instant t. as in [ ] , in order to combine the complex effects of lockdown strategy, a time-dependent exponentially decreasing function can be used to model the transmission rate β(t), with constant parameters β , µ and κ. note that β(t) is constant during the initial stage of implementing effective lockdown strategies such as social distance, quarantine, healthcare, and mask worn. the transmission rate exponentially decreases at rate µ after these lockdown strategies take effect. the transmission rate β(t) can be illustrated in figure . as one of the most critical epidemiological parameters, the basic reproductive ratio r defines the average number of secondary cases an average primary case produces in a totally susceptible population (see [ ] ). as for the model in [ ] , for the considered model in this paper, only the i − individuals transmit all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint the disease to the susceptible individuals during the early phase of outbreak. , the initial number of susceptible individuals exceeds the critical threshold to allow the pandemic to spread. thus the initial basic reproductive rate is when the transmission rate β(t) and s(t) evolve as time goes by, one dynamic reproductive rate that depends on time is introduced and known as effective reproduction number r(t) in [ ] . in this model, it is defined as, for similarly, when r(t) < , the number of secondary cases infected by a primary undetected infected case on day t, dies out over time, leading to a delay in the number of infected individuals. but when r(t) > , the number of undetected infected individuals grows over time. therefore, by the control of the transmission rate β(t) that can constrain r(t) to be less than , the number of infected individuals grows slowly to ease the pressure on medical resources. when s(t) is bellow a threshold, the epidemic goes to extinction (see e.g., [ ] ). the required level of vaccination to eradicate the infection is also attained from the effective reproduction number. the compartmental model introduced in figure exhibits a large number of unknown parameters ( if we consider λ = ). the uncertainty on these parameters can not be neglected. as an example, let us propagate uncertainty at the scale of the region auvergne-rhône-alpes. the vector of unknown parameters is: we take into account the uncertainties on these parameters by considering that each parameter is uniformly distributed with bounds consistent with typical all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . reported values (see, e.g., [ ] and references therein). lower and upper bounds for each parameter are reported in table hereafter. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint figure shows that the prior uncertainty is pretty high, since for example the difference between the % and the % quantiles for the number of people in hospital is more than at the end of the lockdown period. on figure we propagate the parameter uncertainty on the maximum number of people in intensive care units, on the date at which this maximum value is attained and on the total number of reported cases. note that the total number of reported cases is obtained from the daily number of reported cases, dr, which is driven by the following equation: the we see fpr example on these boxplots that the median for the maximum number of people in intensive care is more than with the iqr greater than . in view of the importance of uncertainties propagated from the model parameters to the quantities of interest (e.g., number of infected people at hospitals), it appears necessary to calibrate the model. our calibration procedure is described in the next section. in this section, regional scales of france are considered and all age classes are summed to calibrate the parameters of the pandemic model ( )-( ) during all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint the following weighted least square cost function is minimized for parameters optimization: where p is a vector which consists of calibrated parameters; z meas (t i ) is the measured values of the corresponding observed state vector z(p, t i ) at time t i , i = , . . . , n, with n the number of days considered for calibration. this optimization problem is solved using levenberg-marquardt algorithm (see [ ] ). since it is a local algorithm, we adopt, as in [ , chapter ] , a multi-start ap- (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . is calibrated on daily data for h, u , d and r + on the lockdown period - - to - - from two data sources: the first one is a public and governmental data source [ ] and the second one is a dedicated national platform with a privileged access [ ] . the time step is chosen as ten percentage of one day for the numerical discretization. a general solver for ordinary differential equations is used to in order to characterize the dynamics of the pandemic transmission processes during the confinement, the epidemiological model ( )-( ) was described in the all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . previous section. we now consider the government action of unlockdown after confinement, there is a pandemic transmission effect between each region in france. considering n a age groups, the following pandemic network model of all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . where transmission rate β ijk (t) depends only on (i, j), and is piecewise continuous depending on the scenario: lockdown or no-lockdown, for all t; for age group j, l kij (t) is the proportion of individuals moving from region k to region i in the age class j; the other parameters depend on the location, and also on the age group j; σ(j, k, t) is periodic (space dependent period t j,k ), satisfies t j,k σ(j, k, t)dt = , and takes value in the interval [− , ]; c i is the set of all regions that have pandemic transmission with region i. as the fast periodic switching policy in [ ] , we consider the inverse of the (same) exponential function of infection transmission rate β(t) in ( ) to denote β ijk (t). even though the end of confinement, the social strategies still go on, so a continuous function β(t) is used for the whole transmission process of covid- from the start date of infection, with the end time of lockdown t end . the transmission rate β(t) for the whole transmission process is illustrated in (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . table : third part of components l ki in the mobility matrix l. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint is th of may in france. in this section, we use the parameter identification method developed in where l ki (t) is the proportion of individuals moving from city k to city i, and is derived from the real data of insee, and c i is the set of all cities that have all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint pandemic transmission with city i. all the other parameters are chosen as the ones of the region to which each city belongs. to simulate this system of * . differential equations, we now specify initial conditions. to simplify, the epidemic start date of each city is taken as the same as the epidemic start date of the region to which it belongs, and the initial condition for the undetected infected individuals i − for the capital of (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint figure : the maps of the transport effect between cities in france (undetected infected plus detected infected from % (blue) to % (magenta) of the population for each commune): the date for the map on the left is - - and the one for the map on the right is - - . figure : the maps of the transport effect between cities in france(undetected infected plus detected infected from % (blue) to % (magenta) of the population for each commune): the date for the map on the left is - - and the one for the map on the right is - - . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint which could be included in the modelling of the transmission rate β(t). . discussion and a new integro-differential model in this section, the general form of an integro-differential model capable of integrating different age classes and areas is introduced to discuss the transport effect of covid- in france after lockdown. by "areas" we mean a given geographical scale as the set of metropolitan regions (as considered in section ), or the set or all french departments, or all cities (as considered in section ), or other geographical areas. for each age class a ∈ ages in area x ∈ areas, we consider the following integro-differential equations, for any time t ≥ after confinement, ∂ t x(a, x, t) = f a (x(., x, t)) + areas σ(a, x, y, t)(Λ in (a, x, y, t) − Λ out (a, x, y, t))x(a, y, t)dy • areas, the set of different areas of population under study, depending on the considered geographical scale. as an example, considering all all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . • x(a, x, t) ∈ r is the -vector consisting of compartments of the age class a, in the area x, at time t; • for all age class a, f a (x(., x, t)) is the pandemic transmission dynamics for age class a from all other age classes in the area x at time t. without considering the age effect, it is given by the right-hand side of systems ( )- ( ) . inspired by the contact matrix approach developed in e.g. [ , chapter , page ], by considering multiple age classes, the transmission term is the following integral where β a,b,x (t) is the contact function between age classes a and b, in the all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint area x, and at time t. therefore the function f a is given by where all parameters depend on the age class a and the area x; • Λ in (a, x, y, t) ∈ r is the density of people coming (in) area x from area y ∈ areas at time t, for age class a; • Λ out (a, x, y, t) ∈ r is the density of people going to (out) area y ∈ areas from area x at time t, for age class a; • σ(a, x, y, t) is the lockdown function for the age class a, between the areas x and y at time t. as an example, before the th of may, it was forbidden to travel for more than km in france. such a policy could depend on the age classes and on the areas, e.g., to control so called "clusters" of covid- ; • areas σ(a, x, y, t)Λ in (a, x, y, t)x(a, y, t)dy provides the total number of people coming into area x from all the other areas. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint equation ( ) describes the network dynamics of covid- pandemic after lockdown and the transport effect on different age class on the basis of the regional pandemic transmission dynamics during lockdown. the proposed structure makes it easier to understand different forms of the kernel. the interest of this model is that it could be adapated to any geographical scales, and to all age classes. for a control point of view, the most important term is σ(a, x, y, t) which defines the lockdown policy that defines the mobility between areas x and y at time t for the age class a. many control problems could be studied for this model, as optimal control to reduced the pandemic effect, or to minimize the mortality in particular. it is of great importance for the mobility dynamics of the pandemic. beyond that, inspired by advection-diffusion modelling of population dynamics (as considered in [ ]), it is natural to model the displacement inside a given area by a diffusion term (see [ ] ). the corresponding model is formulated as follows: ∂ t x(a, x, t) = f a (x(., x, t)) + d(a, x, t)∂ xx x(a, x, t) + areas (Λ in (a, x, y, t) − Λ out (a, x, y, t))x(b, y, t)dy +f ext (a, x, t), where the diffusion coefficient d(a, x, t) is a function that depends on age class a, areas x and time t. this -order partial differential equation predicts that for age class a in the area x, how diffusion causes the number of individuals in the different compartments, especially undetected infectives and deaths, to change with respect to time t after lockdown. as long as one susceptible person is infected after directly or indirectly contacting disease carriers in the area x, diffusion takes place. when the number of infectious individuals in a local area is low compared to the surrounding areas, the pandemic will diffuse in from the surroundings, so the number of infectives in this area will increase. conversely, the pandemic will diffuse out and the number of infectives will increase in the surrounding areas. the process of diffusion is influenced by distance, nearby individuals or all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint areas have higher probability of contact than remote individuals or areas. finally, gender differentiation or other properties may be taken into account to characterize types of populations and to study the optimal lockdown control of pandemic dynamics based on our previous work. it is worth stressing that, in the long run, optimal lockdown strategies should consider the balance between the lower number of deaths and minimum healthcare and social costs. in this paper, we investigated an extended model of the classical sird pandemic model to characterize the regional transmission of covid- after lockdown in france. incorporating the time delays arising from incubation, testing and the complex effects of government measures, an exponential function of the transmission rate β(t) was presented for the regional model. by fitting the regional model to the real data, the optimal parameters of this regional model for each region in france were derived. based on the previous results of the extended model, we introduced and simulated a network model of pandemic transmission between regions after confinement in france while considering age classes. regarding the transmission rate β(t) for the network model, we selected the inverse function of the previous β(t) to contribute to the transport effect after lockdown. by using the same model and method, we simulated the pandemic network for all cities in franc to visualize the transport effects of the pandemic between cities. considering age classes, we discussed an integro-differential equation for modelling the network of infectious diseases in the discussion part. because of the large volumes of data and complicated calculations needed for parameters calibration and simulation when considering many geographical areas and many age classes, the requirements in terms of computer hardware and software are rather high. in order to achieve accurate results, appropriate and efficient data processing methods will be applied. moreover appropriate dedicated theoretical work is needed to study the integro-differential model derived in section . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint in future works, we will formulate and study optimal control problems in order to balance the induced sanitary and economic costs. the lockdown strate- gies implemented in france should be evaluated and compared to the proposed optimal strategies. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint early dynamics of transmission and control of covid- : a mathematical modelling study asymptomatic coronavirus infection: mers-cov and sars-cov- (covid- ) covid- : identifying and isolating asymptomatic people helped eliminate virus in italian village estimated effectiveness of symptom and risk screening to prevent the spread of covid- coronavirus : personnes en quatorzaine après des cas de covid- déclarés dans uneécole de lyon an seir infectious disease model with testing and conditional quarantine effect of a one-month lockdown on the epidemic dynamics of covid- in france population modeling of early covid- epidemic dynamics in french regions and estimation of the lockdown impact on infection rate covid- pandemic control: balancing detection policy and lockdown intervention under icu sustainability basics and trends in sensitivity analysis predicting the number of reported and unreported cases for the covid- epidemic in south korea modeling infectious diseases in humans and animals infectious diseases of humans: dynamics and control stochastic epidemic models with inference on the distribution of points in a cube and the approximate evaluation of integrals the levenberg-marquardt algorithm: implementation and theory a comparison of three methods for selecting values of input variables in the analysis of output from a computer code gouvernement français on fast multi-shot covid- interventions for post lock-down mitigation modeling infectious diseases in humans and animals modeling fish population movements: from an individual-based representation to an advection-diffusion equation the mathematics of diffusion the authors are very greatfull to sébastien da veiga, senior expert in statistics and optimization at safrantech (france) for the r codes used for calibration and uncertainty calibration. key: cord- -mg mix authors: bitar, dounia; tarantola, arnaud; capek, isabelle; barboza, philippe; che, didier title: risques d’importation des maladies infectieuses exotiques en france métropolitaine : détection, alerte et réponse date: - - journal: bulletin de l'académie nationale de médecine doi: . /s - ( ) - sha: doc_id: cord_uid: mg mix summary the french public health institute is responsible for promoting and coordinating threats the detection and assessment of health risks, and for suggesting possible responses. transmissible diseases affecting both human and animal health are the focus of surveillance networks. early detection of potential infectious threats is based on the screening of “ alert signals “ identified through routine surveillance networks and other systems. the quality and accuracy of these signals is first verified, before assessing, through a multidisciplinary approach, the risk of introduction and dissemination. this article examines specific cases illustrating the process of detection, risk analysis and response, with respect to infectious threats that are endemic in tropical regions and have the potential to be imported into metropolitan france. for both novel pathogens and exotic diseases — which, not being endemic in france, are less well known — the analysis and response process must regularly be adapted to the latest epidemiological, clinical and biological findings, taking interactions between the pathogen, host, and environment into consideration. the need to improve reaction times and risk assessment is also discussed. dans le cadre de ses missions de surveillance et d'alerte, l'une des priorités de l'institut de veille sanitaire (invs) est d'anticiper l'introduction et la diffusion d'une menace de santé publique sur notre territoire afin d'apporter les réponses les plus appropriées [ ] . À cette fin, la veille prospective, la surveillance et l'expertise sont associées dans une démarche multidisciplinaire. l'organisation de la réponse est facilitée lorsqu'un phénomène infectieux est identifié au-delà de nos frontières et que sa vitesse de diffusion est relativement lente. ce fut le cas avec le sras en : bien qu'initialement peu précises, des informations cliniques et épidémiologiques étaient disponibles dans les pays du sud-est asiatique avant l'alerte internationale par l'organisation mondiale de la santé (oms). cette circulation des informations en amont d'une confirmation par une source officielle a permis aux pays européens d'anticiper l'introduction de cas importés sur leur territoire et d'organiser la mise en place des mesures de prévention et de contrôle, dans un délai de temps suffisant [ ] . de même, la réactivité de la réponse est améliorée lorsque des plans de lutte sont déjà élaborés et mis à disposition des différents acteurs. ainsi, les plans contre la pandémie grippale et les nombreux exercices conduits depuis plusieurs années aux échelons local, national et international [ ] ont permis aux pays européens de mettre en place des mesures de réduction de risque et de contrôle dans un délai très bref, suite à la diffusion rapide du nouveau variant de grippe a(h n ) depuis le continent américain en avril . dans ces deux exemples, la stratégie de l'autorité sanitaire ne vise pas nécessairement à « éviter à tout prix » l'introduction de cas individuels sur le territoire car la faisabilité et l'efficience de cette stratégie sont limitées. il s'agit surtout d'empêcher, de retarder, voire d'atténuer un cycle de transmission autochtone d'un pathogène par le diagnos-tic, l'isolement et la prise en charge précoces des patients et de leur entourage. un autre scenario pouvant être envisagé est celui d'un patient porteur de fièvre hémorragique virale arrivant en france métropolitaine, pour lequel les mesures d'isolement seraient retardées faute de diagnostic précoce. enfin, dans l'hypothèse de pathogènes non identifiés ou mal connus, il importe d'évaluer très rapidement le degré de menace pour la santé publique, malgré des informations cliniques, biologiques et épidémiologiques initialement peu précises. dans cet article nous nous attacherons à décrire quelques exemples de détection et d'analyse de risque concernant des phénomènes infectieux endémiques dans la zone intertropicale et potentiellement importables en france. il faut toutefois préciser que ces exemples n'illustrent qu'une partie du champ de la surveillance des maladies infectieuses telle qu'elle est organisée en france. par exemple la surveillance des infections nosocomiales ou les spécificités de la surveillance en fonction des zones géographiques ne sont pas abordées. le dispositif de veille et de surveillance des maladies infectieuses est basé sur le recueil d'informations visant à décrire un problème de santé, à détecter des épidémies ou des augmentations anormales de cas, à alerter l'autorité sanitaire, à aider à la gestion et enfin à évaluer l'efficacité des mesures (figure ). dans une optique de prévention et de contrôle des maladies infectieuses -que ces dernières soient importées ou qu'elles surviennent sur le territoire métropolitain -cette surveillance ne peut être dissociée de celle concernant les phénomènes émergents. ces derniers sont en effet définis comme '' des phénomènes infectieux ou présumés comme tels, inattendus (en référence à leurs propriétés intrinsèques ou aux connaissances de leur biologie), touchant l'homme, l'animal ou les deux. il peut s'agir d'entités cliniques d'origine infectieuse nouvellement apparues ou identifiées, d'entités pathologiques infectieuses connues dont l'incidence augmente dans un espace ou dans un groupe de population donné ou d'une modification qualitative et/ou quantitative des caractéristiques de l'agent, de la maladie, de la population touchée et de son environnement. dans une optique d'anticipation, il peut s'agir d'une maladie identifiée dont les conditions d'expansion deviennent favorables. habituellement, une incertitude réelle ou perçue quant au potentiel évolutif, la maîtrise du phénomène et l'impact en santé publique humaine et/ou animale est présente '' [ ] . les signaux d'alerte repérés par la veille sanitaire peuvent inclure des évènements non préalablement suivis ou la modification d'indicateurs provenant de systèmes de [ , ] . l'un des objectifs des systèmes de surveillance organisés est de détecter des épidémies ou des phénomènes inhabituels. la surveillance des maladies à déclaration obligatoire et celle issue des centres nationaux de référence permettent le recueil de données cliniques, biologiques et épidémiologiques concernant des entités cliniques précises. le choléra ou les fièvres virales hémorragiques sont des exemples de pathologies non endémiques sur le territoire français métropolitain, soumises à la déclaration obligatoire, de pronostic potentiellement sévère, pour lesquelles des mesures rapidement mises en oeuvre peuvent prévenir la diffusion secondaire. la « surveillance syndromique » complète le dispositif en recueillant de manière systématique et continue des données d'activité médicale regroupées en syndromes : consultations aux urgences ou admissions hospitalières codées selon la classification internationale des maladies, pour lesquelles le tableau clinique détaillé et l'étiologie ne sont pas disponibles. une augmentation inhabituelle du nombre d'évènements par rapport à des valeurs de référence représente un signal qui doit être exploré. ces systèmes organisés ne permettent pas de détecter des phénomènes rares, peu connus ou insuffisamment caractérisés. pour ces derniers, il est demandé aux soignants, biologistes et acteurs de santé publique de '' signaler tout syndrome infectieux dont la fréquence et/ou les circonstances de survenue et/ou la présentation clinique et/ou la gravité sont jugées inhabituelles ''. le signalement peut être effectué par téléphone pour plus de réactivité comme illustré dans l'encadré avec l'exemple de la fièvre de la vallée du rift (fvr). un autre axe de la surveillance est celui de la veille bibliographique basée sur l'étude des publications scientifiques. dans une optique d'anticipation, cette veille scientifique permet par exemple d'analyser le risque potentiellement lié à des modifications de l'environnement comme le changement climatique, de s'informer sur les éventuelles adaptations des arthropodes vecteurs ou sur les mutations de certains agents pathogènes, de surveiller l'évolution et la rapidité de diffusion des résistances aux anti-infectieux, etc. l'anticipation s'appuie également sur une combinaison de réseaux d'information et d'alerte qui constituent la veille internationale et qui couvrent notamment l'importation de maladies infectieuses exotiques. les dispositifs les plus fréquemment utilisés incluent la veille issue des media, particulièrement structurée par le système canadien gphin d'accès payant [ ] et le réseau promed, plus largement accessible [ ] . des réseaux d'accès limité complètent ce dispositif, notamment le « global outbreak alert and response network (goarn) » de l'oms et le « early warning and response system (ewrs) » de la commission européenne, géré par le '' european centre for diseases control '' (ecdc) [ , ] . ces réseaux sécurisés permettent aux institutions partenaires de s'informer mutuellement en temps réel et de lancer des alertes de portée régionale ou internationale. ils s'appuient sur le nouveau règlement sanitaire international de dont le but est « d'aider la communauté internationale à éviter les risques aigus pour la santé publique susceptibles de se propager au-delà des frontières et de constituer une menace dans le monde entier, en prenant les mesures qui s'imposent » [ ] . en , le centre national de référence des fièvres hémorragiques virales (institut pasteur) signalait par téléphone à l'invs un diagnostic de fvr chez un enfant habitant aux comores et transféré pour une hospitalisation à mayotte. une enquête téléphonique immédiate auprès des cliniciens de mayotte permettait de retracer le parcours et les dates de séjour de l'enfant aux comores. au cours des heures suivantes l'équipe de la veille internationale complétait l'investigation en interrogeant les partenaires sur la survenue d'épizooties, de vagues d'avortements chez le bétail, de cas suspects ou confirmés aux comores et à madagascar. cette enquête mettant à jour la circulation du virus dans l'archipel des comores, une alerte a été immédiatement émise. les partenaires au niveau national et local (invs, cire, ddass, cliniciens, biologistes, etc.) ont été mobilisés pour la détection rapide des cas et la mise en place de mesures de contrôle. un dispositif de détection des cas à mayotte a été mis en place, complété par une intensification de la surveillance syndromique. en parallèle une analyse rétrospective des prélèvements a été effectuée, à la recherche d'anticorps anti-fvr parmi des patients ayant présenté des symptômes similaires mais pour lesquels le résultat initial était négatif pour la dengue, le chikungunya, la leptospirose et le paludisme. la grande richesse et la diversité des systèmes de surveillance assure une complémentarité des informations. certains systèmes sont plus sensibles au risque de générer de nombreuses fausses alertes. d'autres sont plus spécifiques et basés sur la confirmation étiologique mais peuvent engendrer un retard avant l'alerte. il importe donc de vérifier de manière rapide, rigoureuse et systématique les signaux réceptionnés à l'invs. cette étape est d'autant plus importante que la source d'information est peu spécifique, comme la veille issue des media. les éléments de validation portent sur plusieurs critères : la source de données et leur degré de fiabilité, les informations initiales sur la description clinique des cas (sévérité, tableau clinique compatible avec le diagnostic évoqué, éléments de confirmation biologique ou d'orientation étiologique, etc.), les données épidémiologiques (modalités de diffusion du germe), l'existence éventuelle de phénomènes similaires rapportés dans d'autres zones géographiques ou dans un passé proche, etc. la possibilité d'une origine bioterroriste doit être évoquée à ce stade. un nouvel évènement infectieux, une nouvelle souche bactérienne identifiée ne signifient pas nécessairement un risque pour la santé publique. ce dernier est évalué en fonction de nombreux critères : données cliniques et épidémiologiques (taux d'attaque, incidence, gravité, létalité, population touchée, classes d'âge touchées) ; nature de l'agent et modes de transmission supposés ou avérés (une transmission interhumaine par voie respiratoire faisant suspecter un phénomène hautement contagieux) ; interactions animal-homme (maladies vectorielles, zoonoses) ; facteurs environnementaux (changement climatique, pullulation vectorielle) ; modifications des comportements humains (flux migratoires, échanges commerciaux licite, ou non, entre pays) ; capacité épidémique et risque d'émergence ou d'extension (notamment en fonction de la sévérité du tableau, des difficultés de diagnostic biologique et de traitement spécifique) ; perception sociale et politique du risque ; enjeux économiques ; risque de diffusion internationale. les disciplines impliquées dans l'analyse reflètent la diversité de ces critères d'analyse. elles associent cliniciens, biologistes, épidémiologistes, vétérinaires, entomologistes, sociologues, etc. ainsi que les gestionnaires, afin de s'assurer de la faisabilité et de la cohérence de la réponse. l'expertise intègre de plus en plus souvent les modèles mathématiques qui permettent par exemple d'estimer le risque d'importation du sras ou de la dengue en europe [ , ] ou d'évaluer l'impact des mesures de contrôle aux frontières pour la réduction de la diffusion d'une pandémie grippale [ ] [ ] [ ] . l'analyse de risque peut être effectuée conjointement par plusieurs pays frontaliers ou sous la coordination et l'impulsion des institutions internationales : le risque d'implantation du chikungunya en france métropolitaine et sur le pourtour méditerranéen a fait l'objet de diverses expertises nationales et européennes [ ] [ ] [ ] [ ] . nous proposons ci-dessous un exemple d'analyse multidisciplinaire du risque d'introduction et implantation de la fvr en france métropolitaine. -modalités de l'expertise l'invs a mis en place dès un groupe d'experts multidisciplinaires (cliniciens, épidémiologistes, biologistes, vétérinaires, responsables de santé publique, chercheurs, gestionnaires) chargé de définir les priorités de surveillance concernant les zoonoses non-alimentaires [ ] . ce groupe définit les zoonoses à surveiller en priorité en fonction de l'importance de la maladie humaine (incidence ou prévalence, sévérité, mortalité, potentiel épidémique, modes de transmission, existence de mesures de prévention et de contrôle), de l'importance de la maladie ou du portage chez l'animal (mammifère ou insecte) et du contexte environnemental qui peut évoluer au cours du temps. entre et , le groupe d'experts a notamment été interrogé sur le risque de survenue de fvr en métropole. -analyse de risque pour la france métropolitaine ( ) l'incidence de la maladie est nulle en mais le risque d'importation et d'implantation (i.e. d'émergence) existe en raison de l'épizootie dans des zones géographiques ayant des liens particuliers avec la france métropolitaine, de la présence de vecteurs compétents et du rôle potentiellement aggravant du réchauffement climatique. il n'y a pas, en théorie, d'importation de bétail en métropole à partir de ces zones. le risque serait essentiellement lié à une arrivée de voyageurs infectés et virémiques, pouvant être à l'origine d'une chaîne de transmission en cas de piqûre par l'un des vecteurs présents. sur la base de ces critères, les experts ont classé la fvr comme hautement prioritaire en matière de surveillance humaine (niveau sur une échelle de à ). en parallèle il importe de renforcer et adapter la prévention et le contrôle (niveau ) et de développer des recherches pour une meilleure connaissance clinique et épidémiologique (niveau ). par ailleurs, la surveillance et le contrôle dans le milieu animal sont également jugés prioritaires car l'infection peut provoquer des avortements spontanés et la mort de jeunes animaux d'élevage avec un impact économique important. les connaissances concernant le risque de fvr doivent être approfondies avec les entomologistes, les vecteurs potentiels étant nombreux (rapport en cours). les résultats de l'expertise ont été soumis à l'autorité sanitaire (direction générale de la santé) et à l'agence française de sécurité sanitaire des aliments (afssa) (lien http://www.afssa.fr/cgi-bin/countdocs.cgi). la première étape de la réponse a consisté à intensifier la surveillance biologique et à renforcer les capacités de détection des cas dans l'archipel comorien, avec une mise à dispostion de fonds. concernant la métropole, l'afssa devrait évaluer le risque d'importation pour le volet animal et recommander des mesures pour éviter l'implantation. ce travail est en cours. une alerte est émise lorsqu'une menace pour la santé publique est retenue comme plausible ; cette alerte doit s'accompagner d'informations sur les mesures de réduction de risque ou de contrôle. un exemple désormais classique est celui du sras au début de l'alerte mondiale, lorsque le germe était encore inconnu [ ] . en cas d'urgence, des réunions téléphoniques sont rapidement organisées avec les experts des différents secteurs concernés sous la coordination de la direction générale de la santé. des conduites à tenir peuvent être proposées. par exemple, un guide de gestion autour des cas importés de choléra a été élaboré entre institutions (lien ). ce dans le cas de menaces avérées par leur sévérité ou leur diffusion, mais insuffisamment caractérisées, l'évaluation, l'alerte et la réponse doivent être conduites rapidement malgré les nombreuses incertitudes. ainsi, la mise en quarantaine de personnes asymptomatiques exposées à un cas de sras n'est plus considérée comme justifiée car le risque de diffusion à l'entourage est infime avant la survenue de symptômes [ ] , mais cette notion n'était pas clairement établie dans les premiers temps, justifiant que des mesures drastiques aient été mises en place. le dispositif que nous avons décrit doit également fournir des informations permettant l'évaluation de l'impact et l'adaptation des mesures mises en oeuvre. la mise à jour régulière des plans et conduites à tenir est une preuve de ces ajustements effectués en fonction des nouvelles connaissances épidémiologiques, biologiques, cliniques dans les domaines humain et animal, et en particulier dans le domaine des vecteurs. la mise à jour des connaissances par la veille scientifique est complétée par des retours d'expérience organisés au niveau national ou international. toutefois, ces retours d'expériences sont rarement publiés [ , [ ] [ ] [ ] ). enfin, l'ensemble du dispositif de surveillance concernant un pathogène spécifique peut être évalué [ ] . du fait de la grande diversité des sources d'information, les épidémiologistes chargés de la veille en maladies infectieuses font face à un volume croissant de signaux d'alerte, issus d'outils et de réseaux de veille très diversifiés. il importe d'observer avec un regard critique ces outils visant à détecter et à retarder l'implantation des agents importés. selon une étude récente [ ] , parmi les milliers de signaux émis par le réseau promed entre et sur une période d'un an, vingt-sept signaux avaient été sélectionnés par l'institut de santé publique néerlandais en raison d'un risque potentiel d'importation et de diffusion pour leur pays. deux signaux étaient exclus car non validés et une menace possible était retenue et analysée de manière approfondie pour cinq des vingt-cinq signaux retenus. toutefois aucune de ces cinq menaces possibles n'a finalement donné lieu à une alerte ou à la mise en place de mesures de contrôle immédiates. d'autres auteurs [ , ] soulignent également les limites de ces outils de veille dont la valeur ajoutée serait marginale en termes d'alerte et de réponse pour les pays européens ; ils indiquent néanmoins que ces signaux restent utiles et nécessaires pour connaître le « bruit de fond ». la combinaison de différents systèmes de surveillance apportant une meilleure sensibilité, des projets de recherche opérationnelle visant à améliorer les modalités de détection et à définir des critères plus pertinents de sélection des signaux d'alerte pourraient éventuellement permettre d'améliorer la spécificité. en parallèle, le renforcement des capacités de diagnostic biologique et des capacités de réponse est essentiel. enfin, le renforcement de la surveillance doit impérativement englober les pays de la zone intertropicale : le risque relatif de survenue et de diffusion d'une émergence infectieuse est en effet particulièrement élevé dans ces pays alors que les efforts de veille et d'anticipation sont généralement développés dans les pays plus riches [ ] . À daniel eilstein et christine saura (institut de veille sanitaire) pour leur relecture attentive du document surveillance des maladies infectieuses : principes et organisation en france en l'épidémie de sras en en france. rapport sur la gestion épidémiologique du sras par l'invs different approaches to gathering epidemic intelligence in europe emergence des maladies infectieuses animales et humaines. inra productions animales the role of evolution in the emergence of infectious diseases gowtage-sequeria s. -host range and emerging and reemerging pathogens the global public health intelligence network and early warning outbreak detection: a canadian contribution to global public health promed-mail: an early warning system for emerging diseases emergent pathogens, international surveillance and international health regulations the early warning and response system for communicable diseases in the eu: an overview from implementing the international health regulations ( ) in europe desenclos j.c. -an approach to estimate the number of sars cases imported by international air travel -assessing the risk of importing dengue and chikungunya viruses to the european union international travels and fever screening during epidemics: a literature review on the effectiveness and potential use of non-contact infrared thermometers delaying the international spread of pandemic influenza entry screening for severe acute respiratory syndrome (sars) or influenza: policy evaluation invs -cas importés de chikungunya et de dengue en france métropolitaine: bilan de la surveillance à partir des données de laboratoire imported cases of chikungunya in metropolitan france: update to chikungunya risk assessment for europe: recommendations for action définition de priorités et actions réalisées dans le domaine des zoonoses non alimentaires severe acute respiratory syndrome: an update rapport de la mission d'évaluation et d'expertise de la veille sanitaire en france fièvres virales hémorragiques. bulletin Épidemiologique hebdomadaire bulletind'alerteetdesurveillanceantilles-guyane -la surveillance du virus west nile en france -the value of promed-mail for the early warning committee in the netherlands: more specific approach recommended epidemic intelligence in the european union: strengthening the ties surveillance sans frontieres global trends in emerging infectious diseases quelle est la place des centres nationaux de référence (cnr) dans l'élaboration de l'alerte sanitaire lorsqu'une maladie est inconnue en france ? en effet, face à un agent inconnu, il est difficile pour les cliniciens et biologistes de pouvoir identifier d'emblée vers quel cnr s'adresser en première intention. le renvoi des prélèvements ou souches vers un autre cnr lorsqu'un diagnostic alternatif est évoqué peut provoquer différents dysfonctionnements : délais prolongés d'acheminement, risque d'égarement des prélèvements ou mauvais rendement de prélèvements répartis dans plusieurs laboratoires, etc. l'autorité sanitaire a mis en place un laboratoire de niveau p à même d'effectuer un premier triage '' à l'aveugle '' sur des prélèvements de nature inconnue : la cellule d'intervention biologique d'urgence (cibu) basée à l'institut pasteur. ce laboratoire a pour mission d'intervenir lorsque la classe d'agent biologique n'est pas encore identifiée. l'intervention de la cibu est déclenchée par l'autorité sanitaire après analyse du risque. dès que l'orientation le permet, les cnr correspondants sont sollicités les plans d'action devant une alerte sanitaire sont-ils harmonisés en europe ? les modalités et déclinaisons des interventions restent adaptées à chaque pays, selon la situation et l'organisation du système sanitaire des pays respectifs. les plans '' pandémie grippale '' des pays européens ont été inspirés d'une trameélaborée par l'oms et adaptée par les différents pays de la communauté. par la suite de nombreux exercices inter-pays ont été organisés sous la coordination de l'union européenne, comme le '' common ground exercise '' conduit en (suivi d'autres exercices). ces exemples de mise en commun ne concernent pas exclusivement la pandémie : les alertes bioterroristes ou celles liées à des agents inconnus font également l'objet de discussions la réponse face à une alerte est également coordonnéee ou concertée, autant que faire se peut : -pour une alerte prévisible (exemple : pandémie grippale) dès le début de l'alerte l'union européenne a organisé des réunions téléphoniques de coordination et concertation, accompagnées d'échanges sur le site internet de l'ewrs. ces échanges permettent tout d'abord une mise à jour des connaissances et un partage sur les arguments ayant conduit chaque pays à faire un choix stratégique particulier. par la suite une mise en commun des données est organisée. ainsi pour la grippe h n , l'invs fournit chaque semaine à l'ecdc un bilan des cas de grippe, selon un format standard. l'ecdc se charge ensuite du transfert de ces données agrégées à l'oms pour les bilans mondiaux key: cord- - poerd authors: vermeulen, tom d; reimerink, johan; reusken, chantal; giron, sandra; de vries, peter j title: autochthonous dengue in two dutch tourists visiting département var, southern france, july date: - - journal: euro surveill doi: . / - .es. . . . sha: doc_id: cord_uid: poerd we report dengue virus (denv) infection in two dutch tourists who visited département var, southern france, in july and august . as some autochthonous dengue cases have occurred in europe in recent years, awareness among physicians and public health experts about possible intermittent presence of denv in southern europe is important to minimise delay in diagnosis and treatment. quick diagnosis can lead to timely action to contain the spread of vector-borne diseases and minimise transmission. we report dengue virus (denv) infection in two dutch tourists who visited département var, southern france, in july and august . as some autochthonous dengue cases have occurred in europe in recent years, awareness among physicians and public health experts about possible intermittent presence of denv in southern europe is important to minimise delay in diagnosis and treatment. quick diagnosis can lead to timely action to contain the spread of vector-borne diseases and minimise transmission. travel-related diseases may serve as sentinels of transmission of disease in the visited area. prompt diagnosis and notification of such diseases may assist in the detection and control of disease outbreaks. when we diagnosed dengue in a dutch tourist who visited southern france, we coordinated joint action between the patient and clinical and public health experts. this led to rapid international notification and consecutive outbreak control efforts by french authorities. a second, related, dutch patient with a recent fever was retrospectively also diagnosed with dengue. a previously healthy woman in her s (patient ) spent a -week holiday with relatives in la croix valmer, france, from to july . in the first week of august, patient stayed with other friends in another house nearby. on august, she developed fever accompanied by myalgia in her calves and neck, as well as a painful skin (day of the disease episode). on day post onset of symptoms (pos), she was nauseous and vomited once. on day pos, she returned to the netherlands. a test for severe acute respiratory syndrome coronavirus (sars-cov- ), on a nasopharyngeal swab, was negative. on day pos, the patient noticed an itchy erythematous rash on her hands and lower legs. on day pos, she consulted her general practitioner who, suspecting petechiae, referred her to our hospital. in addition to the reported signs and symptoms, she mentioned a blurred, colourful spot in the field of vision of her left eye. during childhood, the patient was vaccinated according to the dutch national vaccination scheme; she had never received any vaccinations for yellow fever, japanese encephalitis or tick-borne encephalitis. interestingly, one of her family members upon our clinical suspicion of dengue virus infection spontaneously acknowledged having seen tiger mosquitoes (aedes albopictus) around their holiday home. physical examination showed normal vital signs and revealed slight erythematous exanthema on her hands and upper limbs and a confluent petechiae-like exanthema on both legs. the presumptive diagnosis of dengue was made, common laboratory tests including dengue virus (denv) serology were ordered and she was referred to the ophthalmologist. fluorescein angiography of the eyes showed an inflammatory foveolitis in her left eye. laboratory results on day pos showed a mild thrombocytopenia and leukocytopenia with plasmocytosis, and moderately elevated serum levels of the liver enzymes (table ). on the same serum, comparative igm and igg serology (immunofluorescence arbovirus fever mosaic , euroimmun ag, lübeck, germany) against chikungunya virus (chikv), denv and japanese encephalitis virus (jev) was performed at the dutch national institute for public health and the environment (rivm) laboratory. high concentrations of denv-specific igm and igg antibodies were detected ( table ). there was a slight igg response, without igm response, against jev, interpreted as non-specific cross-reactivity. rt-pcr was not done. patient spent the holiday in la croix valmer, together with patient , from to july. he returned to the netherlands week before patient . on august, he noticed a mild pain in his right ear (day of the disease episode). on day pos he had a high fever ( . ° c). on day pos, suspecting bacterial otitis, he was prescribed amoxicillin. on day pos, he noticed a mild pain behind his eyes. on day pos, day after defervescence, he noticed a slight rash on his trunk and extremities and interpreted this as allergy to the amoxicillin. patient 's blood sample of september (day pos) tested positive for igm and igg antibodies against denv, with high titres ( table ). the igg response against jev was interpreted as non-specific cross-reactivity. also this patient had been vaccinated according to the dutch national vaccination scheme. on august , upon confirmation of the serological results, patient was reported by the rivm to the french authorities through the early warning and response system of the european union as an autochthonous denv infection probably acquired in france with cross-border implication. the french authorities contacted the patient and announced the case by press release on september [ ] . santé publique france advised that also other family members with symptoms should be tested. that identified patient . the other seven dutch family members visiting the holiday home between and july did not develop any disease symptoms and neither did the individuals with whom patient stayed during the first week of august in the other house nearby. none of the household members had recently travelled outside europe. notification to the french authorities led to a prompt local public health response including mosquito control around the holiday home (with deltametrine and bacillus thuringiensis israelensis on a m radius) and door-to-door investigations in order to identify other cases and raising awareness among local healthcare professionals and the public [ ] . here we describe two patients with dengue from the same family, who acquired the disease in department var, southern france. the signs and symptoms, as well as the plasmocytosis, of patient were typical for dengue [ ] . the list of differential diagnoses was therefore very short and the high igm and igg titres for denv were considered confirmative, even though definite confirmation would require demonstration of virus or serodiagnosis on paired samples [ ] . pcr was not conducted to detect denv in blood or urine because the chance for a positive test was considered low in this rather late stage of disease and it was not deemed necessary for confirmation of disease, clinical management, notification or public health measures. dengue is endemic in large parts of the world and a common illness among returning travellers from (sub) tropical regions [ ] . because of globalisation in travel and trade and under changing ecological conditions, the geographical distribution of the vector of denv, ae. albopictus, gradually expanded over the last decades and may continue to do so [ ] . imported infections can continue to cause autochthonous outbreaks. a lack of awareness and a long interval between the viraemic episode of the patient with imported dengue and the first registration in the public health system were identified as possible drivers of local outbreaks [ ] . aedes albopictus has been established in france since and currently, the mosquito species is endemic in large parts of the country including one area close to the belgian border [ , ] . the presence of ae. albopictus in multiple sites in europe means that also other diseases can be transmitted. upon introduction by returning viraemic travellers, european cases of chikv, denv and zika virus infection have been reported [ , ] . as recently as august , five patients in vicenza province, northern italy, were confirmed to have a denv infection weeks after a household member infected with denv returned from west sumatra [ ] . autochthonous denv infection was first reported in france in and has since been reported at an almost yearly basis [ , ] . in , by the end of september, six other autochthonous denv cases had been reported by french authorities, one in the department hérault and five in the department alpes-maritime [ , , ] . our case signalled the first evidence of local denv activity in département var in . in the recent past, between and , six cases of autochthonous transmission were confirmed in the départment var [ ] . however, our cases do not seem to have any connection with the other autochthonous cases identified in southern france this year. the cases reported here again illustrate that travel medicine can have a role as a sentinel for detection of silent circulation of infectious diseases [ ] . clinicians should be aware of the possibility of 'tropical' vectorborne diseases acquired by travellers within european areas where competent vectors are present, even when cases have not been reported (yet) by local authorities. rapid notification by clinicians and communication between national authorities is essential to ensure timely local risk management and disease control. none declared. tom d. vermeulen: clinical description of case. johan reimerink: serology and co-authoring manuscript. chantal reusken: international notification, epidemiological perspective, co-authoring manuscript. sandra giron: french public health perspective, co-authoring manuscript. peter j. de vries: clinical case management and description, corresponding author. marseille: agence régionale de santé high incidence of peripheral blood plasmacytosis in patients with dengue virus infection dengue guidelines for diagnosis, treatment, prevention and control: new edition. geneva: who travel-associated illness trends and clusters past and future spread of the arbovirus vectors aedes aegypti and aedes albopictus from importation to autochthonous transmission: drivers of chikungunya and dengue emergence in a temperate area chronology of the development of aedes albopictus in the alpes-maritimes department of france european centre for disease prevention and control (ecdc) vector-borne transmission of zika virus in europe, southern france ongoing and emerging arbovirus threats in europe first autochthonous dengue outbreak in italy Émergences de dengue et de chikungunya en france métropolitaine bilan de la surveillance des arboviroses en : transition vers une surveillance des cas confirmés de chikungunya, dengue et d'infection à virus zika en france métropolitaine. [review of arbovirus surveillance in : transition to surveillance for confirmed cases of chikungunya, dengue and zikavirus in metropolitan france an outbreak of indigenous cases of dengue detected in the alpes-maritimes cinq cas autochtones de dengue détectés à nice a case of dengue type virus infection imported from africa to italy license, supplementary material and copyright this is an open-access article distributed under the terms of the creative commons attribution (cc by . ) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made.any supplementary material referenced in the article can be found in the online version. key: cord- - p rwp authors: nan title: escp th european symposium on clinical pharmacy ‘implementing clinical pharmacy in community and hospital settings: sharing the experience’, istanbul, turkey – october ; abstracts date: - - journal: pharm world sci doi: . /s - - - sha: doc_id: cord_uid: p rwp nan pharmacy, groupe hospitalier pitié salpétrière, paris, france background and objective: strongyloides stercoralis infects each year millions of persons worldwide. in immunocompromised patients, this intestinal nematode can disseminate and cause a fulminant fatal illness: hyperinfection syndrome. oral ivermectin is the principal treatment. since one of the features of s. stercoralis hyperinfection is the development of an ileus and small bowel obstruction, the drug absorption is impaired and thus a reduced efficacy is noted. no parenteral antihelminthic drug is licensed for human use, but parenteral ivermectin is commonly used in veterinary medicine. we report two cases of s. stercoralis hyperinfection syndrome that were refractory to oral drugs and, as a life saving therapy, were treated with a veterinary formulation of parenteral ivermectin (ivomec Ò , merial) after agreement from the french drug administration (afssaps). design: case report. setting: pneumology and neurochirurgical intensive care units, university hospital, paris, france. main outcome measures: case report. results: patient a -year-old african man has been hospitalized in august for a degradation of his condition in neurosarcoidosis with hydrocephalus, associated with enterococcus faecalis meningitis. he underwent ventriculo-peritoneal shunt and was treated with corticosteroids (prednisone mg/day). his condition worsened on september , with the diagnosis of a disseminated strongyloidiasis with paralytic ileus. he was initially treated with ivermectin ( mg bid) via the nasogastric tube. antibiotics were added on day to control the sepsis. on day albendazole ( mg/day) was added. subcutaneous ivermectin was then obtained and administered on day ( lg/kg) in association with ivermectin via nasogastric tube while albendazole was discontinued. the patient's condition improved during the following days. he completed days of ivomec Ò and days of oral ivermectin. he returned home months later. patient a -year-old african man was hospitalized in november because of a kg weight loss. he was diagnosed with hiv, meningeal tuberculosis, urinary and pulmonary infections and s. stercoralis hyperinfection treated with oral ivermectin. on january he was admitted in pneumology intensive care unit with melena on severe immunodepression. on february he developed a septic shock with ards on an important bowel obstruction. therefore, among other antiinfectious therapies, a veterinary formulation of subcutaneous ivermectin was administered ( lg/kg/day). the sepsis was controlled, but on february he died of an acute haematological deterioration. conclusions: as the occurrence of malabsorption is a frequent complication of disseminated strongyloidiasis, a parenteral formulation of ivermectin would be really helpful, especially as the efficacy of the subcutaneous form has been proved in the literature. keywords: strongyloides stercoralis, hyperinfection, parenteral ivermectin • each pharmacy received three covert sp visits over six weeks. verbal and written feedback was provided to the pharmacy staff after each sp visit. no pharmacy was visited by the same sp more than once. each pharmacy was visited by the same pe throughout the study. background and objective: heart failure (hf) is a common disease with an estimated prevalence of . to % in europe. patients with hf have frequent episodes of exacerbation. non-compliance to medical and dietary advice is a significant clinical problem as is suboptimal treatment. one example of factors influencing the ability to comply with a treatment plan is impaired comprehension. the objectives were to construct a medication assessment tool and to establish face validity for its use in this project, to construct an interview schedule in order to identify non-compliance, poor patient comprehension and suboptimal treatment, to conduct a survey and to report the findings to the clinic. design: a cross-sectional study performed during april -may . setting: the emergency department and medical wards at malmö university hospital. main outcome measures: comparison of compliance, comprehension and optimal treatment on a population basis between men and women, younger (\ years) and elderly ([ years) patients, and patients in different new york heart association (nyha) classes, in order to assess if exacerbation could have been caused by any of these factors. results: of the patients included, % reported high compliance. in the subgroup analysis, women and elderly patients reported a significant higher compliance than men and younger patients. comprehension on self-care was poor. only % weighed themselves regularly and % did not limit the amount of fluids. no more than % reported they would contact a health care provider in case of experiencing more symptoms. suboptimal treatment was also found to be a great concern with only % being treated with angiotensinconverting enzyme inhibitors (acei) or angiotensin ii receptor blocker (arb), % with beta blockers, and % with aldosterone receptor antagonists, but no consideration to other co-morbidities has been taken into account. the majority treated with recommended agents had not achieved target dose as recommended in guidelines. conclusions: poor patient compliance and comprehension as well as suboptimal treatment could contribute to hf exacerbation and efforts should be made to improve these factors in order to reduce hf exacerbation. keywords: heart failure, compliance, sub-optimal treatment background and objective: adherence with inhaled corticosteroids has repeatedly been reported to be poor. poor adherence could lead to inadequate control of asthma complaints. monitoring of repeat prescriptions by a pharmacist could offer an opportunity to reach concordance with the patient and improve adherence. the objective of this study is was to improve asthma control by optimizing use of asthma medicines. design: retrospective follow up study. all pharmacy dispensing records concerning respiratory medication (r ) from st october to th september were collected. between st october to th september monitoring of repeat medication was conducted by a pharmacist. pharmacists discussed asthma complaints and use of asthma medicines with all patients calling for repeat prescriptions. when indicated the pharmacist proposed adjustments of asthma medicines to the gp after this telephone consultation. setting: community pharmacy and one gp practice ( gp's) in leiden, the netherlands, serving a community of . patients. main outcome measures: self-reported use of short-acting betaagonists (saba) by intervention patients. defined daily doses (ddds) of short-acting beta-agonists (saba), long-acting beta-agonists (laba) and inhaled corticosteroids (ics). results: consultations were registered for intervention patients. for patients more than consultation was registered. at the first consultation only of patients ( %) reported use of pharmaceutical sciences, strathclyde institute of pharmacy and biomedical sciences, university of strathclyde, glasgow, united kingdom background and objective: to test a method to quantify adherence of medication use to clinical guideline recommendations in a primary care setting. design: retrospective survey to field-test a -item instrument (mat-cvd) based on earlier studies of quality of medication use in cardiovascular disease (cvd) . setting: a database of patients [ % male, mean (sd) aged ( ) years] coded with circulatory system disease (read code 'g*') was drawn from computerised records of all patients receiving care from a single community pharmacist and general medical practitioner (gp) collaboration (n = , ). the pharmacist worked as a supplementary prescriber and had remote access to the electronic records of the gp. patients had diagnoses of diabetes (n = ), hypertension (htn; n = ), ischaemic heart disease (ihd; n = ), other ischaemic vascular disease (cerebrovascular n = ; peripheral vascular n = ), heart failure (hf; n = ), atrial fibrillation (af; n = ), were anticoagulated (warfarin, n = ) or otherwise identified as potential candidates for primary prevention of cvd (n = ). main outcome measures: adherence (%) to criteria based on guideline recommendations on primary and secondary prevention of cvd, treatment of htn, ihd, hf, af and warfarin therapy; overall applicability of criteria and quantification of insufficient data; interrater agreement of application of individual mat-cvd criteria and of the overall tool (cohen's r) results: a total of criteria were applicable and for ( %) of these there was insufficient data to apply the standard. the guideline adherence ( % ci) overall was ( - )%. highest adherence was to 'primary/secondary prevention of cvd' [ ( - )% adherence, n = criteria]. lowest adherence was to 'treatment of af' [ ( - )% adherence, n = criteria]. non-adherences were found to at least one criterion in ( %) and to c criteria in ( %) patients. inter-rater agreement was assessed on the application of the tool to all patients by two independent raters. all six sections and the overall tool were found to have inter-rater agreement r [ . and a percentage agreement [ %. among the ( %) of individual mat criteria that were applicable to c patients showed r [ . . in two of the remaining seven criteria the base-rate problem was responsible for r \ . and when taken into account the number of individual criteria with acceptable inter-rater agreement was ( %). conclusions: the application of the mat-cvd to routine primary care records in a scottish primary care setting is feasible and reliable; the tool has potential use in continuous quality improvement of prescribing in primary care. pc- self-management of complications in diabetic patients: a pharmaceutical care program in community pharmacies pharmacy, general medicine unit, university medical outpatient clinic, lausanne, switzerland background and objective: seamless care refers to continuity of patient care in the health system across caregivers. the objectives of the present study were ( ) to identify barriers to seamless information between a given hospital and an outpatient clinic in switzerland and ( ) to propose tools for improving pharmaceutical seamless care. design: this is a retrospective study with a convenient sample of patients for mapping the information flow network. the inclusion criteria were: ( ) at least one stay lasting more than hours in the hospital in , ( ) regular checkups with a gp in the outpatient clinic and ( ) medication delivered by the community pharmacy of the outpatient clinic months prior to months after the hospitalization. setting: both hospital and outpatient clinic are independent and run their own pharmacy. the hospital pharmacy is implied in drug production and distribution without generalized pharmaceutical care activities, and the community pharmacy delivers rx or otc medication for outpatients. geographic and computer proximity between both entities constitutes an ideal setting for seamless care projects. main outcome measures: ( ) to map medical or administrative information between community pharmacy, gp and hospital and ( ) to find opportunities to improve pharmaceutical seamless care. results: sixteen patients met inclusion criteria ( women, men, average years, mean visits/patient/year with gp: , and with community pharmacist: , i.e. -time more with pharmacist than gp). we observed that administrative information is computerized on a common database for both hospital and outpatient clinic. in contrast, clinical information is mainly handwritten and difficult to share between hospital and outpatient clinic caregivers. patient medication database is managed by a community pharmacy software not linked to medical information. however administrative information flows in one direction from the administrative to the community pharmacy database. we identified potential tools easily available to the community pharmacy to improve pharmaceutical seamless care in the center: ( ) an alarm through the administrative database connection if a patient is hospitalized to allow pharmacist to contact hospital physician for medication history and ( ) an access to patient discharge letter and lab results to improve rx validation process. conclusions: clinical information is not easily shared between caregivers of the hospital and the outpatient clinic. if global seamless care still remains a long term goal, initial actual steps promoted by community pharmacists can be easily implemented. keywords: seamless, information, community pk- implementation of a protocol for pharmacokinetic monitoring of high-dose methotrexate background and objective: to quantify the impact of the implementation of a protocol for the pharmacokinetic monitoring of patients receiving high-dose methotrexate. design: prospective experimental study, in which the hospital pharmacy department designed a specific protocol for the pharmacokinetics follow-up of these patients and for gathering the data required for a correct rescue. results were compared between three months before and three months after implementation of this new protocol. setting: -hr infusions of methotrexate at a dose of c g/m were evaluated in adult patients admitted to the oncohaematological area of a tertiary level hospital. main outcome measures: number of infusions started at the correct time, number of missed blood extractions, number of missed leucovorin doses, calculation of the elimination half-life, and measurement of the urinary ph (dichotomous variables). degree of compliance with the leucovorin rescue dosage protocol was measured on a scale of - points, with all items carrying the same score (correct loading dose, dosage as function of body surface area, and dosage as function of the concentrations of methotrexate obtained). results: the number of infusions started at the correct time increased from % to %. the number of missed blood extractions fell from . to . extractions per course; and missed leucovorin doses dropped from . to per course. the elimination half-life could be calculated in only % of courses in the first study period versus % of courses after protocol implementation. urinary ph changed from not being measured in any cycle to being measured in % of cycles. compliance with rescue dosage protocol was scored with . points before versus . points after implementation. background and objective: to quantitatively evaluate the safety of the current injectables medication process, from prescription to administration, in the paediatric and neonatal intensive care units. to compare the potential impact of safety measures on the risk. to classify these measures from a pharmacoeconomic point of view. design: assessment by a prospective risk analysis according to the failure modes, effects and criticality analysis (fmeca) method [ ] by a multidisciplinary team: one physician, two nurses, three pharmacists. three drugs chosen as models (gentamicin; morphine; dopamine). failure modes (fm) defined during brainstorming and criticality indexes calculated on the basis of their likelihood of occurrence, potential severity for the patients and detectability. impact of ten safety measures on the criticality indexes of the selected three drugs, extrapolation to all drugs injected daily and calculation for each measure of the investment in euros per year to improve the safety by quali (- point of criticality) per day. setting: university hospital, fifteen nicu beds and ten picu beds. main outcome measures: mean criticality indexes; gain in qualies per day; cost-efficacy ratios for each safety measure. results: in the current situation, the sum of mean criticality indexes of thirty-one identified fm was , for the selected three drugs. we gain , qualies ( , by extrapolation to all drugs injected daily) with civas (centralized intravenous additives services), , ( , ) with a clinical pharmacist, ( , ) with double check by nurses, ( , ) with cpoe (computerized physician order entry), ( , ) with in-line filters, ( , ) with vial of dilution, ( , ) with horizontal laminar airflow hood, ( , ) with intermediate dilution, ( , ) with simple additional measures of asepsis and ( ) with a drug planer. the best cost-efficacy ratios were obtained by a clinical pharmacist ( quali = . euros) or by double check by nurses ( quali = . euros) or by civas ( quali = . euros). the highest ratio was obtained with cpoe, due to the very high costs investment ( quali = . euros). conclusions: the use of a prospective risk analysis allowed us to quantitatively evaluate the relationship between the medication process of injectables and the paediatric patient safety and to build a strategy for continuous quality improvement, by selecting the most appropriate evolutions. based on the results of the pharmacoeconomic analysis, development of clinical pharmacy and civas for some drugs will be discussed with the paediatric department background and objective: studies show that up to % of patients starting treatment with antidepressants fill only a single prescription at the pharmacy, apparently not accepting treatment. the aim of this study was to determine characteristics and reasons associated with non-acceptance of ssri treatment. design: retrospective questionnaire study. patients presenting a gp prescription for a newly started ssri treatment to a community pharmacy were selected. 'non-accepters' were defined as those patients filling only a single ssri prescription, and patients who received at least three prescriptions were defined as 'accepters'. setting: community pharmacies in the netherlands. main outcome measures: characteristics evaluated included sociodemographic (e.g. level of education), disease (e.g. reason for use) and treatment (e.g. type of ssri) characteristics. 'non-accepters' were also asked for the reason not filling a second prescription. results: 'non-accepters' and 'accepters' were included in the analysis. 'non-acceptance' was more common among patients with a low level of education (or . ; ci . - . ) and in patients who reported aspecific symptoms like fatigue, stress and restlessness as the reason for ssri use (or . ; ci . - . ). in addition, there was a trend that 'non-acceptance' was more common among patients over years of age (or . ; ci . - . ) . of all 'non-accepters', . % (n = ) did not start ssri use, while . % (n = ) discontinued ssri use within two weeks. fear of side effects and the actual occurrence of side effects are main reasons for not accepting ssri treatment. in addition, a considerable number of 'non-accepters' indicated that they felt an aversion towards medicine use, were feeling better meanwhile or disagreed the gp's diagnosis. of the 'nonaccepters', . % discontinued treatment without informing the gp. conclusions: acceptance of ssri treatment is a decisive moment in compliance to treatment initiated by gps, and deserves more attention. gps and pharmacists should address issues related to the use of ssris especially in groups who are at risk for non-acceptance. keywords: antidepressants, discontinuation, nonadherence edu- pharmaceutical interventions by pharmacists working within surgery and medicine departments julie prince , stephanie diallo , eric grandsire , anne lecoeur , caroline fijalkowski , michelle lebas-certain , franck le mercier pharmacy, ambroise pare hospital ap-hp, boulogne-billancourt, france background and objective: since , our pharmacy department set up a nominative daily drug distribution system without computarization. in each pharmaceutical unit localized in clinical departments, the prescriptions are screened daily by a pharmacist, then the drugs are delivered by a technician. our objective was to compare the frequency and content of pharmaceutical interventions in surgery and medicine departments. pharmacy, haematology, nurses' management, cancer centre henri becquerel, rouen, france background and objective: while several studies have evaluated the frequency and the consequences of medication errors, few have explored their causes. in particular, knowledge of nurses regarding treatment of their patients has been scarcely studied. this survey has been carried out to determine how nurses master medications prescribed to the patients they care for, and how often they access drugs database. design: this work is a prospective study carried out from february to april . we have decided to focus on the clinical audit method, following french health authorities recommendations. a questionnaire has been elaborated and submitted to nurses during semistructured interviews. setting: french cancer centre: nurses from an oncology department and from a haematology department. main outcome measures: data collected were: nurses' profile (age, length of service, competencies' self-assessment), knowledge on drugs prescribed to their patients (usage, administration, side-effects, drug interactions…), use of existing tools (i.e. drugs database) and possible tools to be developed by the pharmacy ward to help them in their daily practice. results: twenty out of twenty six nurses (mean age: , mean length of service: years) consider their medical knowledge as intermediate level. % of pharmaceutical classes are quite well known ( % of the indications are known). only % of drugs' inn are given and more than half of the generic drugs' names are not mastered. administration conditions and conservation are known for respectively % and % of the products. however, side-effects ( %), contraindications ( %) and drug-drug interactions ( %) are not acquired. in their daily routine, nurses face problems mainly related to: drug administration ( %), drug conservation ( %), and dealing with generic drugs and therapeutic equivalence ( %). % of nurses refer to a drug database several times a week when only % more than once a day. pharmacy ward is considered to give information on drugs on a 'regular' basis. three tools have been identified for their potential to help nurses: summarized data on drugs (card format), drugs administration and conservation tables. conclusions: this study has helped to define nurses' difficulties regarding patients' treatment, and their needs for information on drugs. it is also useful for the pharmacy ward to improve its relationships with clinical wards and feedback on treatments. training sessions will shortly be organised to improve the above results. results: the aim of the vita (vienna transdanubia aging) study is the early detection of alzheimer dementia and the discovery of its risk factors. at basic examination, dementia was diagnosed in out of patients ( %) at an age of years. in % of these cases dementia was classified as alzheimer disease. in addition, a clinically relevant depression was diagnosed in % of patients at basic examination, but only % of them were treated accordingly. the first re-examination after . years included those patients, who showed no or only mild signs of cognitive disorders at basic examination. % of these patients developed dementia within the period of . years. the first reexamination also revealed a rapid increase of patients with depression ( % vs. %). the incidence for the development of dementia was % in patients, who have never suffered from depression. however, in patients with the diagnosis depression at basic examination, the risk for dementia was doubled. we aimed to prove whether there is a statistically significant correlation between long-term medication with selected drugs and the development of depression. eleven classes of drugs were investigated, including calcium channel blockers, beta-and alpha-blockers, corticoids, statines, non-steroidal anti-inflammatory drugs, h -blockers, neuroleptic drugs, benzodiazepines, levodopa and opiates. medication was documented from those patients pharm world sci ( ) : - ( male, female) without dementia and depression at basic examination, and without dementia at first re-examination. at first reexamination of them were depressive ( % male, % female), and had no depression (control group). for each class of drug, patients were divided into groups according to gender and duration of medication. a statistically significant (p \ . ) correlation was found between the treatment with benzodiazepines (c months) as well as beta-blockers (c months) and the development of depression in both male and female. conclusions: in elderly long-term therapy with benzodiazepines and beta-blockers can aggravate the development of depression. background and objective: pharmacogenomic studies aim to elucidate the genetic bases for interindividual differences and use such genetic information to predict the efficacy, response rate and safety of a selected drug. to date, the prognostic value of fccr polymorphisms as markers to predict treatment outcome in nhl is still being studied. our goal was to determine whether there is any correlation between fccriia polymorphisms and clinical response to rituximab in patients with nhl. design: in the present study we analysed fccriia polymorphisms in the genomic dna isolated from peripheral blood of patients with nhl who have undergone immunotherapy with rituximab. genotype analysis was based on a polymerase chain reaction (pcr) method followed by a restriction fragment length polymorphism (rflp) study. data were analysed using the computer software spss for windows (version . ) and treatment outcomes of the patients were compared using chi-square or fisher's exact test. a cut-off p-value of . was adopted for all the statistical analysis. survival estimates were calculated using the kaplan-meier method. the curves were examined by the log-rank test. setting: unit of molecular oncology of instituto português de oncologia, porto, portugal. main outcome measures: the response to therapeutics with rituximab was evaluated according to physical examination and computed tomography images. responses were scored according to international working group consensus. overall response rate (orr) was considered as complete response (cr), unconfirmed complete response (cru) and partial response (pr). overall survival (os) duration was defined as the period of time between st treatment with rituximab and either death or the last clinical evaluation of the patient. event-free survival (efs) was defined as the time interval between st treatment with rituximab and the occurrence of an event (recurrence or death) or the time of the last clinical evaluation of the patient. results: the orr for hh genotype was % and for r allele was % (p = . ). however, our results demonstrate that all patients carrying the hh genotype had complete responses to rituximab therapy. complete response rate for hh genotype was % and for r allele was % (p = . ). when comparing the fccriia genotypes, hh genotype or r allele does not have a significant impact on os at -year (p = . ) or on efs at -year (p = . ). conclusions: this study demonstrates that fccriia polymorphism is predictive of complete response to regimens containing rituximab in nhl patients, but is not predictive of overall or event-free survival. based on the current observation, rituximab has in some way an fccriia-dependent mechanism of action which is ameliorated in patients with hh genotype. we hypothesize that hh genotype increases affinity of fccriia receptor not only for naturally occurring igg , via antibody-dependent cellular cytotoxicity but also ameliorate connection with chimeric igg rituximab. keywords: non-hodgkin lymphoma, rituximab, pharmacogenomics pt- platinum salts, cancer and renal insufficiency. sub-group analysis of the irma study xavier pourrat , nicolas janus , stéphane oudard , isabelle ray-coquard , jean-philippe spano , jospeh gligorov , jean-françois morere , philippe beuzeboc , gilbert deray , vincent launay-vacher pharmacy, hôpital trousseau, tours, nephrology, gh pitié-salpêtrière, medical oncology, hôpital européen georges pompidou, paris, medical oncology, centre léon bérard, lyon, medical oncology, gh pitié-salpêtrière, medical oncology, hôpital tenon, paris, medical oncology, hôpital avicenne, bobigny, medical oncology, institut curie, paris, france background and objective: the irma study reported the high prevalence of renal insufficiency (ri) in solid tumour patients: mean age . , mean weight . kg ( . % between and kg), glomerular filtration rate (gfr) \ ml/min for - % [ ] . we present the results for irma patients who received a platinum salt (ps) as part of their chemotherapy. design: data were retrospectively collected for in and outpatients with cancer presenting over two periods in (february st- th and october st- th). setting: anticancer centers in france. main outcome measures: subgroup analysis of irma patients who received ps. data collected: sex, age, weight, serum creatinine (scr), type of tumor and anticancer drugs. the prevalence of scr [ lmol/l was assessed. gfr was estimated with cockcroft-gault (cg) [ ] and amdrd [ ] formulae. chi-square test was used to compare the prevalence of ri between patients who received ps and patients who did not. results: patients were included: mean age . and weight kg, men. the prevalence of scr [ lmol/l was . %. gfr \ ml/min was . % with cg and % with amdrd. the prevalence of ri was significantly higher in patients who received ps as compared to patients who did not receive ps (p = . ). there were prescriptions: . % carboplatin, . % cisplatin and . % oxaliplatin. . % of patients received carboplatin or cisplatin, the two drugs of this class needing dosage adjustment and being nephrotoxic. conclusions: ri is highly frequent in cancer patients receiving ps. appropriate evaluation of renal function necessitates cg or amdrd calculation. in addition, two third of those patients with pre-existing ri are at risk for iatrogenic acute renal failure still receive nephrotoxic ps. consequently, appropriate methods for the nephrotoxicity prevention of those drugs should be used as recommended for cisplatin by the escp special interest group on cancer care [ ] . [ ] prevalence of renal insufficiency in cancer patients and implications for anticancer drugs management: the irma study. cancer (in press). [ ] prediction of creatinine clearance from serum creatinine. nephron, ; : - . [ ] a simplified equation to predict glomerular filtration rate from serum creatinine [abstract] . j am soc nephrol ; : - . pharmacy, hospital la candelaria, tenerife, spain background and objective: to describe an quantify the pharmaceutical interventions in patients on total parenteral nutrition (tpn)and the drug related problems (drp)in patients on this type of nutritional support. to know the acceptance degree of the interventions and its relevance on patients' care and quality of life. design: prospective longitudinal study for four months (from january to april ). all patients on tpn were included. the registered data were: patients number, hospital departments, type of interventions and modifications on the tpn. setting: the pharmaceutical interventions were classified in: indication, effectiveness, safety and adherence according to the cipolle and cols methodology in order to identify de drp related to the tpn and/ or to the drugs. main outcome measures: all interventions were recorded both in the patient medical record and in a excel database in the pharmacy department. results: patients were evaluated and interventions were recorded. that means an average of interventions per patient and a duration average per nutrition of . ± . days. the drp were: indication . %, safety . %, effectiveness % and adherence . %, being the drp the most representative. the drp were listed in: nutritional assessment ( . %), monitoring ( . %) and individualized tpn ( . %). a total of patients ( . %) was favoured through some type of pharmaceutical intervention, being the most implicated hospital departments the neonatology and digestive surgery departments. a % of the interventions were focus on monitoring and optimization of nutritional support and % on drugs (diuretics, insulin, digoxine, enalaprile, and propofol). the acceptance degree of the interventions was %. conclusions: the individual monitoring of the patients with tpn represents an improvement of their clinical outcome and a lower incidence of drp. therefore, with this method we contribute to a lower hospital stay and it also may prevent the appearance of new adverse effects. main outcome measures: the number of interventions carried out and accepted, items concerned: regulation problems, nitrogenous and calorie intake, electrolytic intake, prescription omissions and eventually other fields. results: altogether, parenteral nutrition prescriptions were analyzed for one month in five pediatric units. the pharmaceutical analysis, which consumes hours a day for pharmacists, generated interventions for prescribers: % concerned electrolytic intake, more than half of which concerned potassium and sodium, the main dangerous electrolytes; % were prescriptions omissions; % about nitrogenous and calorie intake; % about other fields (weight error, incompatibility of lipids with divalent ions). of these interventions, which concern exactly prescriptions, were accepted by prescribers, that is %, leading to prescription modifications. conclusions: putting in place a systematic pharmaceutical analysis of parenteral nutrition prescriptions has ensured the detection and the correction of prescription errors. these errors concern mainly non adjustment of electrolytes to the biological results of the child. pharmaceutical interventions are important for safety of the patient and represent a privileged way to communicate with prescribers and their acceptance is, on the whole, satisfactory. background and objective: the number of elderly intensively increases and the fact that they consume a great amount of pom and otc drugs makes them a significant group of patients that need pharmacy care. unfortunately pharmacists often find their interaction with elderly clients very difficult and determined by many factors such as the sensory and physical limitations that accompany the aging process. to test the readiness of the elderly patients to communicate with the pharmacist, to assess the barriers that hinder the proper communication process and to provide a communication skills training in order to be improved the communication process. design: an experimental design involving two stages -assessment and education. setting: setting: the elderly patient center (hospice) and private community pharmacies both situated in the city of sofia, bulgaria. participants: patients aged + ( community pharmacy patients and patients from the elderly center). main outcome measures: an initial interview with the patients and questionnaire with the selected pharmacists to assess the level of communication and to clarify the hinders. communication skills training leaflets provided to the pharmacists. test of the newly received skills. final interview with the patients to be assessed the level of their satisfaction. results: the trained pharmacists that have passed the education process are more facilitated in providing pharmaceutical care that leads to the elderly patients' satisfaction (about %). additionally, the elderly patients obtained significantly more information from their pharmacists that leads to better care and avoidance of nearly half of the drug-related problems (drps) for this age. conclusions: pharmacist communication skills' training appears to be an effective means of enhancing the communication process in the pharmacy. background and objective: as the hospital has no pharmacists working in the multidisciplinary teams on the wards, we wanted to introduce and evaluate a clinical pharmacy service. design: a month prospective pilot study with three aims: . identify drug related problems (drps) (data collecting period of eight months). . design drug related information sheets and teach nurses. . evaluate the service by questionnaire. suggest cost-effective measures. setting: paediatric ward with beds, national university hospital. main outcome measures: the acceptance rate of the drps identified and suggested by the pharmacist, the number of drug information sheets introduced and lectures given to nurses. physicians' and nurses' views on the service. results: the pharmacist identified drps in ( %) of the charts that was screened. immediate action was taken in ( %) of the cases, the physician considered ( %) of the suggestion rational but no immediate action was taken due to various reasons, and ( %) of the suggestions were not approved by the physician. the most commented drp was ''dosage'' ( %), which included too low or too high dose, non-optimal administration time or inappropriate formulation. the pharmacist designed six drug information sheets and gave five lectures. cost-effective measures were suggested for drug handling and specific drugs. seven out of eight physicians and all nurses (n = ) considered the pharmacist a natural participant in the multidisciplinary team. conclusions: quality assurance of drug treatment may be performed by a clinical pharmacist, not only by the traditional way of identifying drps, but also by designing drug information sheets and teaching. the clinical pharmacist is also capable of suggesting cost-effective measures. physicians and nurses considered the clinical pharmacist a natural participant in the multidisciplinary team. as a result of this project, the clinical service will continue and also be introduced to one of the other paediatric wards. keywords: drp, paediatrics, quality assurance pc- iatrogeny and drug dispensations for outpatients: implication of a hospital pharmacy emilie degris , isabelle peyranne , anne laure sarda , nadine malric , brigitte bellon pharmacie, hôpital paule de viguier, chu toulouse, toulouse cedex , france background and objective: in france, some drugs are not available in community and outpatients have to go to hospital to obtain their treatment. our objective was to assess the role of the pharmacist in prevention of iatrogeny when dispensing drugs, in particular medication errors at high risk for the patient. design: a month retrospective study, from december to may setting: pharmacy of paule de viguier, teaching hospital of toulouse, france main outcome measures: each error encountered was recorded and analysed. first, we determined the number of errors avoided and the number of errors effective (divided into groups: non avoided and created by pharmacy). then, we quantified the frequency ( = once, = from twice to ten times, = more than ten times) and the severity ( = no risk, = weak, = moderate, = high) of each error. the multiplication of those two parameters gave us the level of the risk of error for the patient ( = no risk, to = weak risk, and = moderate risk, = high risk). finally, for each type of error we noted the actors. results: we made dispensations during the period of the study. we recorded errors ( . %): ( for dispensations) were avoided by the evaluation of the pharmacist, were not avoided ( for dispensations) and were created ( for dispensations). among the avoided errors, ( . for ) were at high risk ( ), ( for ) at moderate risk ( or ), ( . for ) at weak risk ( or ). the actor of of them was the prescriber (mainly lack of information on the prescription like no dosage). among the effective errors, ( . for ) were at moderate risk ( or ), ( . for ) at weak risk ( to ), ( . for ) had no risk ( ). the actor of of them was the pharmacy. conclusions: the errors for the activity ''retrocession'' are not numerous. the majority of them are stopped by the evaluation of the pharmacist, in particular those at high risk for the patient. we implemented curative and preventive measures to decrease the number of errors made both by prescribers and pharmacy. background and objective: despite improved treatments and guidelines, asthma control remains suboptimal. in a recent observational study, we described the asthma control test Ò (act) as an easy tool to measure asthma control of patients presenting at community pharmacies ( ) . the present randomised controlled trial was set up to study the hypothesis that a pharmacist intervention, focused on optimal use of asthma medication and tailor-made to the patient's current asthma control, would result in improved asthma control in adult patients. design: a -month randomised controlled trial in asthma patients: patients in the control group (c) and patients in the intervention group (i). patients in the control group received usual care. patients in the intervention group received a protocol defined pharmacist intervention, mainly focusing on inhaler technique and adherence to controller medication. setting: randomly selected community pharmacies in flanders (the dutch speaking part of belgium). main outcome measures: primary outcome was the level of asthma control, as measured by the asthma control test Ò . secondary outcomes included rescue medication use, night-time awakenings due to asthma, patients' peak expiratory flow, inhalation technique, adherence to controller medication, quality of life, knowledge on asthma and smoking behaviour. results: mean act scores did not change from baseline for both study groups (act at baseline for c: . , i: . -act at months for c: . , i: . ). however, a predefined subgroup analysis of patients having insufficiently controlled asthma at baseline showed that the intervention significantly increased act scores during the course of the study compared with usual care (p = . ). the intervention also significantly reduced reliever medication use (p = . ) and the frequency of night-time awakenings due to asthma (p = . ). inhalation technique (p = . ) and adherence to controller medication (p = . ) were significantly better in the intervention group. these findings suggest that the more effective use of asthma medication is responsible for the improvements in symptom control. there were no differences between control and intervention group in peak expiratory flow, quality of life, knowledge on asthma and smoking behaviour. conclusions: a pharmacist intervention can significantly improve outcomes for asthma patients (clinicaltrials.gov number nct ). background and objective: patients admitted to surgery departments receive multiple drugs before, during and after surgical procedures. drug-related problems (drp) are the most common cause of injury to hospitalized patients. in pharmacotherapeutic follow-up (ptf) a pharmacist is responsible for drug-related patient needs by detecting, preventing and solving drug-related problems (drp) aiming at specific results to improve patient quality of life. drp are pharmacotherapy negative outcomes leading to failed therapeutic goals or undesirable events. when a drp appears, it affects not only older hip fracture patient health, but also the effectiveness of hospital health care. the general objective of this study was to demonstrate that ptf improves the hip fracture assistential process quality, comparing some quality indicators of this process between patients in study group (sg) and control group (cg). design: cuasi-experimental study with control group. ptf was the intervention. setting: two traumatology wards in a large teaching hospital, ''hospital san cecilio'', granada, spain. the period of study was from january to july (sg) and the same period but in (cg). main outcome measures: incidence and types of drp; drp solved in sg; differences in lengh of stay, six-months mortality and threemonths readmissions between study and control groups. results: the incidence of drp was % in sg (n = ) and . % in cg (n = ). in sg, more than % of drp were resolved. in sg and cg the % and % of drp were related to medication need, % and % to effectiveness, and % and % to safety, respectively. mean length of stay was days in sg and . in cg. in general, patients with drp had a significative longer length of stay ( . d) than those without drp ( d); but in sg, patients in which drp were solved had the same length of stay than those without drp. sixmonths mortality was . % in sg and . % in cg, and readmissions was . % and . % respectively. . % (n = ) of patients reported an aspirin allergy that triggers their asthma attacks. . % (n = ) had never used aspirin before and did not know whether they had any sensitivity to aspirin. . % (n = ) of subjects could correctly describe and distinguish between preventor and releiver drugs. . % (n = ) confused the terms, while . % (n = ) had no idea about these terms. % (n = ) of asthmatics had received previous inhaler usage education from a specialist (doctor, nurse or pharmacist). of these patients had ineffective inhalator usage although they had ostensibly received education. the inhaler technique of the remaining who had been previously educated was accepted as successful. % (n = ) of the patients had never received inhaler usage education before a specialist. of these patients demonstrated successful technique but of failed. conclusions: the results of this pilot study indicate that some asthmatics are ignorant of their condition. in addition most of them seem to have no comprehension of the concepts of preventor or reliever therapy. despite prior education about half (n = ) were unable to demonstrate successful technique. furthermore cigarette smoking may be a detremental factor to the lives of asthmatic patients. this results of this study suggest the potential benefit of an innovative pharmacist led patient education service among asthmatic patients in turkey. background and objective: according to a recent meta-analysis, drug-related morbidity leads to . % of preventable hospital admissions causing enormous expenditures. in austria, there are only data on the incidence of adverse drug reactions (adrs) of psychiatric drugs. clinical pharmacy is not widely practised at hospital ward level. with this study, we aim to evaluate and document adrs leading to or occurring during hospital admissions. to improve the co-operation of doctors and pharmacists in an austrian hospital, to enhance doctors sensitivity in detecting drug-related morbidity, to increase patient safety and lower costs by reducing hospital admissions. design: two study nurses especially instructed about typical symptoms of adrs identify and document these cases prospectively in cooperation with doctors on selected internal wards for a period of three months. these cases are evaluated by a clinical pharmacist by means of a computer tool and data-base specialised on detecting causality and severity of adrs. results and outcomes form the basis for structured feedback to doctors. setting: university teaching hospital. main outcome measures: quantity and quality of adrs connected with hospital admission. results: during the first six weeks, patients were screened. sixty three adrs ( female) were identified ( . % of admissions). more than % of adrs occurred in patients more than years old. reasons: polypharmacy (mean number of drugs on admission . ) and reduced renal function (mean creatinine clearance . ml/min). diuretics, oral anticoagulants, nsaids, digoxin and antibiotics were most frequently associated with drug-related problems. water-electrolyte imbalance, overantigoagulation with or without bleeding, gastrointestinal problems and bradycardia are some of the most common problems. results concerning the severity of adrs will be available in september . conclusions: adrs are frequent in austria. incidences are comparable to numbers given in the literature. mainly older patients are affected. the impact on clinical practice is yet unknown. background and objective: adherence to cardiovascular treatment, particularly in the first year, is low and can result in serious complications. depression is associated with a fold increased risk of nonadherence with medical treatment. therefore, our aim was to investigate whether illness and treatment perceptions were associated to depressive symptoms in patients starting treatment for cardiovascular diseases. design: cross-sectional study with mailed questionnaire. setting: patients, who were dispensed at least a first prescription for a cardiovascular disease (anti-thrombotics excluded), were selected from pharmacies in the netherlands. main outcome measures: the questionnaire comprised the illness perception questionnaire-brief (ipq-b), beliefs about medicines questionnaire (bmq), the medication adherence report scale (mars) and the centre for epidemiological studies depression scale (ces-d). descriptive statistics and associations between depressive symptoms and the other study variables were assessed by bivariate correlations. results: sixty two ( . %) of eligible patients returned our questionnaire. the mean age was . yr ± . (range - ) and . % was female. patients reported to have hypertension ( . %), cardiac arrhythmia ( . %) and hypercholestereamia ( . %). the mean score on ces-d was . ± . and median self-reported adherence (mars) was . reports of depressive symptoms increased with emotional response (ipq-b emotional response, r = . ), the perceived consequences (ipq-b consequences, r = . ) and increased experience of symptoms (ipq-b identity, r = . ) attributed to their cardiovascular disease. depressive symptoms correlated with concerns about medication (bmq, r = . ), but not with self-reported adherence. adherence was relatively high, as . % of the sample had the maximum mars score of . conclusions: in patients who started cardiovascular treatment, perceptions about cardiovascular disease and concerns about medication are associated with report of depressive symptoms. depressive symptoms did not correlate with self-reported adherence. the majority of patients reported excellent medication taking behaviour, which might reflect their awareness of the importance of adherence or reluctance to report deviant behaviour rather than their actual behaviour. further research is needed to clarify this finding. results: fifteen nurses completed the questionnaire. % of the nurses were aware of the purpose of controlled release formulations. pharmaceutical codes added to brand names such as uno, zok, la and ocas related to prolonged activity were not recognised in % of cases. in contrast, retard and cr were linked to slow release by % of the responders. the purpose of enteric coated (ec) drugs was only known by %. in general, the nursing staff did not pay a lot of attention towards the prevention of drug-nutrient and/or drug-tube interactions. the recommended time interval between administration of enteral feeding and drugs was not respected. % of the responders would crush drugs together (in the same mortar) when multiple drugs are prescribed. based on the results of the survey, an intervention plan has been developed. this consisted of information rounds, a poster related to the topic and implementation of the use of a website dedicated to crushing medication developed by the flemish association of hospital pharmacists. background and objective: the detection and solution of drugrelated problems is an important activity within pharmaceutical care. this study focused on drug-related problems (drps) detected during dispensing of new prescriptions in community pharmacies and aimed to explore frequency as well as nature and the pharmacist's management of them. design: during their pharmacy internships fifth-year pharmacy students collected consecutively hospital discharge and primary care prescriptions. after training, they documented drps and interventions on an adapted pcne classification form. inclusion criteria were: age over , at least one new medication, at least prescribed drugs. setting: swiss community pharmacies affording the opportunity of internships for fifth year pharmacy students. main outcome measures: prevalence, nature and management of drps in community pharmacies assessed with an adapted pcne classification form. results: the patient's median age was years (iqr ) and they received a median of (iqr ; range - ) different drugs. prescriptions of patients ( ( . %) discharged from hospital) were analysed. in ( . %) of all prescriptions at least one drp was detected. the most frequent drps were potential interactions ( . %), wrong/improper application or time of drug intake ( . %), inappropriate drug ( . %) or inappropriate drug form for indication ( . %), no clear indication for drug use ( . %) and too high or too low dosage ( . %). these drps led to a total of interventions (multiple answers): patient counselling ( ); request of information from prescriber ( ); change of drug ( ; there from after consultation with physician), drug form ( ), dosage ( ), instruction for application ( ) or deliverable drug amount ( ); drug stopped ( ); start with new drug ( ); referral to a physician ( ); others ( ). out of all interventions . % could be managed by the pharmacist without any contact to the prescriber. there were no differences between hospital discharge and primary care prescriptions. conclusions: in the delivery process of new prescribed drugs drps are frequently observed prompting many interventions. most drps can be managed by the pharmacy. further studies are needed to analyse relevance of the problems and impact of according interventions. the main selection criteria were clinical relevancy (patient centred initiatives) reproducibility of clinical and economical outcome, outcome indicators and multidisciplinary approach. an approval by the hospital board and medical council must underline the willingness to integrate the clinical pharmacy in the patient care team. results: projects has submitted (on a total of hospitals). a total number of hospitals were selected to receive funding for clinical pharmacy activities. projects were quoted for a full time equivalent and projects for a half time clinical pharmacist. the projects described different fields or a combination of different aspects of pharmaceutical care like e.g. the transfer of information on medication use on admission and discharge conclusions: the funding of the belgian health authorities triggered a very high response rate, which proves the increasing attitude from the belgian hospitals to the positive impact of clinical pharmacy. the funding was complementary to other national projects to improve overall safety of medical treatment. also, many hospital administrators took the opportunity to enhance more economical and rational use of drugs. financial support by the belgian authorities of clinical pharmacy and the results of the projects could trigger a further integration of the hospital pharmacies into a patient care team. background and objective: the task of assisting patients in selfmedication practice is an important component of pharmaceutical care in spain. in order to provide appropriate self-medication counselling pharmacist should be able to distinguish between a minor ailment and one that it is not, and should, consequently, refer patients as necessary to gps. nevertheless, there are no criteria for referral to gp in spain. the objectives were:( )to identify the most relevant minor ailments, agreeing on the specific criteria for referral to the gp.( )to select the non-prescription drugs, with evidence of safety and effectiveness, for the treatment of the identified minor ailments design: qualitative study with an expert panel which was made up of primary care physician from semfyc and six community pharmacists (two members of sefac and four members of giaf-ugr). the expert panel held two meetings, of five hours each. it was established which minor ailments were considered most relevant within the framework of community pharmacy in spain. subsequently, the expert panel, reach an agreement on the general content that should be included in the protocols for the management of each selected minor ailment. finally, a working team composed of gps and three community pharmacists prepared the protocols, which were compiled into a guide for self-medication counselling. setting: university of granada, spain during . main outcome measures: identified minor ailments, content of the protocols for each minor ailment, non prescription drugs selected. results: it was selected minor ailments, allocated as follows; respiratory (rhinitis, cough, cold, flu), pain (period pains, sore throat, headache, backache, toothache), gastrointestinal (heartburn, diarrhea, constipation, vomiting, hemorrhoids), skin and mucous membrane (aphthae, acne vulgaris, cutaneous wounds, burns, stings, urticaria, herpes labialis, eczema lesions) and others (vaginitis, varicose veins, fever, conjunctivitis, insomnia). the following sections were specified in each protocol: banal and serious reasons or conditions that can lead to the symptom (including drugs); referral criteria according to the duration of the symptom and associated signs; drug treatment and non-pharmacologic therapies. it was selected a total of different non prescription drugs. conclusions: a total of minor ailments were identified as the most frequently demanded in community pharmacies in spain. referral criteria were based mainly in the duration of the symptom and other associated symptoms that are indicative of illness. for the treatment of these minor ailments, different non prescription drugs were selected. keywords: non-prescription drugs, minor ailment, community pharmacy services mareike kunkel , matthias ganso , irene kraemer pharmacy, johannes-gutenberg-university hospital, mainz, germany background and objective: in clinical pharmacy service was implemented in three surgical clinics (inclusive icu). drug related problems (drp) were identified by medication review and discussed with the physicians. from january to june all pharmaceutical interventions (pi) from pharmacists ( fte) were recorded (paper based) and classified according to drp (with the pi-doc Ò -system, which was modified to comply the requirements for hospital use ), intervention type, outcome and clinical relevance. the pis were documented and evaluated with an access Ò database. design: retrospective study of pis in surgical patients, identification of drp by medication review. setting: departments of neurosurgery, accident surgery and general/abdominal surgery ( , and beds, respectively), university hospital. main outcome measures: drp, intervention type, outcome, clinical relevance, drugs and admission diagnoses being at risk for drps. results: within six month patients were admitted. drps were identified in % (n = ) of the patients. patients with drps were older (mean = y sd ± vs. y sd ± ) and had an increased length of stay (mean = d sd ± vs. d sd ± ). pis were made. the acceptance by the physicians was . %. pis were classified to the outcome subcategory patient safety and clinical relevance was estimated as major (n = ) or moderate (n = ) by pharmacists. further data are based on these pis. the most often addressed drps categories were overdose ( %), no or insufficient drug monitoring, when necessary ( %), untreated indication ( %) and increased risk of an adverse drug reaction ( %). the type of recommended intervention varied: change dose/time of application ( %), stop drug ( %) and conduct drug monitoring ( %). drps related with the outcome patient safety and at least moderate clinical relevance were caused by drugs ( %). the most affected drugs were vancomycin ( %), diclofenac ( %), potassium ( %), acetaminophen ( %), digitoxin ( %), phenytoin ( %) and theophylline ( %). the incidence of the most frequently admission diagnoses of patients with relevant drp differed from the incidence of diagnoses of all admitted patients (incidence icd- (cost-)effectiveness has started to emerge ( ) . a literature review was carried out that a) summarized the findings of pharmaco-economic studies; b) evaluated the methodology employed by studies; and c) suggested how future research has to be designed to meet the requirements of a pharmaco-economic analysis. design: studies to be included are identified by searching electronic databases. due to limited relevance of older studies, the scope is limited to studies published between and . mainly three techniques can be used to conduct an economic evaluation: costeffectiveness analysis, cost-utility analysis and cost-benefit analysis. ( ) all studies are reviewed regarding results and methodological quality. nineteen out of studies met our eligible criteria. setting: clinical pharmacy services provided in a hospital setting. main outcome measures: results were analyzed in terms of number of preventable adverse drug events (ade), length of stay (los) and financial savings. methodological quality was assessed with respect to perspective, scope and measurement of costs and consequences, sources of data on costs and consequences, and application of an incremental analysis. results: a) nearly all studies conclude a financial benefit based on direct cost saving and estimated cost avoidance as a measure of prevented ade or shortened los. b) methodologically there are a number of shortcomings: e.g. not including the wage of the personnel, lack of control groups, use of expert panels to estimate savings and costs, possible selection bias, no valorization of health effects. c) a methodology for conducting a prospective economic evaluation of an observational study is proposed. conclusions: it is not obvious to calculate the net savings of a clinical pharmacy program or to compare different programs because there are no common guidelines for this type of assessment. the ideal protocol is hard to achieve, so best practice will be more realistic. addition of direct cost saving, labor cost and economic value of prevented ade and shorter los results in a lucrative service. these savings are higher for specific inverventions (like preventing ade, switch therapy) or disciplines (e.g. intensive care unit versus geriatrics background and objective: the contribution of pharmacists to the delivery of public health in scotland is recognised in national pol-icy , . the new community pharmacy contract with its emphasis on public health will provide a new framework in which the contribution of community pharmacy to improving health in scotland can be delivered. the objective was to define the core public health competencies applicable to community pharmacy practise, using the 'skills for health public health practice competency framework' . design: a web based delphi methodology was used to achieve consensus on which competencies, from the 'skills for health public health practice competency framework', should be met or aspired to by practising community pharmacists using a multidisciplinary group of expert stakeholders. two rounds took place. setting: primary and secondary healthcare and academia. main outcome measures: panel members rated their extent of agreement/disagreement that each community pharmacist should achieve or be striving to achieve that particular competency. consensus was defined as c % rating a competency as strongly agree/ agree. results: ten organisations ( % of those invited) and organisation members ( % of those invited) agreed to participate. responses were received from ( %) individuals in round and ( %) in round . consensus was achieved for / ( %) competencies in round and a further / ( %) in round . competencies achieving consensus predominantly focused on health improvement activities at individual and local community levels and ethical management of self, rather than those relating to surveillance and assessment, strategic leadership or research and development. conclusions: this research has identified that many of the competencies in the 'skills for health' document can be applied to community pharmacy. research has since been carried out, using focus group and questionnaire methodology, to investigate the views of practising community pharmacists. background and objective: in spain, off-label drug utilization (nonapproved indications, patient population, doses, administration route, association), must be derived to compassionate use, which requires a prior national health authorities (nha) approval and a monitoring plan and follow up information provided to them. request to nha includes circumstances of case and patient protection measures, including: physician assessment, informed consent and institutional clearance. the objective of this study is to analyse the strength of recommendation, strength of evidence and clinical efficacy of drugs prescribed outside the terms of product licence (off-label) in paediatric patients of our hospital. design: literature review to evaluate the evidence level: micromedex healthcare series, cochrane library, pub-med, embase, expert opinion or consensus. sample: % off-label drugs used in at least paediatric patients (prior spanish nha treatment approval required for every patient). years, retrospective observational study ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . setting: paediatric hospital ( beds) and pharmacy department ( pharmacist) in a large general teaching hospital ( beds, pharmacists). main outcome measures: categorisation of evidence-based medicine according to thomson ratings of recommendation (class i-iii), evidence (category a-c) and efficacy (class i-iii). results: paediatric patients ( % total patients: adults and paediatrics) used off-label drugs ( % total drugs: adults and paediatrics). out of this off-label drugs, % ( / ) only approved for adults, % ( / ) outside of license in terms of indication for adults and paediatric patients and % ( / ) both causes. off-label drugs for indications were used in at least paediatric patients: % anti-infective agents, % haematopoietic growth factors, % cytokines, % hormone therapy, % antiarrhythmics, % other therapeutic groups. the categorisation according to evidence-based medicine was: a) strength of recommendation class: % i, % iia; % iib, % iii and % indeterminant; b) strength of evidence category: % a, % b, % c and % no evidence; c) clinical efficacy class: % i, % iia, % iib and % indeterminant. conclusions: a higher proportion of off-label prescriptions is observed among paediatric patients, most of them related to nonapproved indications in this population. there is a broad range of therapeutic groups involved. the evidence of most off-label therapies are based on meta-analyses of randomized controlled trials with conflicting conclusions, small numbers of patients or significant methodological flaws or nonrandomized studies and, although the weight of evidence favors efficacy of the treatment for a specific indication, the therapy may be useful and indicated in some, but not most, cases. keywords: medicine-based evidence, off-label, paediatrics background and objective: there is an increasing demand towards the involvement of the community pharmacists in health promotion. it has been reported that community pharmacists have a successful role in providing services, which help to improve and promote health with regard to smoking cessation, coronary heart disease, skin cancer prevention, drug misuse, sexual health, immunization, mental health, diabetes, nutrition and physical activity [ ] . the aims of this study were to describe the current practice of community pharmacists with regard to their provision of health promotion activities, identify their willingness to participate in health promotion and identify the barriers that may limit their participation. design: a descriptive cross sectional study, which included community pharmacies that selected via stratified and systematic random sampling. data were collected via face-to-face structured interview of the respondents using a pre-tested questionnaire. setting: community pharmacies in khartoum state. main outcome measures: the extent of the pharmacists' involvement in counselling patients about health promotion topics, their preparation to counsel patients in health promotion topics, and their success in changing the patients' health behaviour. results: the response rate was . %. seventy five ( . %) of the study participants were strongly involved in counselling patients on health promotion related to medications, but less involved in counseling them on the other personal health behaviours such as tobacco use ( . %), alcohol use ( . %) and exercise habits ( . %). seventy two ( . %) of the respondents perceived themselves as very prepared to counsel patients on taking drugs and less very prepared to counsel them on other personal health behaviours. fifty two ( . %) claimed a high level of success in helping patients to change their behaviour with regard to medications, but not in relation to other personal health behaviours. ninety eight percent of respondents indicated their willingness to participate in continuing education programs to gain more knowledge and skills about health education and promotion. the main barriers facing the community pharmacists' participation in health promotion as perceived by respondents were lack of information and/or training ( . %) and lack of pharmacists' time ( . %). conclusions: community pharmacists reported to achieve considerable success in helping patients to change their behaviours in relation to medications, but were less successful of their ability to change personal health behaviours. the majority of the respondents have the interest and willingness to be a prime source of advice and support on health promotion. results: during the first study period, inpatients were exposed to major or moderate pddis. ( %) of these pddis were judged clinically relevant by the pharmacist. recommendations including pddi information, and simply information leaflets were handed out to the physicians. % ( of ) of the recommendations were accepted. at hospital discharge, in % ( of reviewed instances, which were accepted) the drug changes due to the recommendations were implemented. during the second study period, patients at hospital discharge were exposed to major and moderate pddis. ( %) pddis were assessed as clinically relevant by the pharmacist. recommendations including pddi information, and simply information leaflets were sent to the physicians. % ( of ) of the recommendations were accepted. one year after hospital discharge, of drug changes due to recommendations were still existent. overall, in % and %, respectively, of all major and moderate pddis detected by pharmavista, clinical management was adapted accordingly. conclusions: the management of clinically relevant pddis can be improved by physicians' advice of clinical pharmacists. changes in medication due to pddis were found to persist up to one year after hospital discharge. background and objective: current treatment options for metastatic renal cell carcinoma (mrcc) are limited and there is a need to identify novel and effective therapies. sunitinib is an oral multitargeted tyrosine kinasa inhibitor, which has shown activity in cytokinerefractory metastatic rcc patients. this agent inhibits vascular endothelial grown factor receptor and platelet derived growth factor receptor. the purpose of this study is to analyse the efficacy and safety profile of this agent in patients with mrcc. design: retrospective assessment in seven patients treated with sunitinib as second-line treatment in mrcc. data were obtained from clinical histories and informatic records from the oncology pharmacy department. setting: oncology and pharmacy department. la paz university hospital. madrid. spain. main outcome measures: assessment of clinical response and adverse events. results: seven patients were evaluated ( men, women), median age was ( - ). six of them presented bone, lung, brain or liver metastases, all patients were treated with vinblastine and ifn-alpha as first-line therapy. patients received sunitinib at a starting dose of mg per day in repeated -week cycles for consecutive weeks followed by weeks off treatment. they started therapy with sunitinib because of progressive disease in patients and adverse events in patients on previous therapy. sunitinib was discontinued in four of them, causes were: adverse events ( patient), volunteered dropout ( patient) and progressive disease ( patients). the median progression-free survival was . months. the median number cycles received was six and of the patients are still in treatment at the time of data analysis. dosage was reduced mg daily because of unacceptable toxicity: hand-foot syndrome( patient) and hypothyroidism ( patient). the most common adverse events experienced were: asthenia ( patients), diarrhea ( patients), damaged nails( patients), insomnia ( patients), dermatitis ( patients)and dehydration ( patients). conclusions: in our experience, sunitinib has demonstrated an acceptable efficacy and safety profile as a single agent in second-line therapy for patients with mrcc. (ii) validated questionnaire to guide discussion; (iii) fostering group interaction to generate data; (iv) post-interview analysis of verbatim transcripts with specialized software (qsr nvivo . for windows Ò ), based on the grounded theory approach (classification of emerging themes). setting: groups: prescribing physicians ( ), nurses ( ) , and laboratory technicians ( ) , all involved in antibiotic tdm as performed in orthopaedic surgery, general surgery, neurosurgery, vascular surgery, haematology, and pulmonary wards in a beds teaching hospital. main outcome measures: (i) issues causing poor antibiotic overall tdm performance, (ii) approaches for optimizing tdm performance supported by group consensus results: key identified issues: (i) nursing work overload; (ii) insufficient education to pharmacokinetics and lack of specific training; (iii) insufficient information communication and lack of coordination and involvement of all stakeholders; (iv) conflicting guidelines; (v) lack of perception of positive benefit/risk ratio. approaches for optimization (consensus): (i) continuous education of all stakeholders; (ii) daily multidisciplinary collaboration with infectious disease physicians and clinical pharmacists; (iii) simplification and uniformization of guidelines and procedures; (iv) implementation of a simpler administration scheme (v) increased staffing. conclusions: correct performance of tdm and its implementation in routine clinical care needs to be critically assessed and appears to be mainly dependent on non laboratory-related parameters. background and objective: new data published at the end of and the beginings of the , suggest not to exceed a haemoglobin level of g/dl to avoid cardiovascular morbility-mortality in patients with anaemia and chronic kidney disease (ckd) treated with recombinant human erythropoietin (rhuepo). before these evidences the optimal target haemoglobin levels was greater than g/dl. our aim is to evalue if these new published evidences have changed the clinical practice in pre-dialysis ckd patients. design: retrospective observational study. all the pre-dialysis patients who received rhuepo were including. in order to evaluate the possible changes in clinical practice, we measured the levels of haemoglobin prior to the publication of evidences (march-may of : group ) and after the publication of these evidences (march-may of : group ). we made a descriptive analysis of independent data. setting: department of hospital pharmacy. main outcome measures: the main outcome measures were: age, sex, mean glomerular filtration rate (gfr), mean haemoglobin level, mean haematocrit and type of rhuepo used. results: we studied patients ( in group and in group ). patients age ranging between to years (median = years). the proportion of women was . %. mean gfr for both years located around ml/min and the most frequent stages of renal injury were and . the most rhuepo used was darbepoetin alfa ( . % of patients). mean haemoglobin level for group was . g/dl (sd = . ) and . g/dl (sd = . ) for group . mean haematocrit was . % (sd = . ) and . % (sd = . ) for group and , respectively. conclusions: our nephrologist are cautious about of prescribing rhuepo, not only after the publication of the new scientific evidences on this subject, but before this too. it s worth questioning if clinical practice in ours hospital is different from the published evidences. background and objective: tacrolimus (tac)-based immunosuppression is effective in adult renal transplant patients with acute or chronic rejection or cyclosporin (cya)-related toxicity. the conversion from cya to tac resulted in improved cardiovascular risk profile and increased prevalence of post-transplant diabetes mellitus (ptdm)compared with treatment with cya. the aim of this study was to review clinical documents for renal transplant patients and assess patients' outcomes. design: a retrospective review of clinical data. excluded from the study were patients converted to tac less than months posttransplantation. statistical analysis (one sample paired -tailed t test) was performed using microsoft office excel . the graft survival was analysed with kaplan-maier survival curve using xl stat software. the study was approved by the northern ethics committee, auckland, new zealand. setting: tertiary care setting. main outcome measures: mean serum creatinine, incidence of ptdm, mean total cholesterol, hdl cholesterol, ldl cholesterol, total/hdl cholesterol ratio, mean blood pressure and antihypertensive scores, graft survival censored for death. results: forty-four patients were converted to tac more than months post-transplantation from to june . mean serum creatinine (scr) increased in the months prior to conversion from lmol/l ( % ci - ) to lmol/l ( % ci - ) at months post conversion to tac (p-value = . ). thirty-four patients were taking cya for more than months. the mean scr increased from lmol/l ( %ci - ) at the months prior to conversion to lmol/l ( %ci - ) at months post conversion (p-value . ). if scr for seven patients who had an acute rejection episode were excluded, the mean scr did not show any change in slope after conversion and showed a tendency to gradually increase from lmol/l ( %ci - ) to lmol/l ( %ci - ) months post conversion (p-value . ) eleven out of patients were affected by diabetes mellitus. six patients were diabetic pre transplantation and remained diabetic post transplantation and post conversion to tac. two patients developed new onset ptdm post transplantation and two became glucose intolerant. after conversion to tac, glucose intolerance resolved in one patient and one patient ( %) developed new onset ptdm. in patients converted to tac more than months post transplantation, mean total cholesterol was reduced from . to . mmol/l (p-value . ) and mean ldl cholesterol from . to . mmol/l (p-value . ). in june , / patients ( . %) were taking tac with a mean scr of +- lmol/l. four patients ( %) lost their grafts. mean graft survival time was . months. -year graft survival was . %. conclusions: conversion from cya to tac was beneficial with respect to renal function and cardiovascular risk profile. the conversion had no added benefit on renal function in patients with stable renal function taking cya more than months post transplantation. the reported incidence of ptdm was found to be low ( %). background and objective: most of the cancer patients suffer from severe pain especially during the terminal phase of the disease. it is essential to monitor these patients to achieve adequate and successive pain management, not just because of the importance of the effects, side effects and overdose problems; but also to improve quality of life. the aim of the study was to evaluate oncology pharmacist interventions on pain management. design: numeric pain scales was conducted prospectively among the cancer patient who were over years old and were selected randomly. patients were separated into two groups: of the patients was control group, pharmacist had been effectively included to the rest patient's pain management strategies, which was pharmacist intervention group. all of the patients had been evaluated by numeric pain scales (time ). the patients who were on pharmacist intervention group were monitored by pharmacist on treatment effectiveness and side effect profile every three days during the study. after one month, numeric scales were repeated (time ). the interventions that pharmacist done were pain evaluation, suggestion on appropriate pain reliever, dose management, patient education and patient monitoring. our therapy recommendations were made on the basis of the world health organization's analgesic ladder following the results of assessments. setting: oncology outpatient unit of a university hospital main outcome measures: the demographic and diagnostic information of the patients were collected. the results of the evaluations via numeric pain scales were calculated by using arithmetic mean value. results: the mean of pain intensity was significantly decreased in pharmacist intervention group when compared with control group ( . ± . vs. . ± . , p = . ) and between time and time ( . ± . vs. . ± . , p = . ). the mean of pain's effect on daily activity was significantly decreased in pharmacist intervention group when compared with control group ( . ± . vs. . ± . , p = . ) and between time and time ( . ± . vs. . ± . , p = . ). the mean of drug effectiveness was significantly increased in pharmacist intervention group when compared with control group ( . ± . vs. . ± . , p = . ) and between time and time ( . ± . vs. . ± . , p = . ). conclusions: the harmonious working of the pharmacist with the other health care staff working in oncology unit, helped patients to achieve more effective pain management. in this study; pain intensity was decreased, pain interfered less with daily activities was, and the reported effectiveness of drugs was increased in the pharmacist intervention group compared to the control group. all these outcomes show that the clinical pharmacists have an important role in oncology services, especially pain management. background and objective: to investigate and compare the frequency and nature of prescrbing errors requiring contact with the prescriber at community pharmacies in norway, estonia and sweden. design: a protocol, based on a scheme originally presented by rupp ( ), revised and developed by kennedy ( ) and translated and transformed to the nordic context by haavik ( ), was used in all three settings. in norway the protocol was self-completed by the pharmacists; in sweden and estonia observers (trained students) recorded and classified the interventions. setting: norway - community pharmacies in southern and western norway; estonia - community pharmacies in three cities; sweden - community pharmacies in swedish cities and public pharmacies at hospitals in sweden. main outcome measures: prescriptions with errors or ambiguities where the pharmacist decided to contact the prescriber to correct, clarify or complete the information on the prescription. results: the total numbers of dispensed prescriptions were: norway , , estonia , , sweden , (community pharmacies) and , (public pharmacies at hospitals). the proportion of handwritten prescriptions and prescriptions where pharmacists contacted the prescriber was higher in estonia than the other countries. administrative problems -reimbursement issues; prescriber data and distribution and licensing issues -were the reason for more than one third ( - %) of all contacts with the prescribers in all settings. however, the patterns of prescription problems with potential clinical hazards varied -in estonia and norway, errors concerning strength, administration form and number of doses were the most common errors and constituted and % of the problems. in sweden, errors concerning the prescribed dosage were the most common reasons. conclusions: the proportion of problem prescriptions requiring a clarifying contact with the prescriber was higher in estonia compared to norway and sweden. the main reason may be that most prescriptions in estonia were handwritten. administrative problems (reimbursement and availability of prescribed products) constituted a similar large portion in the three countries. however, prescription problems with potential clinical consequences for the patients, varied. , presenting with salmonella bacteremia and neurological deterioration due to cerebral toxoplasmosis was admitted to the intensive care unit. he was immediately intubated. to treat toxoplasmosis, cotrimoxazole was started in a dose of mg smx/ mg tmp qd. days later the patient developed leucopenia (absolute wbc count: . /l, neutrophils: . /l). folinic acid mg od was associated to restore white blood cell count. neutrophils further dropped to attain its nadir ( . /l) on day of cotrimoxazole therapy. cotrimoxazole was stopped and clindamycin mg td was used instead. neutrophil count restored, normalizing on day after stopping cotrimoxazole. this event was attributed to the administration of cotrimoxazole. the time relation between the administration of cotrimoxazole and the onset of neutropenia as well as the normalisation of neutrophils was clear. other explanations, such as the contribution of concomitant medication (ranitidin, ceftriaxon, ethambutol, isoniazid, rifampicin, aciclovir, amphotericin b, enoxaparin)could be ruled out. the naranjo score, which estimates the probability of adverse drug reactions, is . the use of folinic acid as rescue therapy in association with cotrimoxazole is controversial, as it can theoretically antagonise the anti-infective action of cotrimoxazole. therapeutic failure in aids patients, receiving this combination for pneumocystis jiroveci pneumonia, has been reported. nevertheless, we decided to start folinic acid to further prevent nosocomial infections in this severe immunocompromised host. we don't know whether folinic acid contributed to quick recovery of neutrophil count in our patient. further studies are necessary to clarify its role as rescue agent during treatment with folic acid antagonists. conclusions: this case report illustrates that cotrimoxazole, frequently used in opportunistic infections, can be associated with agranulocytosis. this dangerous complication in immunocompromised patients with severe infections must be prevented, although the effectiveness of folinic acid as rescue therapy is still a matter of debate. background and objective: intravenous immunoglobulin (ivig) therapy is increasingly used in inflammatory and autoimmune disorders, because of its therapeutic benefit and its good safety profile. cutaneous adverse events are rare and include prurit, rash, alopecia and eczema. in the literature, about cases of eczematous skin reactions have been reported. most of the cases were treated for a neurological or neuromuscular disease. erythematous eruptions on hands and feet have been notably reported after high-dose infusion. in most of the cases, the eruption was progressively extending to involve the entire body. when ivig were readministered, eruptions were more rapid and more intensive. we report an eczematous skin reaction of the palms after ivig infusion without extensive eruption, in spite of three administrations. design: case report. setting: neurology ward, university hospital, grenoble, france. main outcome measures: a -year-old man was treated with ivig (tegeline Ò , lfb, france) for an inclusion body myositis. he developed a skin reaction, days after the end of a days ivig infusion (dose of . g/kg was given daily for consecutive days). the eruption was a non-pruriginous erythematous maculopapulovesicular rash located on the palms. this reaction occurred th day after completing the second therapy and did not extend. the lesions regressed progressively with topical application of fatty ointment. three days after the third infusion, the same lesions reappeared, and regressed the same way. results: clinical pharmacist with the help of pharmacovigilance experts and doctor worked collaboratively: because of the chronology of exposure to other treatments, intrinsic imputability and recurrence on reintroduction, we ruled out an adverse drug reaction to any other medication. we decided not to interrupt the infusion. we advised the patient to continue fatty ointment application and to tell the healthcare team if the reaction became more serious. conclusions: dermatologic adverse reactions such as eczematous skin reactions are rare and usually mild. there is no reason to limit the use of ivig in a case like this one, as long as the treatment is effective. however, a narrow clinical and biological follow-up is required. if necessary, this adverse effect can be prevented by antihistamines or even steroids. anticoagulants must be monitored closely by physicians, because this products have a narrow therapeutic index. numerous interactions with herbs are documented, either increasing or decreasing the anticoagulant effect. our main objective is to identify this interactions in our surgery and if they are clinically significant. design: six months observational study; interviewing patients with their inr alterated about herbs that they were taking at that moment. literature review. setting: the anticoagulant oral treatment surgery. main outcome measures: the two oral anticoagulant drugs available in spain are acenocoumarol and warfarin. the international normalized ratio (inr) is the laboratory test used to measure therapeutic efficacy and safety of vitamin-k antagonists. a control test is done every four weeks and if necessary it can be done earlier. results: among patients with inr [ , six of them were taking herbs at the same time, and we could relate the increase of the effect of oral anticoagulants to those products. one of this patients who was taking dandelion (tarxacum officinale) had a inr. an other one who was taking chamomille (matricaria capensis) and passion flower (passiflora incarnata) had a . inr. all of these products have coumarins compounds. two patients who were taking equinacea (equinacea purpurea, equinacea angustifolia) also had their inr test altered: one had a . inr and the other one . . an other one was taking bilberry (vaccinium myrtillus) and had a . inr. both, equinacea and bilberry inhibit different isoenzimes of cytochrome p . the last patient was taking garlic oil (allium sativum) and had a inr. garlic increases the anticoagulant effect. conclusions: it is commonly believed that herbal products are inofensive, that is the reason why mainly of the patients do not take medical advise before starting a treatment with them. however, there can appear interactions with the usual treatment. if we fix on the vitamin-k antagonists the risk resides on the hemorragic or strokes events. in conclusion, we believe that patients should be educated about the potential risk of using herbal products while being treated with vitamin-k antagonists. a year-old man, treated with clopidogrel after coronary stenting, is hospitalized for aa (neutrophils: g/l ( . - g/l); haemoglobin: . g/dl ( - g/dl); platelets: g/l ( - g/l)). his permanent medications were insulin, perindopril, omeprazole, atorvastatine, bisoprolol, and acetylsalicylic acid. clopidogrel ( mg/d) was prescribed weeks before aa occurence. clopidogrel is withdrawn and aa therapy is started, consisting in the sequential association of anti-thymocyte globulin therapy ( mg/kg) and ciclosporin ( mg/ kg/d) in a filtered-air room. but the severe co-morbidities lead to early stop ciclosporin, then relayed by androgen therapy (norethandrolone). finally, at weeks from the diagnosis, the evolution ends to a resolution of aa, but with platelet-transfusion dependance. after the elimination of the other aetiologies, iatrogenic cause is envisaged. to blame clopidogrel is difficult with regard to the other drugs, especially perindopril and omeprazole known to induce bone marrow failures. four arguments lead to target clopidogrel: (i) the length of treatment by perindopril and omeprazole without complications, (ii) the timing between the onset of aa and the addition of clopidogrel to treatment, (iii) the resolution of aa whereas neither perindopril nor omeprazole were withdrawn, and (iv) the support of the literature. conclusions: clopidogrel seems to be responsible of this side effect. we unfortunately lack in specific biological tests to prove it. keywords: clopidogrel, side-effects, pharmacovigilance background and objective: informing patients on their medicines is a patient right. what does current information provision on antidepressants to patients with a depression admitted to a psychiatric hospital look like? what is the current practice of health care professionals? what are the experiences of patients? this study aims to explore current practice on drug information provision in psychiatric hospitals. design: a qualitative study consisting of semi-structured interviews with separate interview guides for health care professionals and for patients. interviews were tape recorded, verbatim transcribed and analyzed using nvivo software. setting: eight flemish psychiatric hospitals. main outcome measures: identification and evaluation of current approaches to drug information provision on antidepressants from the point of view of health care professionals as well as patients. results: patients get information on antidepressants, firstly, through psychiatrists and, secondly, through nurses. hospital pharmacists have a supporting role. the approach in giving information depends on patient characteristics and his/her mental state. information is provided mainly orally. leaflets are not frequently distributed to patients. patients also get information on antidepressants during psycho-educational sessions. on request, patients can read a package insert under supervision of a health care professional. health care professionals consider non-verbal cues of patients to verify if information has been understood. information is repeated when the first instruction was not clear for patients. there are no systematic interdisciplinary contacts on information interventions. patients as well as health care professionals are satisfied with current practice on information provision. health care professionals reported lack of time and lack of interdisciplinary contacts as negative aspects. patients indicated that health care professionals take too little initiative to give information about medicines. positive aspects reported by health care professionals are the hospitals' openness and the opportunity for patients to ask their questions to psychiatrists as well as nurses. suggestions for improving practice are: providing more medication information to patients, in particular on side-effects; enhancing the availability of easy readable information; and organizing continuing education for nurses on medicines. patients are informed about their antidepressants through various pathways. however, there seems to be room for improvement as a number of suggestions were formulated to support these pathways of drug information. keywords: medication information, antidepressant, psychiatry claire chapuis , christine chevallier , céline villier , pierrick bedouch , benoît allenet , jean calop , gérard besson pharmacy, pharmacovigilance, neurology, university hospital, grenoble, france background and objective: the use of intravenous immunoglobulin (ivig) is expanding. the risk for adverse effects can be minimized by taking some precautions. there are yet no standardized practice guidelines for prevention and management of adverse effects occurring during the infusion, and there is a need for it. the purpose was to conduct a study of their knowledge among the nurse community and to make a review of the best practices. design: we search in medline and carried out a questionnaire for nurses. we evaluated knowledge and practices of nurses in neurology, pneumology and haematology units, experienced in intravenous immunoglobulins administration, in order to define their role and the prescribers role in ensuring patients safety during therapy. we used all information and synthesized it in a table. for every type of adverse reaction, we indicated mechanism, frequency, seriousness, risk factors, practical guidelines of management and prevention and actor. the guidelines were reviewed by experts (pharmacist in charge of human derivative products, pharmacovigilance experts and neurologists). setting: university hospital, grenoble, france. main outcome measures: formalisation of practical guidelines on prevention and management of intravenous immunoglobulin adverse effects, for prescribers and nurses in the local hospital network and possibly other hospitals. results: nurses always checked the doses before administration ( / ), always prepared the product aseptically ( / ), warmed up the product until it reached room temperature ( / ), but only few recorded the patients tolerability during the infusion ( / ), and very few knew that most adverse events could be minimized first by slowing down the rate of infusion ( / ). all nurses called a doctor as an adverse effect appeared. conclusions: nurses must be involved in the management of adverse effects, even if the prescriber remains the one who makes the prevention by evaluating risk factors, co-medications, dosing and frequency of treatment and the one who makes the decision to interrupt the treatment if an adverse effect occurs. the clinical pharmacist in care units works collaboratively with both prescriber and nurse. he plays a central role for preventing drugs' adverse effects while counselling every member of the healthcare team background and objective: fludrocortisone tablets - lg (f) is mainly used in the treatment of adrenocortical insufficiency. it may also be used in treatment of orthostatic hypotension. f is manufactured by ageps (public special-order manufacturer) and dispensed to outpatients by hospital pharmacies, as a ''hospital preparation''. in order to follow gmp guidelines, a patient information leaflet for f was elaborated in our pharmacy outpatient unit. the leaflet was approved by our regulation unit. to evaluate the usefulness of this leaflet and to improve its quality, we performed a patient satisfaction survey. design: during days, for every dispensation of f, a leaflet was presented to the patient and an anonymous satisfaction survey was performed. setting: pharmacy outpatient unit, agence générale des equipements et produits de santé (ageps) (ap-hp), paris, france. main outcome measures: the questionnaire consisted of three items: general information about patient and its treatment; patient's knowledge of f before reading the leaflet (uses, precautions, adverse events, storage); patient satisfaction of leaflet (general presentation, language simplicity, information volume, utility). results: patients answered the questionnaire. the mean age was years ( - years). mean f treatment duration was years ( months- years). % of patients were already informed about f: % by physicians and % by associations and internet. % knew the indication ( % adrenocortical insufficiency and % orthostatic hypotension). % knew about precautions, % knew about side effects, and % knew about storage conditions. % of patients did not read the leaflet and had no opinion about satisfaction items. % were satisfied of general presentation. language was understandable for %, and non understandable for %. information volume was sufficient for %, insufficient for %, and too large for %. leaflet was useful for % of pts. patients who found the information insufficient suggested the following items: results of clinical trials, management of acute situations due to disease or f, and contacts of qualified centres in case of serious events. conclusions: nearly half of the patients were informed about f by their physicians, but information is communicated orally without written support. the majority of patients treated by f knew about precautions, side effects, and storage conditions. however, patients were satisfied of our information leaflet and find it useful. leaflet appears to be a good tool to communicate information from the pharmacist to the patient when not available in the packaging. of these recommendations taken into account ( %), within a period of days for most (n = ), involved the intervention of a pharmacy student, were given only via the computer software, and via a telephone call. conclusions: computerisation of prescriptions is an indispensable tool in order to make the pharmaceutical distribution circuit safer. however, its use as a vehicle for pharmaceutical interventions is limited, as shown by this study. it is impossible to analyse a nonresponse of our recommendations: is our advice even red, is it considered as inadequate? a discussion with the clinician (via pharmacy students or on the telephone) allowing a constructive exchange of knowledge, leads to a better transmission of recommendations. background and objective: in hospital setting, employees may be exposed to hazardous drugs. risks and protective measures needed when handling parenteral cytotoxic drugs are well described, whereas information related to drugs like monoclonal antibodies or antivirals are lacking. we developed a standardized method to evaluate drugs potential toxicity and occupational risks taking into account the pharmaceutical forms of the drugs to balance the risks. design: development of an algorithm for toxicity evaluation using material safety data sheet (risk and safety phrases), international agency for cancer research (iarc) classification and official manufacturers' data. . evaluation of chronic toxicity (mutagenicity, carcinogenicity), acute toxicity (sensitisation or irritation in contact with skin, eyes or by inhalation) and toxicity to reproduction . balancing of toxicity according to the pharmaceutical forms . assessment of protective measures (centralization of drug preparation in the pharmacy, wearing of mask, gloves and/or glasses) centralization of drug preparation in the pharmacy is recommended only in case of documented mutagenicity and carcinogenicity and when there is a risk of respiratory or cutaneous exposure related to the pharmaceutical form. setting: university hospital ( beds). main outcome measures: • chronic (r , r , r or iarc group , a or b), acute toxicity (r - , - , - ; s - , - ) and toxicity to reproduction (r - ; cat.d,x) • pharmaceutical forms associated with a risk of respiratory (e.g. tablets crushing), cutaneous (e.g. drug in solution) or ocular contact (e.g. inhalation) results: occupational risks of parenteral monoclonal antibodies, oral and parenteral antivirals, oral cytotoxics and other drugs forms were analysed. according to our algorithm, crushing of % of the tablets forms should be done in the pharmacy (e.g. valganciclovir). only parenteral antiviral should be reconstituted at the pharmacy (ganciclovir). monoclonal antibodies were found not to be at risks of mutagenicity or carcinogenicity and only gloves will be recommended for their manipulation. no ''class-effect'' has been pointed out (e.g. only a few antivirals were found to be hazardous). products were at risks for pregnant women. protective measures to be taken by pregnant nurses or those wishing to have a baby will be discussed institutionally. conclusions: toxicity evaluation of hazardous drugs handling in hospital should take the pharmaceutical forms into account as some toxic drugs may not be associated with occupational risks (e.g. coated tablets). our method allows a standardized way to evaluate whether a drug should be treated as hazardous or not. results will be discussed institutionally in order to implement applicable policies and procedures. results: lidocaine % injectable solution is indicated for tracheobronchial anesthesia, via bolus of mg in adults, and to mg in children. the maximal dose is restricted to mg/kg in adults and - mg/kg in children. epinephrine is indicated in hemorrhagic complications occurring during interventions; the solution is diluted to mg/ ml with nacl . %; a lg bolus is injected and can be repeated up to mg. terlipressine can be used in heavy bleedings; the powder should be reconstituted with nacl . %, at mg/ ml; mg is injected, repeated if necessary. mesna is used to dissolve mucous plugs, especially in patients with cystic fibrosis. the solution is diluted to mg/ml with nacl . %; bolus of ml are injected to allow visibility in the lower airways. there are no data available on the maximal dose. all preparations have to be compounded aseptically and extemporaneously by the nurse when requested by the physician, because of the lack of stability studies in those ranges of concentrations. conclusions: these guidelines are intended to standardize flexible bronchoscopy procedures between the different units, in order to minimize the occurrence of medication errors. almost half of the enquiries ( . %) were about stability and incompatibility in intravenous (iv) admixtures. the frequency of these calls was significantly higher than the calls came from other health professionals (p \ . ). the following types of enquiries were about dosage ( . %) and availability of pharmaceutical products ( . %). while . % of enquiries were answered by hizbim in less than minutes, in % of all calls took more than day to gather and tailor which requires more literature search. the mostly ( . %) used references in retrieving information was general references followed by micromedex ccis inc. with . %. • hizbim has been consulted by nurses in a rising pattern. this means that this service is of importance for them because of its rapidity, accuracy and currency • the preparing iv admixtures that has been left to nurses' responsibilities in practice in our country can be reconsidered by pharmacists since this service has been fulfilled by ''drug experts'', namely pharmacists in many countries. • drug information centers can provide not only emergent information for nurses but also education such as seminars and conferences in some topics they need. the establishment of drug information centers in hospital settings will be of use to stimulate the frequency of consultations by nurses and to improve patient care provided by them. background and objective: the concept of pharmaceutical care (pc) is capturing the attention of a growing number of pharmacists. the strategy followed by us to spread out the implementation of pc in pharmacies involves the utilization of internet tool as an innovation of traditional education. design: to do so, we designed an on line pc course with interactive cases. the specific goals of the course are: • the enhancement of the pharmacy practice. • the promotion of practice research. • the personal and professional development of pharmacists. the objective was to analyse the evolution of the program, whose pedagogical design was based in a process of teaching-learning in the field of the clinical pharmacy and pharmaceutical care that permit the training of the future pharmacists in the use of medicines. design: all the classes were designed with practical-theoretical modality including workshops, reading and analysis of the bibliography, discussion of cases and utilization of virtual simulators. setting: clinical pharmacy. department of biological sciences main outcome measures: students evaluation was carried out by tests of the general contents, degree of active participation during the workshops, resolution exposition and discussion of the cases. results: the development of clinical pharmacy education began in . the students appreciated this experience and the % of them are satisfied with the process of teaching-learning employed. this teaching system improved them the adequate and rational use of the medicines. in these two years the % of the students has approved the examination. the students also realized two poster presentations during this period, namely: . ''comparative study of the information supplies in the leaflet of tablets of omeprazol mg'', in and . ''study of the information supplied by the pharmaceutical laboratories in the television media'' in . conclusions: we consider the importance of the incorporation of clinical pharmacy in the new pharmacy curricula, because the students have major task about the practical education involvement patients and related to their professional future. background and objective: clinical trials' managing and dispensation is one of the obligatory missions of a hospital pharmacy. the respect of good clinical practices is necessary to ensure proper trial performance and to be aware of errors in prescription and in drugs dispensation. the aim of our study was to analyse the nonconformities in the steps of prescription and dispensation and to evaluate their severity. design: the pharmacy department manages clinical trials for all clinical wards with a pharmacist and a technician who change every four months. there is no specific computerized system but an excel software for data filing. we realized a retrospective study of ongoing clinical trials. all the prescriptions were reviewed and we focussed on all the items required for the prescription (e.g., identification of the protocol, the patient, the investigator, the dosage) and for the dispensation (e.g., identification of the drug, the quantity dispensed, batch number, expiry date) setting: results: gps participated to the survey. they agreed (median = , iqr = - ) that the following concepts are crucial in a patientcentred approach: the need of a specific training in counselling in under and post graduate education; the necessity of working with patients to develop mutually agreed-upon goals; the role of information in the decision making process, the ability to understand patients readiness to make change, and to identify barriers to change, the importance to recognize that patients are the experts when it comes to their own behaviour related issues. for only one item (time dedicated to the consultation) iqr changed (iqr = - ) indicating some difficulty in implementing this aspect in practice. conclusions: for benevento community gps, a patient-centred approach is a useful way to help change and promote behaviour. knowing how to support it is an important skill for all care professionals, but education is needed to shift from theory to practice. type ii and gestationnal diabetes. the average age was years and for the half of these patients, the pathology developed since more than years. patients were painful and patients presented a positive score to dn questionnaire. symptoms frequently observed were: pins and needles, prickles, and numbness. among these patients, were treated by antidepressants (amytriptiline) or antiepileptics (clonazepam, gabapentine, tegretol, carbamazepine or pregabaline) or both. the average number of molecules received by patients was . . the average number of lines of treatment was: . . first intention treatment insisted of an anti-epileptic monotherapy in patients. second line treatment involved the introduction of another anti-epileptic or an anti-depressant drug. the drugs have been well tolerated except a respiratory depression under clonazepam. among the not-treated patients, only one benefitted from a treatment initiation. conclusions: patients are treated by ''old'' molecules with a large prescription of clonazepam but their efficacy is very variable. the physicians have been sensibilised to dn questionnaire and they concluded that it is easy to use. however there is a lack of physician's informations about the management of neuropathic pain; besides they are hesitant to initiate a treatment. the relationship between pharmacists and physicians and the development of clinical pharmacy seems important to optimize the management of this pain. background and objective: pharmacy education in the faculty of pharmacy and biochemistry of the university of buenos aires is taught as a product-oriented profession with a focus on the basic sciences. however in pharmaceutical care and clinical pharmacy was integrated as an optional course into the pharmacy curriculum by resolution cd / . the object of the emphasis was on the students' ability to provide clinical pharmacy and pharmaceutical care upon graduation. hence, therapeutic plays a significant role in building students' knowledge and skills in preparation for clinical practice. design: theoretical education and program for students and collaborative implication in the hospital activity or in the community pharmacy. the development of the program is carried out in two phases. in the first phase the clinical activities of the pharmacists, the unidose drug distribution, the role of the drug information centers, pharmacoepidemiology and surveillance studies are explained. in the second phase the concept of pharmaceutical care is introduced and its implementation in different pathologies is developed. an active approach of the patient and contact with the treating physician was considered as tool in a strong learning environment. setting: undergraduate pharmacy students at university of buenos aires. main outcome measures: the students find them abilities to identify drug related problems and to assess patient care and follow-up. results: more than students have attended pharmaceutical care and clinical pharmacy to: % passed and % failed. students option was that the strongest aspect are the case discussion and the weakest the very few number of hours not enough to discuss other important illnesses. they also say that many topics should be taught sooner in the career and it was not considered an emphasis an the clinical and patient oriented-aspects of the profession. conclusions: an approach to clinical pharmacy education in which the integration of teaching and learning are collaborative creates an atmosphere that is conducive to effective student learning. moreover the clinical pharmacy is a valued and important tool of the general practice team regarding quality improvement in drug therapy. all of the most popular cpd linked pharmacy activities were from the competency-'participation as a member of the multidisciplinary team'. telephone interview findings showed pharmacists cited 'priority for their service development' as the principle reason for cpd identification. no pharmacist identified a cpd need for: 'supporting patient/family motivation'; 'ecording problems in blood glucose control requiring balancing food intake and insulin dose'; 'sharing reflections of where your performance leaves room for improvement within a pharmacists group' and 'taking part in a local multi-disciplinary mentoring group.' conclusions: currently cpd workbooks appear not to be widely used within the pharmacy profession in the uk although there are examples of successful use of reflective portfolio. , almost half of the participants chose the workbook as a means of support leading to a substantial number of identified cpd issues. for community pharmacists to deliver high quality care for diabetes, more attention is required to forms of training and to both uniprofessional and multiprofessional peer support. setting: we retrospectively reviewed all prescriptions of ivig issued from three infectious disease departments ( beds) in a french bed university hospital. main outcome measures: for each ivig prescription, the following data were collected: patient identification, name of ivig product, quantity of ivig issued, date of ivig release, indication for treatment and level of relevance. these levels are determined as follows. level : indications approved by health authorities or for which comprehensive guidelines have been published (high level of proof). level : relevant indications based on scientific publications. level : off-label indications more difficult to prove on a scientific basis (few or no high quality randomized controlled clinical trials). results: during the studied period, grams of ivig were administered for a total of patients and prescriptions. . % ivig used saccharose as a stabilizing agent, other products contain either glucose ( . %), or maltose ( . %). the purpose of this study is to produce an evaluation focus on the risk profile and counselling activities concerning therapeutic and lifestyle change. design: descriptive study. individual measurements were recorded by pre graduation students on a standardized datasheet. blood pressure was measured using a digital wrist device. blood glucose monitoring systems of two different brands were used. setting: the screening was made in the street using passer-by volunteers. in the course of a single day in may , a total of subjects ( . % males and . % females, mean age . years, sd = . ) had glycaemia and blood pressure measured. main outcome measures: age, gender, medication taken, cigarette smoking, body mass index (bmi), blood pressure, capillary blood glucose results: . % of the sample presented bmi [ kg/m. . % had elevated systolic blood pressure values and . % elevated diastolic blood pressure values. . % had elevated occasional blood glucose. . % are cigarette smokers. systolic (t = - . ; p = . ) and diastolic (t = - . ; p = . ) blood pressure values were significantly higher in smokers than in non-smokers. concerning patients taking diabetes medication, fewer patients with blood glucose controled and more patients blood glucose uncontrolled were found than those expected, suggesting either low compliance or lack of efficiency of medication (v = . ; p \ . ). concerning the hypertension medication, similar results were found. more patients under therapy with blood pressure uncontrolled were found than expected. the concordance within the two measures of the blood glucose with different monitorizing system was found to be strong and significant (k = . ; p \ . ). positive and significant correlation between bmi and diastolic blood pressure was found. however, no significant correlation between bmi and systolic blood pressure was found. conclusions: events such as this screening improves the quality of education, as well as develops the interests and opinions of students. as well as it shows face to face were can be apply their knowledge of clinical pharmacy furthermore, events such as these are found by the students to be invaluable in acquiring training in similar-to-professional setting and expertise in field work. background and objective: nhs education for scotland (nes) has worked with the scottish executive health department (sehd) to develop training packages to support the use of validated needs assessment tools (nat) for several longterm conditions. consequently a specific training package was then developed to support and standardise needs assessment and pharmaceutical care delivery by palliative care pharmacists in the local networks set up in scotland. additional support materials (trainers package) was developed for representatives from the scottish palliative care pharmacists' association to deliver the training to their local network pharmacists in a consistent manner. design: development and evaluation of a training package and course for palliative care pharmacists. evaluation of the outcomes from using a nat for delivery of care. identification of issues %; relevance of questions %; time to complete nat - mins; barrier -time; benefits -care issues identified by nat; care issues -counselling and compliance issues, side-effects identified (nausea, dry mouth, constipation), pain relief not adequate. conclusions: nes are proactively supporting national policy and practice through a process of identifying and meeting the educational needs through direct and self-directed learning for continuing professional development (cpd). the needs of palliative care patients are seen as an appropriate target group for pharmaceutical care. the evaluation and feedback from the courses, training pack and outcomes from the use of the nat in practice have been very positive and amendments will be made for further implementation in scotland. background and objective: pharmacy students represent a broad spectrum of learning preferences and styles. this diversity presents a responsibility for the lecturers and instructors to meet the educational needs of all students. in order to develop appropriate learning approaches the instructors need to know the students learning preferences. therefore, the aim of this study was to identify the learning preferences of pharmacy students. design: the visual, auditory, reading/writing, kinesthetic (vark) questionnaire identifies student's preferences for particular modes of information presentation. the vark questionnaire is freeware that can be completed online. however, we administered the vark questionnaire as a hard copy at the end of the 'clinical pharmacy practices' final exam to the fourth-grade pharmacy students. setting: marmara university -faculty of pharmacy. main outcome measures: the frequency of students' preference for modes of information presentation. results: we administered the vark questionnaire to students and ( %) returned the completed questionnaire. almost half of the students ( . %) preferred a single mode of information presentation. among these students, % preferred visual (learning from graphs, charts, and flow diagrams), . % preferred auditory (learning from speech), and . % preferred printed words (learning from reading and writing), and % preferred using all their senses (kinesthetics: learning from touch, hearing, smell, taste, and sight). the other half ( . %) preferred multiple modes [ modes ( . %), modes ( . %), or modes ( . %)] of information presentation. a total of ( . %) students preferred 'kinesthetic' learning solely or in a multimodal combination. conclusions: the students represented a variety of learning styles. student motivation and performance improves when instruction is adapted to student learning preferences and styles; so, it is the responsibility of the instructor to address this diversity of learning styles and develop optimum learning approaches. escitalopram, fluoxetine and mirtazapine were rarely prescribed. the posology and taking plan were generally respected. however, some improvements in terms of treatment optimization could have been brought. they could have lead to actions of clinical pharmacy within the framework of prospective study: posology optimizations and taking plan optimizations. nineteen patients ( %) had an adjustment in their antidepressant treatment: nature and dosage. for all the antidepressants, a sufficient duration was respected before increasing the doses. an average number of interactions by prescription were . but none was clinically significant. conclusions: therapeutic strategies corresponded to guidelines recommendations. the dosage adjustments and duration before increasing the doses respected the indication of antidepressants. in the future, to optimize the medicinal treatment, decision-making tools carried out could facilitate psychiatrists' prescriptions. the pharmaceutical validation of prescriptions will be facilitated by complying with them within the framework of clinical pharmacy activity. background and objective: al-amal hospital is a bed oncology/ hematology hospital. al-amal hospital is now the first hospital in qatar to be accredited by the joint commission international (jci), the worldwide leader in improving the quality of healthcare. the objective is to implement clinical pharmacy services in al-amal hospital in the state of qatar by training the pharmacists about clinical pharmacy services. design: a pharmacist designed the training program and took the initiative and responsibility for training other pharmacists in aah about clinical pharmacy. the department of medical oncology/ hematology, the hospital administration and the pharmacy department agreed that the pharmacists should have central responsibility for antineoplastic agents and other drugs related problems. pharmacists for the program were selected from the existing staff. the healthcare team is consisting of two pharmacists rotating every months. each pharmacist join teams consisting of a pharmacists, a consultant, a specialist, a resident, a rotating resident, nurses, a dietitian, physiotherapist, social worker and a psychologist. we used to have oncology teams and hematology team. both pharmacists participate in the medical rounds and morning report days per week. the pharmacists provide clinical pharmacy services including chart review, pharmacy patient profile review, laboratory tests, therapeutic drug monitoring, antibiotics monitoring, interviews with patients and/or relatives. drug related problems were identified, resulting in interventions. setting: in patient wards, al-amal hospital, qatar. main outcome measures: to identify drug related problems, which well result in interventions and to help the medical team and the patients to reach their treatment goals. patient outcomes were evaluated by follow up with the medical team or by patient interview. we refer patients to the dietitian, physicians, the clinical psychologist as needed. results: more time is needed to evaluate the clinical pharmacy services provided by the pharmacists as the program was just started. patient and physicians were satisfied by starting the training program. conclusions: hematology/oncology setting provides an excellent opportunity to involve pharmacists. a. leads to encephalopathy and progressive neurodegeneration in the infant who is not treated. early diagnosis and dietary management can prevent complications and may allow for normal intellectual development. however, neurologic function may deteriorate rapidly at any age because of acute metabolic decompensation. these severe episodes are caused by catabolism of endogenous protein, which may be provoked by physiological stress (infections, post-surgery). during these crisis, the patient must have immediately intravenous glucose infusion and enteral nutrition free of b.c.a.a. design: case report. setting: department of pharmacy, hospital charles nicolle, rouen. main outcome measures: case report results: one patient with classical m.s.u.d. is followed in our establishment since many years. his disease has been diagnosed in neonatal period. a diet free of b.c.a.a. has been instaured. this diet is successful, now this patient is years old and had a normal development except myalgia and hypoesthesy of the left leg. however, when the diet is not well followed or when he's infected, acute episode occurs (on average or times per years). as the crises starts, the patient is sleepy and confused. in order to be able to treat him very quickly, the medical staff decided to set up an emergency protocol, which include an adapted enteral nutrition formulation. the pharmacy is implicated in this protocol to prepare the mixture. the formula includes: m.s.u.d mix, dextrin maltose, oligoelements, ions, lipids (sunflower oil), vitamins and water. the pharmacy must be able to carried out the preparation at any time and the components must be always available. conclusions: because m.s.u.d is an unherited disease, published report of treatment are rare and they are no consensus for the treatment of acute decompensation. since years, this protocol is successful: b.c.a.a. levels decrease between to days after the setting-up and the patient always recovered rapidly. this formula is administrated by nasogastric tube and avoid the use of hemodialysis which is the last solution to remove b.c.a.a. this is an example that a personnal follow-up program (with plan for clinical and metabolic evaluations) during common intercurrent illnesses can have optimal outcomes. keywords: leucinosis, metabolic decompensation, adapted enteral nutrition nutr- ensuring phosphorus adequacy of human-milk-fed preterm babies canadell laura , cañete carmen , pardo rocio , albujar mar , valldeperez cinta , carretero juan , gallart m jesus , closa ricardo pharmacy service, neonatology, hospital universitari joan xxiii, tarragona, spain background and objective: human milk is the feed of choice for preterm infants both for nutritional and non-nutritional reasons. phosphorus levels in human milk are insufficient for most premature infants. this deficit is the major cause of osteopenia in prematurity. fortification with a commercial multinutrient product should only be considered after weeks of mother's milk feeding, however, phosphorus supplement must be given initially. to describe a standardized scheme for early nutritional support with phosphorus of very preterm infants (\ - g)and describe the phosphorous oral solution we use as a supplement is the aim of this study. design: clinically relevant reports were reviewed to establish a standardized scheme for early nutritional support with phosphorus of the very preterm infants. a standardized formula of oral phosphorous was established to diminish the medical errors when the addition of this mineral is required. setting: pharmacy service and neonatology unit of a third level hospital. main outcome measures: to describe the scheme of adding phosphorous to human milk as well as the standardized formula we use, ''phosphorous oral solution'' ( mg p/ ml). results: phosphorous oral solution procedure: composition, stability and the scheme of addition to human milk to ensure the requirements for bone substrate needs in preterm infants to avoid osteopenia of prematurity. conclusions: various methods have been tested to decide when additional supplements must be given. individual adjustment is not possible due to the delay of laboratory results on milk analysis and the fast changes in infants' requirements. therefore, it is necessary to make a standard adjustment scheme on the dose of the fortifier that needs to be added. shortly to the pancreaticduodenectomy, total parenteral nutrition (tpn) was started ( kcal in progress until reaching his energy requirements). in addition to parenteral nutrition, supplementation enteral nutrition was delivered via jejunostomy along four postoperative days. on post day , transition to a complete enteral formula was achieved (standard formula, ml = kcal: kcal/d). on day , patient complained of colic pain in upper hemiabdomen. an emergency tc revealed presence of liquid in the abdominal cavity from anastomosis pancreatogastric. with the suspect of a leak from jejunostomy, the catheter was removed. tpn was reintroduced and kept as the only way of nutrition until later when oral tolerance was started. during hospital stay ( days) periodic blood controls were performed. main methabolic complication was high blood sugar, needing the administration of insuline. from day to , mean plasma levels of albumin ( . vs . g/dl), total proteins ( . vs . g/dl), total serum cholesterol ( vs mg/dl), total lymphocite count ( . vs . %) and prealbumin ( . vs . mg/dl) increased significantly conclusions: the leak of artificial nutrition to the abdominal space in patients with jejunal feeding is a frequent complication of ne. its incidence is probably related to the length of the tube inserted into the lumen. protocols are need to prevent complications like tube displacement and to encourage early enteral nutrition. increase in plasma concentrations of nutritional parameters suggests effective uptake admixtures might be either prescribed and made ''a la carte'' according to the newborn's needs or provided by pharmaceutical companies as standard formulations. the aim of the study is to review individual pn prescriptions in a neonatology care unit in order to assess the potential for using standardized pn instead. design: prospective study one day per week during weeks. setting: neonatal intensive care unit, strasbourg university hospital. main outcome measures: the major criteria for the comparison are carbohydrate concentration and then amino acid intake. results: prescriptions were analysed and compared with a standardized formulation, pediaven Ò (fresenius kabi). the first point of comparison based on carbohydrate concentration resulted in an exclusion of % ( / ) of the total prescriptions because their carbohydrate concentration was less than g/ ml or more than g/ ml (pediaven Ò glucose concentration, g/ ml). among the prescriptions retained, only prescriptions whose amino acid concentration was less than . g/ ml were included (pediaven Ò : . g/ ml background and objective: it is known that propofol protect myocardial tissue against global myocardial ischemic-reperfusion injury in the isolated rat heart model. the aim of this study was to investigate whether propofol, at a clinically relevant concentration infused during both preischemia and reperfusion (peri-ischemic) period, also provide protective effect against regional myocardial ischemic-reperfusion injury in vivo. design: mail sd rats weighing between and g were anesthetized with mg/kg of ketamine and mg/kg of xylazine. a haparinized g catheter was placed in the left femoral vein. the trachea was intubated and then mechanically ventilated with room air using a volume-controlled rodent ventilator. a left thoracotomy was performed, and the pericardium was opened. for the ischemia-reperfusion experiments, a snare was passed around a left anterior coronary artery territory to induce regional myocardial ischemia. coronary occlusion was produced by pulling the snare and clamping it with a mosquito hemostat. reperfusion was produced by releasing the clamp. setting: rats were subjected to minutes of coronary artery occlusion followed by hours of reperfusion. propofol or intralipid was administrated during minutes starting minutes before the onset of ischemia until minutes after the onset of reperfusion. main outcome measures: the micro-manometer catheter was advanced into the left ventricle via right internal carotid artery and hemodynamic function was checked after hours of reperfusion. infarct size was determined by triphenyltetrazolium staining after hours of reperfusion. results: propofol administration during both preischemia and reperfusion (peri-ischemic) period showed protective effects on myocardial function and infarct reduction. in the control group, the peak rate of ventricular pressure rise (+dp/dtmax) and the peak rate of intraventricular pressure decline (-dp/dtmin) significantly decreased than sham group. in the propofol group, the +dp/dtmax and -dp/dtmin significantly improved than conrol group. infarct size was . % of the area at risk in control group, and was reduced markedly by administration of propofol during peri-ischemic period to . % in the propofol group (p \ . ). infarct size of intralipid group was . % of the area at risk, intralipid had no effect on infarct size compared with the control group. conclusions: propofol, at a clinically relevant concentration infused during peri-ischemic period, provided protective effect after regional myocardial ischemic-reperfusion injury at in vivo rat heart model. the results showed hemoglobin level of less than g/dl in . % of the subjects, transferrin saturation (tsat) of less than % in . % of the hd patients, tsat \ % and ferritin \ ng/ml in . % of the patients, serum alb level of less than g/dl in % of the patients, serum p level of more than . mg/dl in . % of the subjects, ca p product of more than in % of the patients, parathyroid hormone (pth) \ pg/ml (adynamic bone disease) in . % of the subjects and serum pth concentration of more than pg/ml (uncontrolled secondary hyperparathyroidism) in . % of the subjects. the results showed that more than half of the hd patients need erythropoietin and ferrous dose adjustment or follow up for resistant anemia, more than half of the subjects need phosphate binders dose adjustment or replacement and about % of he patients need rocaltrol dose adjustment. we are planning to compare these results with the findings following the participation of a clinical pharmacist in this hd center rounds and monitoring of hd patients. since enough management of complications of crd patients and their drugs monitoring are necessary to improve quality of life of hd patients, clinical pharmacist may have a major role in hd centers. background and objective: computerization of our drug circuit has been deployed gradually to every hospitalisation units of our hospital since . pharmacists coordinated the extension, the installation and the support for starting. they were the first interlocutors to analyze dysfunctions and to help solving them. difficulties encountered by nurses were often notified to head nurses and transmitted to pharmacists. the objective was to study as a whole difficulties of users and to bring a workable solution to their problems. design: a working subgroup depending on the drug commission was created. it was composed with nurses from different departments (intensive care, infectious, pediatric and rehabilitation departments), head nurses and pharmacists. several meetings, organized between october and december , made it possible to the participants to announce their difficulties. reports were written and diffused for validation. to evaluate the pharmacist -patient communication and the level of counseling for otc and prescription drugs dispense; to improve the professional relationship between the patient and the community pharmacist; to assess the effect of clinical pharmacist intervention over those parameters. design: interventional study (visits done by clinical pharmacists, especially employed for), repeated after weeks and again after months. setting: chain pharmacies from bucharest, romania. main outcome measures: the investigation was conducted using a multiple sections protocol. the assessed parameters were: pharmacist's attitude toward the patients, his/her availability to communication and the level of counseling when otc or prescription medication is released. results: during the first visit, in sixteen pharmacies only ( %) the pharmacists greet the patients. two weeks later after the intervention, this number increased to ( %), although after six moths it decreased to pharmacies ( %). in more than % of the chain pharmacies, the professionals had a positive attitude toward the patients. as an example, an empathic approach has been encountered initially in pharmacies ( %), then in pharmacies ( %) and finally raised up to pharmacies ( %) after months. although the clinical pharmacist's intervention (therapeutic counseling) had positive impact, the extent of minimal counseling at otc or prescription drugs dispensing was found to be low at the first visit, since it increased from to pharmacies ( to %) only, during the study period. the processed data showed a very low level of minimal counseling ( % at the end of surveillance period). by considering the patients benefits (quality of life, better control and management of chronic diseases, reduction of medication costs), the pharmacist interventions are imperatively needed in bucharest chain community pharmacies. background and objective: to compare the level of minimal consultation services in chain community pharmacies located in city center or district of bucharest (capital city) vs. independent pharmacy in a country town (cluj-napoca). design: prospective, months, multicenter study. setting: pharmacies accepted to fill in the study protocols. both shifts were covered, monday to friday (week-end days not included). main outcome measures: the investigation was conducted using a multiple sections protocol. the assessed parameters were: the level of minimal consultation when otc and prescription medications are dispensed and the extent of chronic medication release without a medical prescription. at the end of trial period, the results were centralized on weekly and monthly protocols. the interpretation of the collected data was done using percentile calculations. results: to evaluate the minimal consultation, the percentage of counseled patients from the monthly total was calculated, separately for otc and for prescription drugs dispense. the period of the study (from june to january ) was divided in various slices of consecutive months, when certain pharmacies were compared. the level of otc medication counseling in the five studied pharmacies is different and varies from to % (maximum level reached in the country town pharmacy). the counseling level for medication on prescription varied from to %. by counting separately, the percentage of patients who requested chronic medication without presenting a prescription is as high as up to %. conclusions: the level of counseling, especially for otc drugs (recommended or auto-medication), was generally low in the studied pharmacies and may threat the health state of the patients, due to improper administration. as a third of patients come in pharmacy and request chronic medication without a physician's prescription, this commonly leads to complications which aren't discovered and treated in time. background and objective: the quality of medication use in nursing homes (nhs) is subject to growing concern. focus should not only be on appropriateness of prescribing, but also on correct pharm world sci ( ) : - administration of the medication. the aim of this study was to investigate ) the type and frequency of medication administration errors in nhs, ) their clinical relevance and ) whether a training session by a pharmacist on good medication administration practices can contribute to the prevention of the detected errors. design: the study had a pre-post design. during the first phase (pre), medication administration was observed during days per ward by pharmacists (barker method). phase (intervention) consisted of a general information session on good medication administration principles provided by the pharmacists to the nursing staff. moreover, the observed errors were discussed with the head nurse of each ward. phase (post) took part one month after the intervention and consisted again of a -day observation on each ward. finally, in the last phase (phase ), the clinical relevance of the detected errors was scored by an expert panel (geriatrician and clinical pharmacist). setting: volunteering nhs with different medication distribution systems. in total, medication administration was observed for residents. results: the number of detected errors was considerably lower in nh than in nh . however, the type of errors did not differ. besides the unnecessary or forgotten preparation of medication, most problems occurred during the administration stage. . % of crushed medications indeed were not suitable for crushing. the same applied to . % of the opened capsules. moreover, the crushing hygiene was problematic: all medications for one resident were crushed together and the crushing device was not cleaned between different residents. inhalation techniques were inadequate in almost all cases (insufficient inhalation by the resident, coordination problems or expiration in the device). furthermore, specific administration moments were not taken into account. for example, the administration of alendronate (fosamax Ò ) was observed in a horizontal position after breakfast, while it should be administered minutes prior to breakfast in a vertical position. the nursing staff experienced the training course by the pharmacists as very interesting. . % of the attendants found that the discussed topics were not sufficiently covered during their education. background and objective: the off-label use of intravitreal injection of bevacizumab (iib) for the treatment of macular edema (me) requires the approval of its use by health authorities. pharmacy department (pd) participates in that process, assessing each treatment request and preparing a sterile syringe for intravitreal administration. our aim is to evaluate the short term anatomic and visual acuity (va) response after iib in patients affected of me due to diabetic retinopathy or retinal vein thrombosis. the aim is to give an answer within hours. setting: all three pharmacists are working in the virga jesse hospital, a large peripheral hospital of beds in belgium. main outcome measures: implementation of the clinical helpline in the entire hospital. to make sure clinical pharmacy services are known by every physician and nurse and are easy to contact. results: we made an e-mail address and a schedule, so every day another clinical pharmacist is responsible for answering the questions. to let the physicians and nurses on the ward know we exist, we made flyers with the address and the explanation of the service. on a patient safety congress in the hospital, the clinical pharmacists presented a lecture concerning the advantages of clinical pharmacy services. the main aim is to explore other ways of delivering clinical pharmacy services. in belgium, the hospitals don't have a tradition of clinical pharmacy and there is no governmental support for this pharmaceutical function. with the clinical helpline we try to spread our services without having a clinical pharmacist on every ward. in the pharmacy we prepare the question thoroughly on paper. the clinical pharmacist has computerized access to all necessary medical information and pharmaceutical data. afterwards the pharmacist goes to the ward, to see the patient and to have a discussion with the physician. the physician can decide if he agrees with the given pharmaceutical advice or not. the clinical pharmacist has only an advisory function and doesn't do any therapeutic changes in the prescription. by collaboration of several caregivers, the patient receives a more complete and optimal therapy in our hospital. conclusions: by implementation of a clinical helpline, by an e-mail address, it is possible to spread our clinical pharmacy services over the entire hospital, without having a clinical pharmacist on every ward. the aim is to make an advice and go to the ward to discuss it with the doctor. not only physicians can use this e-mail address, also nurses can ask their questions. in this way we reach every caregiver. % of these pi was followed by modification of the prescription few days later. this study showed that % of the opinions were related to psychotropic drug overdose (often confirmed by psychiatrists after pi), especially neuroleptics, the most prescribed therapeutic class on the establishment. . % of the pi was related to inappropriateness to available guidelines. we note an important proportion of no respect of correct use recommendations of long-acting rispéridone injection: no respect of posological equivalence, patients not stabilized by oral way, insufficient period of co-administration oral/im during the initialization of treatment… . % of pi was guiding to drug management and to clinical and biological monitoring. drug related problems still under estimated without clinical and biological data accessible to pharmacists. however, pi may identify the risks related to therapeutic, to prevent potential problems, to reinforce the clinical and biological monitoring. comparison with a similar retrospectif study in shows that the number of prescriptions was increased ( in vs ), and number of pi doubled. however, nature and type of pi are virtually the same. conclusions: in order to reduce drp of overdose (the most frequent problem), an information strategy targeted to psychiatrists was developed and a updated list of maximal psychotrops posology was diffused and put on line. our study doesn't include problem of second -generation atypical antipsychotics association, this association still increasing despite fewer evidence and lucid guidelines; a second study will be soon conducted to identify pi having a significant clinical impact. conclusions: this study showed that clinical practices don't change even after reminding guidelines. information by fact sheet is not the best tool to spread guidelines. study's results will be submitted to an interactive presentation in medical staff, and clinical trials with iva are discussed and changed with acetaminophen oral route. the proportion of gp's that received, reviewed and returned the patient selection to the pharmacist, and the proportion of long-term users that received the informative letter. results: substantially more pharmacists in the intervention ( %) than in the control group ( %) handed over the patient selection to their gp's. % resp. % of the gp's received (n.s.), and % resp. % of the gp's reviewed and returned the list (n.s.). substantially more pharmacies in the intervention group got back any lists ( % vs %) and sent any letters ( % vs %). % and % of all longterm users received the informative letter in the intervention resp. control groep (n.s.). conclusions: the maximal implementation strategy was effective in getting the pharmacists started. the main outcome measures were not significantly different in both groups, though the realized effect on a large scale was relevant in practice. of the participants, total of . % has heard of the term 'pharmacovigilance' in the period- , which is entirely the academicians, while it is increased to . % in the period- (chi-square test with yates correction, p \ . ), which are mainly expressed by the hospital ( . %) and academician ( . %) pharmacists. during the period- , only % of the participants know where to report any adrs ( . % academicians and . % hospital pharmacists), whereas during the period- , this figure is increased to % ( . % academicians, % hospital and . % community pharmacists) (p [ . ). the participants preferred to report any adrs mainly by the internet ( . % vs %) and by the telephone ( % vs . %) at the period- and the period- , respectively (p [ . ). in terms of having reported any adrs among the participants, it is indicated that none of the pharmacists reported adrs at the period- , but only three hospital pharmacists reported an adr at the period- . conclusions: by the national regulations for pharmacovigilance, the pharmacists are entitled to report any adrs to the turkish pharmacovigilance centre. although this study is limited by the small number of pharmacists and location, it shows that there is an increased awareness and knowledge about pharmacovigilance. by the provision of pharmaceutical care, pharmacists' involvement in detecting and reporting adrs will improve, mainly in hospital and in community settings. background and objective: in france, global drug dispensing (gdd) is the common way to dispense drug inpatient. after an experience of implementation of individual drug dispensing (idd) the objective of the department of pharmacy was to prove the benefit offered by this system in comparing the clinical impact of the intercepted dispensing errors of both dispensing system. design: thirty days prospective study. setting: orthopaedic surgical care unit; department of pharmacy. main outcome measures: data related to drug preparation were collected by two pharm d students over a days period and analysed. identified preparation errors were classified in four groups: discordance between prescription and drug administration, exceeding or missing treatment and, unidentified delivered drugs. then errors were classified in potential or effective errors. at last, pharmacist and prescriber have quote this errors. preparation errors were classified according to their clinical impact from (no clinical impact) to (life threatening). results: drugs units were prepared with gdd vs with idd. eighty effectives errors ( . %) were observed with gdd and ( . %) with idd. clinical impact were ( %), ( %), ( %) for gdd and ( %), ( %) and ( %) for idd. a khi test highlight a difference between the dispensing system for clinical impact and (p \ . ). moreover, the mean value of weighting was about . with gdd vs . with idd (p \ . ). conclusions: this study shows the major benefit offered by idd versus gdd. errors weighting represent a relevant parameter for physicians. the results provided by this study highlight the role of the pharmacy staff, to reduce the incidence of medication errors and to promote a rational use of medicines. this work was the second step of our quality medication process; the next step will be the development of idd in several care unit by use and test automated process for preparing doses. for each prescription we collected information about the patient, the prescribed drug, the steps involved in its preparation and its administration. results: we collected prescriptions ( different drugs) concerning children (average age: years). sixty-two percent of medications were oral forms: % liquids, % tablets, % capsules. thirty-two percent of the tablets were cut; only % of those were authorized. due to the age, % of the tablets were crushed to facilitate administration whereas grinding was allowed in only % of the cases. most of the capsules were opened ( %) and % fractionated for an adapted dose. opening of capsules was possible in % of the cases. intravenous drugs represented %, the average injection was . % of the vial; in %, less than a quarter was given. conclusions: about % of pediatric preparations were inappropriate. the results of this study highlight the need to provide drugs adapted to pediatric care, which is one of the main tasks of the pharmacy. we are thus developing more appropriate pharmaceutical formulations for children, such as oral suspensions. a comparative statement of solubilization rates in liquid of crushed tablets or capsules after opening has to be established. moreover if a pharmacist is present in the care units, most medication error will be avoided. background and objective: the world's population is aging. the elderly have many chronic disorders and consequently use more drugs than any other age group. safe, effective pharmacotherapy is one of the results: fifty patients received antibiotics according to several schemes. nineteen patients received prophylactic treatments during . days on average. the employed molecules were mostly part of beta-lactams' family ( . %) including . % of co-amoxiclav. the prophylactic treatment seemed to be effective in . % of case because it wasn't followed by other treatment. thirty-one patients received probabilistic treatment: . % of them received quadruple therapies, . % received triple therapies, . % bitherapies, and . % monotherapies (only beta-lactams). the average duration of the probabilistic treatment was days. twenty-three patients got curative treatment. compared with the probabilistic treatment or with the treatment at the entry, the curative treatment corresponded to a reduction of the spectrum in % of cases with mainly an arrest of the vancomycin ( %). monotherapy was the most prescribed ( . %) and especially beta-lactams ( . %). then came bitherapies ( . %) and triple therapies ( . %). the most frequent isolated germs were escherichia coli ( cases), staphylococcus aureus ( cases), pseudomonas aeruginosa ( cases) and pneumococcus sp. ( cases) alone or associated, including . % of multiple-drugsresistant bacterias. the monitoring of aminosids and vancomycin was globally well carried out. only . % of vancomycin's dosages and . % of aminosides' dosages were not done. thirty-two patients increased their transaminases and/or their creatininemy. these increases were often physiopathological, related to multiviscérales failures or to septic shocks. the antibiotics could be clearly blamed in only one case. conclusions: in the surgical intensive care unit, antibioprophylaxy is longer than in recommendations. the probabilistic treatments used often associate active molecules on multiple-drugs-resistant bacterias. this can be explained by the gravity of infections, and the increased risk of bacteriologic resistances due to former treatments. moreover, bacteriological tests are systematically done, so antibiotics can quickly be adapt to the germs' resistances. background and objective: most of proton pump inhibitors (ppis) do not have legal mention for a paediatric use. however these drugs are largely prescribed to children. one disadvantage resides in the absence of liquid form which causes problems for their administration in nasogastric tubes. indeed, the absence of use recommendations involves many misuses responsible for inefficiency and/or tube obstruction. we tried to evaluate if ppis can be administered through paediatric nasogastric tubes. design: to quantify the transit of different ppis through paediatric nasogastric tubes and to optimise their modes of administration. setting: laboratory of clinical pharmacy and biotechnics. faculty of pharmacy. main outcome measures: we administered four ppis (mopral Ò , ogast Ò , inexium Ò , ogastoro Ò ) through nasogastric tubes by respecting their positioning in a child in a °elevation. for each ppi a study plan was drawn up to assess the influence of different variables: the volume of water to dissolve or put in suspension the ppis ( or ml), the rinse volume ( . or ml), the length ( or cm) and the diameter ( or french) of the polyurethane tubes. for every tests (n = ) we carried out an analysis of each active ingredient at the tube outlet by uv spectrometry. results: all f tubes were obstructed by ppis. through f tubes, we observed a mean recovery of active ingredient of % for ogastoro Ò , . % for inexium Ò but only . % for ogast Ò and . % for mopral Ò . the length of the tubes had no significant influence on the loss of ppi at the outlet of the tube. a water volume of ml instead of ml increased only the final concentration of inexium Ò (+ %). a rinse volume of ml improved significantly the transit of mopral Ò , ogast Ò and ogastoro Ò (+ . %, + . %, + . % respectively). this rinse volume allowed to obtain a . % recovery of lanzoprazole for ogastoro Ò whatever the water volume employed for its administration. conclusions: the most satisfactory results were obtained with ogastoro Ò : an administration volume of ml and a rinse volume of ml allowed a near-complete transit of lanzoprazole. under these conditions only % of inexium Ò was recovered. it is disadvised using mopral Ò and ogast Ò through f nasogastric tubes because no condition ensure the transit of an efficient concentration of active ingredient. keywords: nasogastric tubes, proton pump inhibitors, children background and objective: feeding tube occlusion is a frequent problem. practices to make the clogging off are very varied and are not the subject of any consensus. no study have assessed the impact of the different products on the inner surface of the tubes. in this context, it seams to be important to evaluate if these products are safe in order to rationalize the practices. design: to study the inner surface of nasogastric feeding tubes after contact with various products used to unblock them. setting: laboratory of clinical pharmacy and biotechnics. faculty of pharmacy. main outcome measures: we have put in contact f nasogastric tubes made of silicone or polyurethane with the following products: water, . % sodium bicarbonate, orange juice, pineapple juice, cola, papain syrup, pancreatic enzymes. an analysis of the inner surface of the tubes was carried out after , and days by scanning electron microscopy (sem). photos of unexposed tubes were used as negative controls. photos of tubes exposed to heat, ether or sodium hydroxide were used as positive controls. results: the analysis by sem shows that the silicone tubes are not altered by the different products tested. on the other hand, the surface of polyurethane tubes is modified in the presence of . % sodium bicarbonate and pancreatic enzymes. the papain syrup seems to settle on the surface of the tubes without altering it. water, fruit juices and cola do not modify the biomaterial whatever the exposure time. conclusions: . % sodium bicarbonate, pancreatic enzymes and even papain syrup should not be used in practice to unblock the feeding tubes. the orange and pineapple juices as well as cola can be recommended because of their harmlessness with biomaterials. keywords: nasogastric feeding tubes, occlusion, scanning electron microscopy pc- assessment of administration practices of extemporaneous formulation pediatric capsule preparations helene richard , anne jalabert , sylvie hansel-esteller pharmacy unit, lapeyronie and arnaud de villeuneuve hospitals, montpellier, france background and objective: to assess administration practices of pediatric capsules made as an extemporaneous formulation preparation by the laboratory of the pharmacy unit, in pediatric units. design: a questionnaire was designed and filled in by asking questions directly to nurses about their administration practices, from february to april . setting: altogether, six pediatric units were consulted, corresponding to the units for which the laboratory carries out the most pediatric extemporaneous formulation preparations. main outcome measures: the questionnaire concerned the ten most made up pediatric preparations, which are: amiodarone, warfarine, captopril, propranolol, ursodesoxycholic acid, omeprazole, fludrocortisone, spironolactone, hydrocortisone and calcium carbonate. items filled in were: hygiene rules, the existence of administration procedures in units, preparation conservation, and practical administration details. results: in months, questionnaires were filled in. concerning hygiene rules, nurses wash their hands before every manipulation, % wear gloves, % wear a mask or a mobcap. no administration procedures were available in units and more than % of nurses would like to have one. in every unit, preparations were conserved at room temperature, in a dry place, and % in a light-free place. for amiodarone, propranolol, fludrocortisone, spironolactone and hydrocortisone capsules, nurses use sweetened water (g %) to dilute capsule contents, sometimes milk (especially in newborns units). for the other drugs, the vehicules the most used were: sweetened water ( %), solid vehicules like yogurt, apple sauce, jam ( %), fruit juice or syrup ( %), milk ( %), and coca-cola ( %). the vehicule volume used fluctuates between ml and cl. nurses administer capsules by syringue into the mouth ( %), per os ( %), or by enteral nutrition ( %). conclusions: most of the administration practices of pediatric preparations are homogeneous in the different units (except the vehicule volume used). in the absence of administration procedures, the administration of preparations is more adapted to the child than to the drug. the aim of this study is to provide pediatric units with guidelines about good use of extemporaneous formulation pediatric preparations. services compared to a pharmaceutical care model. items pertaining to pharmacists' communication with the patient and the physician as well as the pharmacists potential to manage drug therapy were assessed. results: of the patients approached, ( %) response rate was achieved. cronbach's alpha = . (expectations) and . (satisfaction). females were ( %), married ( %) and % had post-secondary education. some % expect accurate dispensing of their medications, % expect pharmacists to simplify their medications and % expect the pharmacist to spend as much time as possible with them. expectations were resolved into components: humaneness, friendly attitude and professional competence. only % were satisfied with the professional appearance of the pharmacy, the rest of the items received dissatisfaction rating. two principal components of satisfaction were identified as humaneness and professional competence. marital status and level of education were associated with satisfaction scores. conclusions: patients' expectations of pharmaceutical care services were high but the satisfaction with current services compared to pharmaceutical care was below average. professional competence and humaneness were important dimensions of both patient expectations and satisfaction. there is a need to introduce pharmaceutical care. results: fourteen criteria were defined. a yes, no or not applicable answer has been given to each criterion according to the guidelines. drawing lots have been realised to set up a representative sample of twenty patients among those included in the study: six males, fourteen females, average age: . years old. two patients are below the average. the average mark of the patients is . %. hemoglobin level of % patients is inferior to the targeted hemoglobin level ( g/dl) at the end of the study. six criteria obtain a score superior or equal to %. five criteria obtain a score under %. the three criteria which obtain the best results concern the monitoring of the hemoglobin levels during correction phase and maintenance phase, and the maximum weekly dose of erythropoetin. the three criteria which obtain the lowest results concern the assessment of iron status before erythropoetin therapy starts ( %), the rate of increase in hemoglobin levels which should be - g/dl per month ( %) and the adjustment of total weekly erythropoetin dose. conclusions: the average grade obtained by the patients highlights the deviation from the guidelines. this indicates the needs of improving the anemia management in our haemodialysis unit. so it is necessary to make another time the team of doctors and nurses aware of the problem and to reinforce the pharmaceutic implication into the haemodialysis unit. a re-evaluation of practices (in the guise of clinical audit) must be planned in order to estimate the impact of the setting up of correctional measures. inclusion criteria were: oral and injectable drugs' prescriptions concerning patients who were present in the ward since more than hours. results: medical records were analyzed. patient age varied from to years old and the average duration of the hospitalisation from . to days. omitted variables were: patient's weight in all cases, prescriber's quality in % of cases, prescription's hour in % and prescriber's signature in . %. for each patient, all drugs prescribed since the beginning of the hospitalization in the unit was studied i.e. lines of drugs. we have noted the inn was absent in % of the cases, pharmaceutical form in % and administration route in %. only one prescription had all variables. thus, the global rate of prescription conformity was %. in the wards, nurses write the original prescriptions on a card-index (ci). then, drugs administrations are written on a temperature chart (tc) and on pharmacy's book (pb) for drugs order. we have compared documents one by one, for each drug. conformity rates are . % (prescription/ci), . % (ci/ tc) and % (ci/pb). the noted differences are multiple(modification of the administration or hour route, increase or dicrease of dosage…). the clinical relevance of non conformity was not studied. conclusions: from this study, it can be concluded that many prescriptions do not comply to the regulation and many retranscriptions are not identical to original prescriptions. it constitutes of course a potential iatrogenic impact. the development of computerized physician order entry (cpoe) in our hospital should be a corrective measure. the next stage will consist of realizing this study with cpoe and compared the studies. anne-claire buire , emilie prevost , françois lebargy , bertrand gourdier pharmacy, pneumology, reims teaching hospital, reims, france background and objective: assessment of antibacterial prescriptions regarding community-acquired lower respiratory tract infections and comparison with national guidelines. design: patients treated with antibiotics were included each day, during the analysis of the computerized prescriptions in the pharmacy. then, a report sheet was indicated in the unit with the prescribers for each patients with pneumonia or exacerbation of chronic obstructive lung disease. one month prospective study (in april ) included all patients hospitalised for pneumonia or exacerbation of chronic obstructive pulmonary disease (ecopd) in a unit of the pneumology department and treated with antibiotics. setting: pneumology department -reims university hospital. main outcome measures: for each patient the following data were assessed: age, hospitalization duration, diagnosis, severity factors, comorbidities and antibiotic prescriptions were analyzed. treatments were thought consistent when following the national guidelines [ ; ] . results: new patients were hospitalized during this month. ( %) were treated with antibiotics: with no pulmonary pathology and with pulmonary infection ( patients with pneumonia, with ecopd, with pleurisy, with exacerbation of asthma, with chest secondary infection, with acute respiratory infection, with thoracic pain and with cough). then, the study focused on patients: pneumonia and ecopd (m/f = / , mean age: years old). hospitalization duration was about days. patients ( %) had at least one co-morbidities factor (the majority: with pulmonary antecedents) and ( %) a severity factor (the majority: with an attack of the vital functions). antibiotic treatments were initiated in the unit and out ( in emergency department and by general practitioner). all treatments initiated in the unit were consistent with the recommendations and revaluated with the bacterial results (except one treatment) or changed because of bad tolerability. bacteriological documentation was always researched and the results were significant for patients ( %): streptococcus pneumoniae, pseudomonas aeruginosa, proteus mirabilis with morganella morganii and haemophilus influenzae. when it was possible, per os relay was realized in the at hours, except for patients (relay in the days). conclusions: in this survey, the management of pneumonia and ecopd was globally consistent with the national guidelines. nevertheless, the bacterial documentation is poor and the antibiotics prescriptions for bronchopulmonary infections are difficult and need using molecules with large spectrum. collaborate with all of the health care professionals and patients and to identify causes of errors. the unique position and possibilities of a pharmacist enable him to follow up the errors, from a fact that he is an expert on medication properties and is basically the last in a row who deals with drugs before their administration to the patients. the aim of this study was to describe and evaluate the role of pharmacist in identification and dealing with medication errors in czech pharmacy. design: pharmacists identified and recorded all medication errors over a period of six months of pharmaceutical care. basic characteristic of pharmacists: mean age . years; mean length of pharmaceutical practice . years; of them worked in the hospital pharmacy. of them got the first grade of attestation in pharmacy (by years of working experience in pharmacy and by passing exams). pharmacists collected the following data: types and causes or errors, their interventions, drugs and time concerning errors, subjects making errors and patient's characteristics as age, gender, other drugs used and co-morbidities. all the data were processed by descriptive statistics. background and objective: there is no standard of care to prevent oral mucositis for patients with cancer treated by chemotherapy. most common treatment is local (mouthwashes). no commercial mouthwash solution is available. special preparations are compounded by clinical units. a new . % sodium bicarbonate solution presentation was recently proposed for local treatment. our hospital drug committee decided to evaluate the clinical practices of oral mucositis prevention related to anti-neoplasic agents. design: an observational study conducted in pitié-salpétrière hospital. interview of nurses and physicians by pharmacy students about mouthwashes prescription and practice. data recorded on pre-established questionnaire and analyzed in pharmacy department using microsoft excel Ò . setting: study performed in oncology, hematology and radiotherapy clinical departments and in clinical units with oncologic activity (gastroenterology…). during one year, more than patients received anticancer chemotherapy. main outcome measures: questionnaire items were: use of a specific mouthwash procedure within the service, use of a single agent or in combination. data collected from the nurse point of view were: preparation and administration practices; from the physicians' perspective: circumstances of prescription and duration of the treatment. results: questionnaires were analyzed (nurses: , physicians: ) from clinical units. there was a standard written procedure in only one clinical unit. several formulations were used: each physician proposed his own, including antifungal prophylaxis (amphotericin b, nystatin), antimicrobial agents (povidone, chlorhexidine), mucosal surface protectant (sucralfate), alkalin solution (sodium bicarbonate), anti-inflammatory agent (aspirin), anaesthetic drugs (lidocaïn) and other agent (glycothymoline). preparations were not systematically labelled with patients name, formulation, date of preparation and stability duration. these were administered from to times daily regardless the stability compounding. for physicians, prescriptions of mouthwashes were done for patients with specific toxic anticancer drugs ( -fu, anthracyclin, capecitabin and sunitinib), were concomitant with chemotherapy and systematic after radiotherapy. conclusions: there are variations among clinical units in terms of mouth care regimen used. treatment efficacy was never evaluated. drug committee worked on guidelines in order to prescribe antifungal therapy only for curative aim or avoid anaesthetic drugs (swallowing difficulties). good practices included, before chemotherapy, dental hygiene. maintenance is realized by the patient himself (mouthrinses with alkalin solution or chlorhexidine). analgesics can be taken orally in case of mouth pain. design: the open, randomized, single-blind two-sequence, two-period crossover study design was performed. setting: under fasting conditions, each subject received a single oral dose of mg olanzapine tablet as a test or reference formulation on treatment days. the treatment periods were separated by a oneweek washout period. main outcome measures: the plasma concentrations of drug were analyzed by a rapid and sensitive hplc method with uv detection. results: the pharmacokinetic parameters included auc - h, auc -infinity, cmax, t / , and ke. the mean auc -infinity of olanzapine was . and . ng h/ml for the test and reference formulation, respectively. the maximum plasma concentration (cmax) of olanzapine was on average . ng/ml for the test and . ng/ml for the reference product. no statistical differences were observed for cmax and the area under the plasma concentration-time curve for test and reference tablets. % confidence limits calculated for cmax and auc -infinity of cefixime were included in the bioequivalence range ( . - . %). conclusions: therefore, the two tablet formulations were considered to be bioequivalent. conclusions: clinical pharmacists effectively validate % of these prescriptions but one third of prescriptions will inevitably remain validated by pharmacist residents during night and week-ends. performance criteria must be proposed to measure impact of pharmaceutical improvement initiatives (validation procedures writing, continuous education for example). we suggest to test clinical pharmacy activities related to economic indicators and to medication events reduction. background and objective: education of patients with type diabetes is a key point of non-medical management of that disease. thus objective was evaluation of patients' knowledge about their disease and its management for further development of pharmaceutical care protocols. design: prospective cohort control study. total patients casually were divided in two equivalent groups: study group (n = ), age . ± . , diabetes duration . ± . who were questioned by face-to-face technique and received pharmaceutical care during the interview; control group (n = ), age . ± . , diabetes duration . ± . and filled in questionnaires without clinical pharmacist. inclusive criteria were: presence of type diabetes mellitus, duration of the disease was not considered as criteria. patients were inquired once following standardized questionnaire. all included patients could read and write. two groups controlled their diabetes mostly by oral antidiabetics, only n = in study group and n = in control used insulin. setting: out-patient setting of lviv clinical hospital no , endocrinologist's office. main outcome measures: assessment of patients' knowledge about diabetes and self-monitoring in study and control groups. results: only . % and . % of inquired patients in both groups respectively stated that they possess good knowledge about diabetes. but as it was shown by evaluation of their level of knowledge through assessment of keeping to diet, regular physical activity, self monitoring it didn't conform completely. diet was implemented by almost % of patients in two groups, while regular physical activity was declared only by . % in study and % in control groups. smoking was reported by . % and . % respectively. next step was evaluation of self-monitoring. it has been revealed that only . % and . % of patients in two groups performed blood glucose monitoring at home; body weight was controlled by . % and . % respectively; blood pressure by . % and . % as well. target level of blood pressure was achieved by . % of subjects in study group and in . % -in control. in study group foot examination was performed everyday by . % of patients when in control only by . %. no one from both groups evaluated glycated haemoglobin regularly as it is recommended by american diabetes association, those they couldn't state their present or previous parameter. conclusions: it was estimated that knowledge about necessity of diet following, regular physical activity, and particularly -self-monitoring was very poor in diabetic patients, no regarding compliance which possibly also will be low. even if patients stated good knowledge about the disease they had problems with self-monitoring. it is obvious that patients of this out-patient setting require adequate education, which should be a component of pharmaceutical care program implemented by the clinical pharmacist. protocols of pharmaceutical care for these patients must be developed and include standard procedure of type diabetes patients' education. pharmacy department, hospital universitari vall d'hebron, pharmacy department, pharmacy department, hospital universitari vall hebron, barcelona, spain background and objective: patients who are admitted for a programmed orthopedic surgery in a tertiary hospital are usually elderly and present high co-morbidity which makes therapy complexity increase. the objective is to evaluate whether a pharmaceutical care is necessary before patients hospital admission. design: we analysed the domiciliary treatment in patients who attended to a pre-surgery visit. a form designed by the pharmacy department and validated by anaesthesiology unit was delivered to the patient to fill in with domiciliary therapy. the average of prescribed drugs was . per patient, being this reduced to . drugs in patients younger than and increased to . in patients older than . over one third of patients ( / ) were taken orally nonsteroidal anti-inflammatory drugs (nsaids). from ai, would need a pharmacological evaluation in the preoperative. those were included in drugs, meaning the . % of the different drugs prescriptions. this percentage belonged to patients. conclusions: the fact that a high percentage of patients over with an orthopedic pathology take a considerable amount of drugs at home makes it necessary to monitories the therapy in order to minimise the iatrogenic problems during the admission. we have seen that this situation is so frequently ( %) in our study, so the pharmacy department has established a collaborative job with anaesthesiologist unit to reduce as much as possible therapy problems. levation. through each tube, a ml nutritive solution was delivered on h each day during one week. after dissolution in ml water the ppis were administered once a day after stopping of the enteral nutrition and rinsing of the tube with ml of water. the tubes were then rinsed with ml of water and the nutrition was started again. during each administration of ppi the suspension was collected at the tube exit in order to quantify the ppi by uv-spectrometry. results: no tube was obstructed. the enteral nutrition mixture did not adversely affect the transit of lansoprazole through f nasogastric tubes. the transit of lansoprazole through the tube was complete and regular during the days of the study ( . ± . ). for esomeprazole the mean recovery of active ingredient was of . ± . (coefficient of variation: %) this variability can be explained by the incomplete and inconstant dissolution of esomeprazole because of the low volume of water usable in paediatry. conclusions: orally disintegrating tablet of lansoprazole can be administered through f nasogastric tubes in a concomitant way to an enteral nutrition mixture. for esomeprazole there is a variability of the administered active ingredient. however enteral nutrition doesn't seem to affect the esomeprazole transit. low volumes of water used in paediatry seem to be responsible for the variability. results: we analyzed pharmacological treatment in patients, with a mean age of years old ( - ). the average of drugs per patients was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . after checking the sources we detected possible pi ( severe, moderate and slight), observed times. pi were detected in of patients and mean per patient was . ( - ) . the following pi were described: increase of sedation ( patients), risk of bleeding ( patients), serum potassium levels alteration ( patients), hypotension ( patients), risk of hepatotoxicity ( patients), increase of creatin kinase levels and risk of myopathy ( patients). from all these, only two were observed: sedation which was observed in all patients and hypotension which permitted the reduction of patient antihypertensive treatment. conclusions: the most frequent pi was sedation which is considered beneficial in order to reduce anxiety in sci patients. arterial tension, electrolytic balance, hepatic and renal function, as well as risk of bleeding, are regularly controlled in the sci unit. this way, if one of these parameters was altered it would be easily detected. to conclude, it would be important to carry out regular checking to detect pi, especially for those drugs that are not usual and for those symptoms that are not controlled regularly in the sci unit. background and objective: pharmacists' individualized counselling of patients has positive impacts on the management of hyperlipidaemia, including improved compliance and better treatment endpoints. the objective was to evaluate patient knowledge on hypercholesterolemia and its treatment and to assess impact of pharmacist intervention at the lipid clinic. design: one hundred and fifty statin-treated patients were recruited by convenience sampling. following completion of a scored pre-intervention questionnaire, the pharmacist provided education on hypercholesterolaemia and the use of statins. a leaflet was prepared, evaluated and distributed to the patients. the patients completed again the same questionnaire after the intervention (post-intervention). results: patient demographics: % ( ) were males, % ( ) were females, mean age was years (range - years). a response rate of % was achieved with patients completing both questionnaires. following the educational intervention by the pharmacist, knowledge regarding the correct action to be taken if muscle pain or tenderness occur during statin therapy increased by % (p = ). the awareness regarding the normal total blood cholesterol level increased by % (p = . ) and the knowledge regarding the need for low-fat diet consumption during statin therapy increased by % (p = ). face and content validity of the patients' leaflet were strong. the average gunning fog index obtained for the leaflet was . indicating good readability for individuals. the leaflet was endorsed by the local health promotion department and copies were printed and distributed to patients. background and objective: in order to assess the performance of bayesian individualization of busulfan (bu) dosage regimens, venoocclusive disease (vod) rate was monitored for paediatric patients undergoing allogeneic bone marrow transplantation (bmt). design: consecutive patients undergoing allogeneic bmt with bu as conditioning regimen during five years period (january to febuary ) were retrospectively reviewed ( patients). setting: vod was major outcome variable. preconditioning risk of vod was estimated for each patient using a scoring system that included type of transplant, recipient cmv-positive status and total parenteral nutrition provided pretransplantation. a risk-adjusted cumulative sum method was used to compare observed versus predicted outcome by assigning a risk score, based on log-likelihood ratios, to each patient. main outcome measures: the cumulative scores were sequentially plotted with preset control limits for ''signalling'' where results were substantially different than expected (doubling or halving of odds ratio). results: sixty-six children received bmt after oral busulfan-based conditioning regimen with median age . years, . % of male. median preconditioning risk of vod was . range ( . - . ). observed vod rate was . % (n = ) which was . % ( patients) fewer than the expected number estimated by the risk score. the resulting risk-adjusted score for each patient was plotted sequentially. this plot adopted early a negative slope, crossing the lower control limit twice, after and patients, indicating improved results compared to those expected. background and objective: geriatric patients use numerous drugs; because they have several concurrent diseases. % of those years old and over have at least one chronic disease, % have or more chronic diseases ( ) . the purpose of this study is to evaluate data on the geriatrics' drug usage, assess the appropriateness of their drug treatment and identify their pharmaceutical care requirements. design: patients, who were years old and over and live in a nursing home in the anatolian part of istanbul, were included in our study. by interviewing the patients, individualized information was obtained regarding the drugs they used, dose and frequency of drugs, the purposes of medication use and side effects, and who suggested or prescribed the drug. the patients at risk of drug induced problems were defined and a risk map was developed. patients who have or more risk factors were accepted as being in a high risk category. setting: a nursing home. main outcome measures: the demographic, clinical and drug data of the patients were recorded. the pills count that patients used daily and totally; side effects of the drugs; knowledge of patient's diseases and drugs; risk category of patients were assessed. results: polypharmacy was identified in % of those included in the study (total of patients who take drug therapy). generally it was observed that the drugs were prescribed at an appropriate dosage and time; however % of the patients didn't know for what they were taking drugs. just ( . %) of the patients were aware of the conditions under which they should take drugs. of the patients ( . %) were not aware on how to and when they should receive their drugs. patients ( . %) were using their drugs by self-administration. the drugs of patients ( . %) were administered by their nurse. . % of the patients were receiving no medication. mean of number of drug used by patients was . ± . . . % of patients were in a high risk category. conclusions: as a result, drug effects alter due to polypharmacy, physiological and psychological changes. drug treatment should be individualized and monitored in geriatrics. according to our results, patients have lack of knowledge on drug use and they have never been educated in this matter. it is necessary to begin meeting their factor analytical technique can be either exploratory or confirmatory. exploratory factor analysis (efa) remains one of the standard and most widely used methods to demonstrate construct validity of new instruments. it is used to help development of the instrument by revealing items that may be made redundant from the questionnaire because they contribute little to the presumed construct. the seip consists of items in five domains and is scored on a four-to-fivepoint likert scale. the aim of the study was to assess further psychometric property of the seip using principal component factor analysis (pca). the strength of the inter-correlations among the items will be assessed by the presence of coefficients greater than . in the correlation matrix. if few correlations above this level are found, then factor analysis may not be appropriate. issues were identified including related to inr controls not performed and due to a poor/no adjustement of the oat dosage. % of the issues resolved following the pharmacist recommandations conclusions: computerized follow-up system allows to check inrs at the right date and to reduce the loss of results. pharmacists' recommendations were well accepted by physicians. the impact of the follow-up on the reduction of the overdose incidence will have to be evaluated. indications were hip (n = ) and knee (n = ) total replacement, other orthopaedic surgery of lower limbs (n = ) and pulmonary embolism treatment (n = ). indications were ''off label'' for patients with allergic reaction to heparine and were not documented. at least one of the haemorrhagic risks factors was identified in patients ( %). conclusions: fondaparinux is mainly used in approved indications. within the patients with haemorrhagic risks factors, no haemorrhagic accident was notified in the pharmacovigilance records of our hospital. nevertheless, fondaparinux has to be administred cautiously in this population of patients for whom the . mg dose will be necessary and hopefully available in a few month. diagnose group a streptococcal (gas) acute pharyngitis. this study aimed to evaluate the service; examine whether current clinical records were adequate; and test hypotheses investigating the association between clinical prediction rules (center criteria, cc), rapid antigen detection tests (radt) and antibiotic prescription. design: initially, a semi-structured interview was conducted to gain an insight into the service. data was retrospectively obtained from a standardised template (apef), completed by a pharmacist at the time of each patient visit. patient demographics and symptoms, radt results, cc scores and follow up rates were analysed. chi squared analyses were performed to investigate the aforementioned hypotheses. setting: jones pharmacy, spokane, washington dc. main outcome measures: establishing adequacy and effectiveness of clinical records and relationships between clinical prediction rules, radt and the antibiotic prescription. results: out of a total of patients (mean age = ; male = ) there were children (mean age = . ; male ) and adults (mean age = ; male = ). ten ( . %) of the negative radt results amongst children were referred to a primary healthcare provider. out of patients, ( . %) needed a radt to be performed. only ( . %) out of patients were followed up. significant association existed between radt outcomes and the prescription of antibiotics (p = . , p \ . ). conclusions: the findings from this study indicate that pharmacists need to be educated on the importance of a comprehensive clinical record. inconsistent practices occur amongst pharmacists due to the conflict between us guidelines, particularly relating to the referral of children with negative radt results to a primary care provider. the template was an efficient tool for data collection. jones did not tailor their data collection for the purpose of the study: with improved data collection, jones can yield information demonstrating the value of this service in future studies. only % of the rhophylac prescriptions. lg of anti d ig were administered when natead was available, whereas lg with rhophylac excepted times where , or lg were administered. times the pharmacist has induced a modification of the posology. however, none prescription was made according to the afssaps recommendations. prescriptions were made for children in hematology unit concerning boys and girls. among prescriptions of lg natead, % were correct and % for the rhophylac. the dose administered was not standard ( , or lg) and none prescription was conformed to the recommendations. conclusions: this study shows that since rhophylac was commercialized (more expensive than natead), the doses administered have doubled and the national recommendations (existent for adults only) are not followed. the reason is that hematologic patients may have a lot of transfusions so they want to protect them from any immunization, despite they are immunocompromised patients. new recommendations are needed, more applied to the practice and precising doses for children. the role of the pharmacist is then to remind physicians good practices of prescription. pharmacy, chu bicêtre, le kremlin bicêtre, france background and objective: computerized prescriptions are settled in clinical wards of our hospital. as pharmacist we have to validate those prescriptions and make recommendations to change part of the prescription such as dose, drug drug interactions. the objective of this study is to evaluate what kind of pharmaceutical interventions physicians really pay attention to. design: the prescriptions had been analysed for three months in three medical care units by two pharmacists. one of them take part in the physician round in order to integrate particular medical practices. pharmacist's interventions were recorded and categorized. the ratio of accepted interventions by the physicians was assessed. setting: three medical care units: internal medicine services (acute care unit: beds and long term hospitalisation: beds) and acute geriatric unit ( beds) in a -bed french university hospital. main outcome measures: description and analysis of pharmacist's interventions in clinical wards. results: we analysed prescriptions. interventions were performed and categorized. it should be noticed that % of pharmaceutical interventions were for misused of the new software: wrong selection of unit ( %) and redundant order ( %). the other % interventions were related to the prescription, our suggestions were as follow: time of administration ( %), adequate drug formulation ( %), dose adjustment ( %), therapeutic drug monitoring or biologic follow up ( %), to stop treatment ( %), route of administration ( %). among these recommendations, % were the consequence of drug-drug interaction. % of the interventions led to change in the prescription. % of the physicians maintained their prescription despite the recommendations, mainly for staggered administration to avoid drug interaction. background and objective: sliding scale insulin therapy (ssi) is a commonly used method of adjusting insulin in an attempt to control a patient's blood glucose levels. previous research investigating ssi use in the hospital setting has determined that ssi therapy leads to poor glycaemic control and poor patient outcomes. therefore, a corrective schedule for ssi therapy is recommended by the american diabetes association (ada). the aims of this study were therefore to determine, for the first time whether ssi recipients experience problems in the home care setting and whether ada guidelines on ssi use are being adhered to. design: eligible patients were identified through electronic records of patients admitted from st january to st december . the list was utilised to obtain medical charts of the patients. relevant information including patient demographics, blood glucose readings and documented problems were recorded using a standardised data collection form. chi-squared statistical analysis was determined using spss version . . setting: the visiting nurses association (vna) home care agency, spokane, washington state, usa. main outcome measures: use of 'traditional ssi therapy' versus 'corrective' version recommended by ada. results: of the total (male = , female = ; mean age = . , age range = - ) patient medical records examined in this study, . % (n = ) had at least one problem documented with their insulin regimen. the most common problem that affected over a quarter of the population, . % (n = ) was 'lack of control' which included any hyper-and hypoglycemic events. more than a third of sliding scale recipients, . % (n = ) had preprandial blood glucose levels above mg/dl. in total . % (n = ) of patients were prescribed the non-recommended traditional ssi therapy and only . % (n = ) were using the recommended corrective ssi therapy. conclusions: the findings of this study support previous concerns that ssi use is prone to problems and poor glycaemic control. furthermore, this study has established the lack of adherence to the ada recommended use of the corrective ssi schedule in the home care setting. it is hoped that this study will influence use of ssi in hospital and home care agencies as well as national and international guidelines. background and objective: prolonged (more than hours) mechanical ventilation (mv) is the most important factor associated with nosocomial pneumonia ( ) . nosocomial pneumonia (np) is differentiated in to ventilator-associated pneumonia (vap) if the process arose after the patient has been receiving at least h of mv ( ) . vap is defined as an inflamation of the lung parenchima caused by infectious agents not present or incubating at time mv was started ( ). design: longitudinal, prospective and observational study. setting: we made a prospective evaluation of the clinical files of a patient population of cases with vap diagnosed between april and december , who were assisted in hospital garcia de orta. a total of patients, % male and % female, aged ± years were diagnosed with vap. vap was defined as new positive respiratory culture after at least hours of mv. main outcome measures: these data sugest that the increase of pcr for documented vap caused by pseudomonas aeruginosa ocurred more frequently with ceftazidim than other antibiotics. results: in all vap episodes, an aetiologic microrganism, was isolated from hemocultures and bronchic secretions. the gramnegative bacteria were the most commonly isolated microorganisms ( %). we collected pcr and leucocytes data of seven documented schedules (n = ) for erradication of pseudomonas aeruginosa: ( ) ceftazidime ( g q h) + gentamicin ( mg/kg qd), ( ) ceftazidime ( g q h) + ciprofloxacin ( mg q h), ( ) piperacillin-tazobactam ( g/ mg q h) + aztreonam ( g q h), ( ) piperacillintazobactam ( g/ mg q h) + gentamicin ( mg/kg qd), ( ) piperacillin-tazobactam ( g/ mg q h) + amikacin ( mg/kg qd), ( ) imipenem-cilastatine ( mg q h) + gentamicin ( mg/kg qd), ( ) meropenem ( g q h) + gentamicin ( mg/kg qd). the mean duration of antibiotic therapy was days. there was an increase of the pcr values of the patients who were scheduled with ceftazidim but in the other groups there was a decrease of this parameter. the number of leucocytes didn't have any impact of the pcr variation (p [ . , t-test). considering the group of the patients, % ( ) of the positive cultures for pseudomonas aeruginosa which were sensitive to either ceftazidim or a carbapenem or piperacillin-tazobactam were treated with ceftazidim instead. conclusions: this approach provides useful information on the relation of host defenses and the clinical outcome. it is also useful to study the prevalence of acquired resistance to several antibiotics that may be used in documented antibiotherapy for pseudomonas aeruginosa. background and objective: rfviia is increasingly used as rescue therapy in uncontrolled bleeding, however little information is available regarding its safety and efficacy in this indication. -indication for use: massive bleeding when first-line treatment (surgical control of bleeding, use of blood products) has failed; -to achieve the correction of factors that may interfere with coagulation (hypothermia, severe acidosis, hypocalcemia); -before administration of rfviia the patient or his family should be informed about the treatment; -the prescription of fviia should be initialized by a referent physician -to conform the dose of mu/kg in cardiac surgery patient. conclusions: several solutions have been proposed to the physicians to improve their professional practices: to develop an algorithm for use of rfviia, to fill in a checklist before administration of rfviia to be sure to follow the guidelines, … this study will be extended in others departments using rfviia as gastroenterology and traumatology. pharmacy, administração regional de saúde de lisboa, pharmacy, administração regional de saúde de lisboa, lisboa, portugal background and objective: the pharmacist can contribute to the maintenance and recovery of population health conditions participating in patient house visit s health teams. pharmaceutical care at this level may also have a real impact on the health system costs. the aim of this project is to establish a pathway at this care level in which all aspects of pharmaceutical care are explained and defined. design: program description. participation of the pharmacist in health care teams. definition of field areas, between the pharmaceutical hospital care and the community pharmacy and health care centers. articulation between the hospital pharmacist, the health care center pharmacist and this new ''home pharmacist''. definition of the different types of pharmaceutical care to be implemented in this setting. background and objective: hip fracture is a major public health problem with a high incidence and prevalence in people aged years and older. changes in body composition and organ function, drug-drug interactions, and co-morbidities should be taken into account in the pharmaceutical care of this group of patients. the aim of this study is to analyse pharmacological treatment of elderly patients ongoing hip fracture in order to improve pharmaceutical care in this group of patients. design: a prospective pilot-study was performed during one month, (may-june ) in patients admitted in a tertiary hospital ongoing hip fracture. these variables were recorded for each patient: sex, age, body mass index (bmi), diseases antecedents, serum creatinine and creatinine clearance estimated by cockroft-gault formula, serum albumin levels, lymphocytes count, sodium and potassium levels. drug treatment was recorded from pharmacy database. setting: patients with hip fracture admitted in a tertiary hospital. main outcome measures: prescription profile in elderly patients with hip fracture. results: of patients, were female. mean age was . years old ( - ). mean bmi was . (n = , range - ). albumin levels were lower than . g/dl in patients. sodium levels were out of the normal range in patients. five patients had creatinine clearance lower than ml/min, of them less than ml/min. we analysed prescriptions which included drugs. they were classified in categories: not adjustment required ( drugs), adjustment required ( ) , inappropriate based in beer's criteria ( ), precaution in elderly people ( ) and not enough information available in geriatric population ( ) . mean number of drugs per patient was ( - ). of prescriptions revised, required adjustment and of them were correctly adjusted. of prescriptions in precaution group were correctly prescribed. conclusions: dosage adjustment or precaution was required in % of prescriptions. of these, % ( / ) needed dosage adjustment according to renal function. besides, % of patients had renal function alteration. thus, it is important to improve pharmaceutical care in this group of patients specially for those drugs that need dosage adjustment in renal failure. keywords: pharmaceutical care, hip fracture, elderly patients pc- hospital pharmacists and community pharmacists: an experiment of pharmaceutical information transmission carried out in an anticancer center anne lebreton , erwin raingeard , christelle audeval , sophie rochard anticancer center, centre rené gauducheau, nantes, france background and objective: to evaluate a programme of pharmaceutical information transmission from hospital pharmacists to the community pharmacists about drugs, particularly anticancer drugs, dispensed until then by hospital pharmacy and now distributed by them. design: while doing the last dispensation by the hospital pharmacy, an informative fax was sent to the community pharmacists indicating the name of the patient, prescribed drug and its posology, date and quantity dispensed, approximate date of the next dispensation and general information about the delivered drug. one month later a questionnaire was sent to the pharmacists to get their appreciation about this document. in the case of having no answer from them, the same questionnaire was re-sent. setting: pharmacy of french anticancer center, centre rené gauducheau, nantes. -appropriateness of the way of transmission -pertinence of information sent -efficiency of the programme results: out of patients treated ( vinorelbine oral, erlotinib, sorafenib, sunitinib), ( . %) were registered in this study, involving pharmacists. six pharmacists ( . %) answered after the first sending of the questionnaire and ( . %) answered after they received the remainder. fourteen pharmacists ( . %) did not answer. all the pharmacists were satisfied with the way of transmission. however, one of them suggested having the information sent by e-mail. seventeen professionals ( %) thought information was useful and ( %) thought that it was sufficient for their practice. only pharmacists ( %) encouraged us to continue the programme; the others did not express any opinion about its efficiency. moreover, none of them called us even though we suggested so. conclusions: this experiment seems to be interesting and to correspond to the needs of the pharmacists. it would also be an easy way to make dispensation safe. nevertheless, many problems appeared: much time spent and the difficulty making an exhaustive follow-up of all the patients thus limiting the application of this kind of programme to larger cohort. background and objective: to make a proposal of software that facilitates the analysis of clinical relevance of antiretroviral drug interactions, in the medical prescription, dispensation and dader methodology of pharmaceutical care study phase. design: pubmed and other databases evaluate revision. antiretroviral drug interactions were classified in four levels according to probability and severity of the interaction. the probability was grouped in categories: defined, probable and possible. so, severity was grouped in categories: serious, moderate, and slight. the levels are: level (serious and defined or probable); level (serious and possible, moderate and defined or probable); level (moderate and possible, slight and defined or probable) and level (slight and possible). we used the pubmed and database review for identified and organized the information to software elaboration. were by the enzymatic inhibition. antiarrhythmics, antihistaminics, ergot alkaloids, prokinetics, benzodiazepins, statines, calcium channels antagonists, phosphodiesterase inhibitors, azoles antifungics, selective serotonin reuptake inhibitors, opioid analgesic, immunosuppressants, macrolides, classic anticonvulsivants, and riphamicins were the most common drug therapeutic groups with anti-retroviral drug interactions. conclusions: interactions were classified, which % were drug-drug interactions; as well, . % were pharmacokinetics interactions and in their majority ( . %) were mediated by the enzymatic inhibition. about . % of interactions were level and , this levels of greater clinical relevance and whose the alert generated by software are contributions that help to analyze and take decisions with respect to the handling from the same ones. outcomes (e.g. mortality, morbidity, adverse drug reactions.); a profile of patients and clinical activities; an update of task description based on evidences and context. results: studies demonstrate that pharmacists have an impact on selected clinical outcomes following clinical pharmacy activities (e.g. drug therapy monitoring, pharmacokinetics and education for medical and paramedical professional). patients admitted in the intensive care unit have a higher level of complexity of care than average patients in the hospital, but a similar length of stay. for pharmacy services, their drug cost per admission is higher ( $cad vs $cad), as the number of pharmacist paid hours per admission ( . vs . ) and the number of pharmaceutical interventions per admission ( . vs . ). the approach helped us to identify solutions to problems like the nonparticipation to the cardiology patient's round, the absence of a medication reconciliation process or the inconstant documentation of interventions. a revised task description will be tested by both clinicians. conclusions: this study illustrates an approach for the evaluation of a pharmaceutical care model in a pediatric intensive care unit. background and objective: from , an increase in the consumption of biological and synthetic glues was noted in the cardiac service of surgery. these are drugs and expensive medical devices, which represent an expenditure of more than k€. the impact of their use was evaluated on the post-operative bleedings, the duration and the cost of stay by the way of a economic medical study. design: an observational exploratory study was carried out between january and march . all the operated patients were included. setting: the computerized consultation of the patient files made it possible to identify the following criteria of judgement: indication, surgeon, type of glue used, catch of platelet aggregation inhibitors or oral anticoagulants, volume of drainage, number blood transfused, duration of hospitalization. the statistical analysis related to two groups of patients, treated or not by glues, with a stratification on the hemorrhagic factors of risk. main outcome measures: the comparison of the averages of the various parameters was carried out by tests of student, for the large samples presenting comparable variables, and of fisher in the other cases. the cost of each hospitalization was calculated from the numbers of stays. results: during the three months of study, patients, whom average age was years [ - ], were operated. the two principal indications were the valvular replacement ( . %) and aorto-coronary bridging ( %). on all six surgeons, % of the interventions were dealt with by three. on the whole, patients receive a glue ( %), of biological type with tissucol Ò ( . %), and synthetic with bioglue Ò ( %), arista Ò ( %) and the grf Ò ( %). some patients received two types of glues ( . %). no significant difference between the two groups appeared in the total analysis. among the patients who received a pre-operative anticoagulant treatment ( %), only % were treated by a glue, for which the number of transfused globular bases and the duration of stay in reanimation were significantly lower (p \ . ; p \ . ), compared to untreated patients. conclusions: in general, the use of glues in cardiac surgery does not decrease the post-operative bleedings, but increases the cost of the stay. it however finds its utility with patients at the hemorrhagic risk. a standardization of the local practices of the surgeons is in progress because there is not any national consensus. these results should be confirmed by randomized studies on a large scale. although za is times more expensive than pa, it seems more effective with a shortened administration time ( minutes vs - hours). in respect of the contract of good use (cbu), our objective was to evaluate the real cost of a daily hospital (hdj) session per patient treated with bp, and to compare it with the one obtained from the national study of costs (enc) in order to determine if whether our hospital gained or loosed benefit from this new refunding status. design: retrospective study. setting: conception hospital, bd baille, marseille ce-dex , france. main outcome measures: for every hdj session, we accounted pharmacy direct expenditures (including implants, medical devices or drugs), medical technical acts, and structure and logistic supports costs. moreover, we evaluated matching of prescriptions with the cbu criteria. we analyzed data together with the public health and medical information ward and the department of management control. results: real costs of hdj session amounted to € without bp, and increased to € and € with pa and za administration, respectively. according to enc results, each session including intravenous bp was refunded for . €. in , on a total of hdj sessions ( patients), sessions had bp administration ( . %) and concerned patients ( . %). real costs of hdj were € ( € and €, for and hdj sessions with pa and za, respectively). if bp administrations were integrated in the ghs at this time, the sessions would have been refunded for € ( € and € with pa and za, respectively) resulting in a profit of € for the hospital. among bp prescriptions, % matched approved indications, . % were part of temporary protocol of use and . % were medical publication-based. internal medicine ward initiated % of the prescriptions. conclusions: using pamidronic acid compensates the deficit generated by zoledronic acid use. in respect with the cbu, zoledronic acid use will be restrained to approved indications in outpatients and pamidronic acid will be preferred in all others indications. keywords: biphosphanate, refund, economy pec- evaluation of the implementation of automated medication-dispensing system in an intensive care ward gregory gaudillot , marie antignac , fabien heck , nadine casimir , robert farinotti pharmacy, groupe hospitalier pitié-salpétrière, paris, france background and objective: the development of the implementation of automated medication-dispensing system in french hospitals is the main aim, defined in the ''good use'' contract voted in . the purpose is to improve the safety around the drug dispensing by the nurses. the objective of this work was to evaluate this implementation in thrusts: safety, economic and organization. design: comparison between two different organization ways: before the implementation (pharmacy order by nurses chief) and months afterwards (automated order and arrangement by a chemist assistant). setting: surgery intensive care unit ( beds) in the pitie-salpétrière hospital (ghps), a large teaching hospital. main outcome measures: study ''before and after'': follow up of prescribing and dispensing matches, analysis of time repartition between nurses and chemist assistants, follow up of line of emergency order, analysis of the results of a nurses satisfaction survey and costs study of drug consumption and drug storage. results: this study, performed over days, showed very small differences between prescribing and administered medications ( % before versus % afterwards = no significant difference). organization: implementation of automated device allowed a better division of activities, the management by nurses chief of the pharmacy order decreased (from % to % of their weekly working time), because this part was attributed to the chemist assistant ( % of their weekly working time). satisfaction: % of nurses preferred the automated system and especially because they find that this system was safe. in the same time, % of them appreciate with the new relationship with the pharmacy department. costs study: a huge decrease of the storage: - % of cost ( , € versus , €) and - % of references number ( versus ). in the same time, over months, the drug consumption of the unit has been reduced by % (- , €). conclusions: even if the study did not demonstrate a decrease of the number of medication errors (due to the tiny number of them as much before than afterwards the implementation), this automated system allowed a safety access to the drugs (biometric system) and contributed to reduce the risk of medication errors. the other great interest is the real involvement of the pharmacy in the clinical wards. the presence of the chemist assistant ensures a better management of drug storage (decrease of emergency order), a direct follow up and allowed a contact with nurses. results: in in france, intracranial stents with various technical characteristics are available (steel or nitinol, learning curve: to procedures, follow-up of patients is from months to years). stents with a hight radial force are used for intracranial artery angioplasty. more precisely, those devices are implanted in patients with recidive stroke, with an intracranial stenosis c % and who had failed medical therapy. two stents are identified in this therapeutic use: wingspan Ò (boston) and pharos Ò (micrus). about implantations are estimated for in france, whom implantations (cost = , €) in paris hospitals. the need is not actually clearly identified: the number of eligible patients is not already known and not systematically searched by neuroradiologists. so number of implantations will evolve. intracranial stents are used in combination with detachable coils embolization in patients with wide-necked cerebral aneurysms: léo Ò (balt), neuroform Ò (boston), entreprise Ò (cordis) and pharos Ò (micrus, double therapeutic use). for , about to implantations are estimated in france ( to % of endovascular treatment of intracranial aneurysm), whom implantations (cost = €) in paris hospitals. those data might increase. indeed, there is evidence of the effectiveness of stent implantation in intracranial aneurysm which generates lower rate of aneurysm recanalisation. conclusions: intracranial stents has been identified in precise therapeutic uses. however, an exact quantitative assessment can not be realised: those medical devices are innovating and are actually changing the management of patients concerned. those quantitative results might evolve. further similar studies will be necessary in order to follow up those innovating therapeutic uses in neuroradiology departments and to estimate their cost impact, actually negligible. keywords: stent, intracranial angioplasty, intracranial aneurysm, implantable medical device, cost evaluation pec- feasibility of economical impact of management of cytotoxic remainders in a centralized cytotoxic unit hélène corneau , déborah schlecht , sébastien bauer , sylvie froger , jacqueline grassin background and objective: the french law forecasts to reimburse the most expensive cytotoxic drugs to the real quantity administered to the patient. so to set up a secured procedure to use remainders of cytotoxic drugs in a centralized cytotoxic preparation unit in order to conform to the french law. design: prospective study. setting: clinic unit of oncologic pharmacy. main outcome measures: this procedure was tested during ten weeks and had concerned the three most expensive cytotoxic drugs used in digestive cancers. generated remaining quantities are conditioned inside isolator in radio-sterilized bags, and identified with the number of register of prescriptions, the drug's name, the remaining quantity, the conservation' conditions, the opening date, the duration of physico-chemical stability according to the data of the literature. bags are joined when they are gone out of the isolator. a theoretical differential is daily established between the doses which are prepared with or without management of remainders. results: the management of remainders involves that a rigorous manipulation during the conditioning and a daily management of the out-of-dates. the average of realized savings € a week, that represents , € extrapolated to one year and a decrease of . % of expenses for these three drugs. pharm world sci ( ) : - our software of the preparations of drugs is adapted for the use of the remainders. but the invoice-software allows only one invoice for one flask, which involves with the management of the remainders an unequal invoicing system for the patients conclusions: the management of remainders generates a real financial profit. but the really administered quantity cannot be imputed to the patient because the software of inventory control and invoicing does not manage the fraction of flasks. nevertheless, according to the french law we must express under fractional shape the quantities of cytotoxics, which are administered by stay to the patients for the most expensive drugs. background and objective: since july , financing of drugs in belgian hospitals is based on a lump-sump system. this decision favours efforts leading to more rational use of medication like for example the sequential treatment. efforts like posters, recommendation letters and information rounds were part of the strategy. since - - a clinical pharmacist puts also attention on this subject by contacting physicians and nurses regarding individual drug therapies. documenting the results of interventions suggested by a clinical pharmacist is often quite difficult. the number of ddd of intravenous administered drug versus the total amount of ddd [oral + iv] of the particular drug administered has been described. this parameter can be disturbed by the use of the oral form over a long period of time or by the early discharge of patients combined with a continuation of the therapy at home. to follow up the sequential treatment on the emergency department, this parameter seems to be accurate by the fact that the stay of the patient here is between and days. cost savings by sequential treatment of paracetamol and levofloxacin were euro and euro, calculated from the start of the activities the clinical pharmacist (for months). old habits are difficult to change among them the administration of drugs by intravenous route. almost every medical and surgical patient admitted to the emergency department receives an intravenous line, so the threshold for intravenous administration is low. furthermore a wide variety in patients' medical conditions added to a rapidly changing timetable for technical examinations and surgery necessitates an individual approach of the most suitable route of drug administration. official letters and posters may be useful. however this report confirms the importance of a clinical pharmacists' permanent presence on the ward for maintaining awareness of sequential treatment. results: applications for compassionate use treatment were processed ( . processings/ inhabitants). the higher number of applications has corresponded to gynecology unit, processings of misoprostol to use in delayed curettage. secondly, botulinum toxin was processed in cases, . % of them corresponding to anaesthesic and reanimation unit for miofascial pain. active substance that has caused a higher impact on the hospital s budget was inhalated tobramicin, processed for a total of patients with bronchiectasias colonized by pseudomonas aeruginosa. the expenses for this indication has added up to , € during . secondly, as regards to economic impact we found botulinum toxin which raised up to , €, expenses that were attributed in % to anaesthesic and reanimation unit. the highest cost/treatment by patient during corresponded to inhalated tobramicin that has increased up to , €, followed by infliximab approved for hydrosadenitis treatment which accounted for , €. total cost of treatments for compassionate use has involved approximately . % of total consumption for drugs during . conclusions: the higher number of processings in corresponds to misoprostol requested by gynecology unit although its economic impact on the consumption of medicinal products for compassionate use is very low. inhalated tobramicin utilization for colonized bronchiectasias involves the highest global cost and the highest cost/treatment on total medicinal products for compassionate use. the percentage accounted for medicinal products for compassionate use on total consumption of medicinal products is low. it would be interesting to perform a multicenter study in order to value economic impact in last years of medicinal products for compassionate use. the vacuum-assisted closure (vac) therapy: a -months medico-economic retrospective study stéphanie roche , nathalie herment , sandrine havet , amélie pruvost , willemin jean-claude , frances carole pharmacie, reims teaching hospital, reims, france, background and objective: in the management of wounds care, spectacular results have been achieved through the application of negative pressure wound therapy. this approach known as vacuum-assisted closure (vac) involves the use of a defined controlled negative pressure (delivered by an ambulatory motor) over a polyurethane or polyvinyl sponge (which are considered as consumables) placed in the wound. in our hospital, this therapy was first introduced for acute traumatic wounds. in june , the interdisciplinary wound and cicatrisation group decided to extend indications to chronic wounds. to improve management regarding this larger and complex use through all units, a specific prescription form associated with recommendations was set up. this document is available as well as paper or electronic form. the purpose of this study was to evaluate conditions of use and global costs of vac therapy. design: a -months retrospective study was based on the analysis of nominative prescriptions of vac consumables (canisters with gel, small, medium or large foam dressing kits and y connectors . the cost of consumables was estimated to . euros per day per patient. total cost over the study period amounted to euros, including the hiring of the vac system ( . euros per day). these data indicate that the use seems to be appropriate and optimized without overuse. conclusions: facing the high cost of this technical therapy, its use must be closely managed. this study suggests that the multidisciplinary collaboration in our hospital between medical staff and pharmacist unit contributes to guarantee the optimal use of this specific therapy. to analyze the effectiveness of an educational programme to increase the adrs reports during this period. design: clinically relevant events possibly caused by exposure to drugs have been analysed in a retrospective study for a period of five months. we analysed the prevalence of adrs reported, the medical conditions of the patients, the relationship between adr and the suspected drug using karch-lasagne algorithm and the severity of the reaction using who criteria. during this period, we implemented a pharmacovigilance programme in order to increase the reports and to estimate the prevalence of adr in this unit. setting: pharmacy service and critical care service of a general hospital main outcome measures: to evaluate prevalence, characteristics, nature and severity of adrs in uci. to measure the effectiveness of an educational programme to improve the awareness and detection of adrs. results: a total of patients were hospitalized during this period, adrs were detected ( . %). severe sepsis and cardiac arrest were the most frequent diagnosis in this group of patients. . % males and . % females, . years in average. dermatologic effects as urticaria rashes and haematological effects as pancytopenia were the most frequently noted, with more than % each. therapies most often associated with the reported events were antibiotics (piperacillin/tazobactam, ertapenem, azitromicin) in . % of cases and nitroglycerin in . % of them. the adr reported were classified as low severity in . % of cases, medium in . % and high severity in % of them. level of causality more frequent was ''probable'' in a . % of the reports followed by ''possible'' in . % of them. since the implementation of the educational programme the number of reports of adr have increased from . % to . %. the high level severity of adrs reported has increased in this period from % to %. conclusions: communication and educational programmes should be implemented to promote detection, identification, reporting and evaluation of adrs. the analysis to determine the probability, causality and severity of adr is necessary to establish the measures needed to improve the security and the quality of health attention. background and objective: ace inhibitors are used for controlling blood pressure, treating heart failure and preventing kidney damage in people with hypertension or diabetes. although ace inhibitors are generally well-tolerated by the most individuals, they are not free of side effects. dry cough is one of the most common side effects seen in patients during ace inhibor therapy. in this study, we have evaluated the incidence of dry cough that appears during the ace inhibitors therapy and relationships with the other coughing factors and the other side effects that may appear. design: the questionnaire was applied on ambulatory patients ( m/ f) who have used or been using ace inhibitors and hypertension patients as control group who use also anti-hypertensive drugs except ace inhibitors. ace inhibitors, and the reactions against the dry cough were determined by pharmacist's questionnaire on the patients who come to the community pharmacy. results: dry cough was observed on the patients out of , during their using of ace inhibitors ( %). % of male patients and % of female patients were having a cough. the patients out of ( %) from control group were having a cough. the incidence of dry cough that appears on the patients who use ace inhibitors were; silazopril %, ramipril %, lisinopril %, fosinopril %, qinapril %, perindopril %, enalapril % and trandolapril %. the treatments of patients out of who complain from coughing during the therapy of ace inhibitors were changed with angiotensin receptor antagonists and calcium antagonists by their physician. treatment changes were resulted in increasing in the cost by . ytl monthly if it was calculated on the base of generics. conclusions: as a conclusion, the incidence of dry cough from ace inhibitors was found to be % in the blacksea region of turkey. the pharmacist can play an important role in determining side effects such as dry cough and refer the these patients to physician. patient counselling and drug therapy monitoring in the community pharmacies will increase the compliance and provide better outcomes in many chronic diseases. setting: community in _ istanbul main outcome measures: visual analouge scale (vas) was used for assessment of pain. the universe of the study consisted of randomly selected women and men (n = ). a pilot study was run on individuals from different occupation groups to determine the validity and intelligibility of the questionnaire. results: the data were analyzed using a spss . program. the level of significance was accepted as p \ . . the results show that women experience more intense pain than men; their mean vas score is higher than men ( vs ). headache is the most common type of pain ( . %) and also its vas scores reached the highest level ( ) ( ) . this pain is especially caused by migraine and hypertension. in addition, % of questionees prefer to take an analgesic drug in order to manage their pain problem. it was recorded that especially non-steroidal antiinflammatory drugs (nsaids) and preparations containing paracetamol are the first choice in pain management. most respondees took analgesics when pain begun ( . %), when pain increased ( . %) or when was intolerable ( . %). conclusions: the majority of those who participated in the study took their analgesic medication without consulting a health professional. this indicates a high level of self medication in our population. this gives rise to a number of potential drug problems such as nsaids usage in gastrointestinal disorders, hypertension and renal failure. although many participants used their drugs in a rondom and improper way, unfortunately the pharmacist was rated second to last as a drug consultant. background and objective: many patients visit the pharmacy for their oral problems like toothache and ask for appropriate pain relievers. the purpose of this study is to examine the attitude and role of pharmacists, dentists and non-health workers towards solving of dental and oral health (like management of toothache). design: different questionnaires were applied randomly on pharmacists, dentists and non-health workers. setting: canakkale -turkey. main outcome measures: dentists' and pharmacists' approach to patients with toothache, drug usage evaluation in dental problems, the type of information given by pharmacists, patient behaviors. results: % of non-health workers (n = ) indicated that if they complain about toothache they choose their pharmacist as their consultant. % of non-health workers (n = ) indicated that they use various medications without consulting a dentist ( % naproxen sodium, % paracetamol, % methimazole, % flurbiprofen, % amoxicillin, % carnation essence). the most common suggested antibiotics was amoxicillin ( %) by pharmacists and amoxicillinclavulanate ( %) by dentists. the most common suggested pain relievers were naproxen sodium by both pharmacists ( %) and dentists ( %). only % of dentists declared that they consult with a pharmacist about drug usage. % of dentists indicated the importance of consultation of patients by pharmacists. conclusions: according to our results; pharmacists must take an important role in prevention and management of oral and dental health problems including informing the patients about convenient drug usage. also collaboration between dental staff and pharmacists need to be improved. background and objective: in hibbard and smithells suggested a link between inadequate maternal intake of folic acid and neural tube defects in their offspring ( ) . consequently, it has been recommended that all women planning to become pregnant should consume additional folic acid before conception and during the first weeks of pregnancy. despite these recommendations, periconceptional intake of additional folic acid is still low in many developed countries and a substantial percentage of women are not aware of its benefits ( ) . design: a questionnaire was used in a face-to-face encounter. setting: postnatal wards of a teaching and a private hospital in iran. main outcome measures: awareness of the effects of folic acid on the fetus was evaluated among women. the questionnaire included questions about demographic information, folic acid supplementation before and during pregnancy, its effects and the most susceptible periods in pregnancy and the source of information regarding the drug's effects during pregnancy. results: the mean age of women was . (± . ) years old. the majority of the subjects had more than high-school education ( % vs. %). out of subjects, ( . %) took folic acid supplement before and during pregnancy. only . % believed that its usage was unnecessary, . % believed in its positive effects. in the subjects' opinion, the most important time for taking this supplement was the first trimester ( . %), then prior to pregnancy ( . %). the second and third trimester were noted important by . %. . % believed in the importance of this supplement during all nine months. the advisor for taking this supplement was doctors ( . %), health visitor ( . %), self-medication ( . %), tv and radio ( . %), family members and friends ( . %) and pharmacist ( %). conclusions: awareness of the value of periconceptional folic acid was high among women of iranian nationality compare to similar studies ( ) . the majority of the participants believed in the positive effects of folic acid. the advices provided by doctors and pharmacists had the greatest and least effect on the use of this medication. regarding the best time of usage of this supplement, the most emphasis was on the first trimester and next on prior to pregnancy. background and objective: diabetes mellitus type-ii is a metabolic disorder that is primarily characterized by insulin resistance, relative insulin deficiency, and hyperglycemia. before type ii diabetes stage, people almost always have ''pre-diabetes''; in which blood glucose levels are higher than normal but not yet high enough to be diagnosed as diabetes. while some people with type-ii diabetes have symptoms, the majority may go - years without apparent symptoms. because some of the symptoms for diabetes mimic other diseases or conditions, makes it harder to predict an precise diagnosis without any additional information. the purpose of this study is early diagnosis of pre-diabetes, prevention or delay of the complications with a collaboration of community pharmacists and patients. design: pharmacists used a structured questionnaire containing questions concerning demographic data and informations which indicate prediabetes symptoms. setting: four community pharmacies in istanbul. main outcome measures: data: age, gender, body mass index (bmi), genetic predisposition, blood pressure, pysical activity and hypoglycemia symptoms. results: one hundred people were screened for undiagnosed diabetes. and the risk for pre-diabetes is evaluated according fasting plasma glucose (fpg) and total oral glucose tolerance (ogtt) test results. blood glucose level between and mg/dl with the fpg test and and mg/dl with ogtt test are considered as pre-diabetes. conclusions: diagnosis of pre-diabetes can prevent the development of type ii diabetes and making changes in nutrition and increasing the level physical activity may even be able to return the elevated blood glucose levels to the normal levels. pepi- awareness among pregnant women of the effects of drugs on the fetus and mother maryam dilmaghanizadeh , simin mashayekhi , masoud naghizadeh , zahra fardiazar , roghaiieh bamdad moghaddam pharmaceutics department, the faculty of medicine, tabriz university of medical sciences, tabriz, iran (islamic republic of) background and objective: since the talidomide catastrophy [ ] concern about the safety of drugs in pregnancy has been increasingly evident. studies have revealed that pregnant women continue to take considerable quantities of drugs. however, all pregnant women worry about whether to take any medications. because of an estimated % of birth defects resulted from maternal drug exposure [ ] , this fear is well justified. little is known about the knowledge of pregnant women regarding the safety of medications during pregnancy. to our knowledge the present study is the first performed in iran. design: a questionnaire was used in a face-to-face encounter. setting: postnatal wards of a teaching and a private hospital in iran. main outcome measures: awareness of the safety of drug use during pregnancy among women. the questionnaire included questions about demographic information, drugs use before and during pregnancy, information regarding the safety of drugs during pregnancy and the most susceptible periods in pregnancy, the source of information regarding drugs' safety during pregnancy. results: the mean age of women was . years old. only an . % and . % used conventional medications and herbal remedies during pregnancy, respectively. a great percentages ( . %) believed in harmfulness of drugs during pregnancy, but only a . % believed in harmfulness of herbal remedies. the first trimester and the second trimester were believed to be the most and the least susceptible period, respectively ( . % vs. . %). the sources of information for the subjects regarding the safety of medications was specialist doctors ( . %), general practitioner ( . %), pharmacist ( . %), midwives ( . %), health center ( . %), media and books ( . %) and friends and family member ( %). discussion: a common concern about the care of pregnant women involves the medications, which led us to establish a special initiative to review available knowledge among our general population. the present study highlights weakness of the role the pharmacists play in providing the information to this vulnerable and eligible group of people, who nourishing our next generation. conclusions: a common concern about the care of pregnant women involves the medications, which led us to establish a special initiative to review available knowledge among our general population. the present study highlights weakness of the role the pharmacists play in providing the information to this vulnerable and eligible group of people, who nourishing our next generation. ayce celiker , nergiz nemutlu , gulru ozkaya hacettepe drug and poison information center, hacettepe university, ankara, turkey background and objective: poisoning casualties require be approached with utmost attention due to their ''medico-social emergency'' nature. children are more vulnerable because of their inherent interests for knowing the environment besides ignorance and carelessness of adults. drugs are among the leading offending agents in children poisonings. the services of drug and/or poison information centers have been regarded as one of the main challenge areas of clinical pharmacy practice. being a pioneer in turkey, hacettepe drug and poison information center (hizbim), has been run for years in working hours basis. objective: the objective is to evaluate the demographic and epidemiological characteristics of drug poisonings in children, thus, to contribute to clarify the actual ''intoxication profile'' and identify necessary measures for the children in turkey. design: the data of childhood (aged \ years) poisoning enquiries received by hizbim between january , and december , were collected retrospectively and analyzed with spss . Ò setting: hizbim is affiliated to hacettepe university, faculty of pharmacy main outcome measures: categorization and comparison of data of children intoxicated with drugs during ten years results: children were involved in % of all poisoning cases and % of those cases were due to drugs. there was no gender difference among very young children, however, girls dominated as the age increases (p \ . ). % of the cases were accidental, and analgesics were involved in % of the accidental poisonings (p \ . ). while the most offending agent group was analgesics in children younger than years (p \ . ), multiple drug ingestions were the main causes of the cases involved older children (p \ . ). multiple drugs were mostly encountered ( %) in suicidal attempts where the dominant gender was girls ( %) (p \ . ). the most frequently reported symptoms indicated central nervous system involvement almost in all intoxication cases and in all age groups. conclusions: pediatric poisonings are rather high in turkey like many other countries and drugs are accounted for mostly in those injuries whether accidental or suicidal exposures. some regulations, the attitudes of physicians, pharmacists, and parents and other care givers make contributions to that outcome. in the prevention of childhood drug intoxications it is essential to make cooperation between drug manufacturers, regulatory authorities, health professionals, and families, besides increasing social awareness. drug and/or poison information centers stimulate rational drug use through providing accurate and rapid information to health care providers, educating people directly, documenting current epidemiological data. recently, we noticed a great interest of pharmaceutical companies in drugs such as monoclonal antibodies or in diseases such as inflammatory rheumatism. the aim of this study is to put in evidence the orientations of biomedical research. we particularly analysed the pathologies concerned, the type and the target of the experimental drugs, and the aim of the biomedical researches. design: analysis of the protocols and of the investigator's brochures of the ongoing clinical trials in january . setting: clinical trials sector of lapeyronie-arnaud de villeneuve hospital, montpellier. main outcome measures: methodological aspects were first analysed: aim of the study, phase, type of sponsor, type of binding, inclusion rate. then, we focused on the experimental drugs involved: type of drug, target, therapeutic area concerned. results: % of clinical trials are promoted by pharmaceutical companies. % are phase trials, % are open-label trials. objectives can be found: testing a new molecule on a new target ( %), testing a new molecule in an known pharmacological group ( %), testing a new formulation ( %), testing an known molecule in a new indication ( %), evaluating a therapeutic strategy ( %). % concern rheumatology, % pneumology, % infectiology, % haematology, and % nephrology. the most frequent pathologies concerned are inflamatory rheumatism ( %), malignancies ( %), asthma/cobp ( %), hiv infection ( %), and post renal transplantation immunosuppression ( %). the percentage of patients included (number of patients in the trials concerned/total number of included patients) and the inclusion rate (real number of inclusions/ number of expected inclusions)are respectively % and % in rheumatology, % and % in pneumology, % and % in haematology, % and % in infectiology and % and % in nephrology. % of experimental drugs are injectable. % are little molecules obtained by chemical synthesis, and % are issued from biotechnologies: monoclonal antibodies ( %), peptide ( %), and gene therapy ( %). the new targets ( % of clinical trials) are receptors ( %), enzymes ( %), gene transcription ( %), cytokines ( %). conclusions: this analysis gives an idea of the future commercialized drugs. it puts in evidence the development of drugs in therapeutic areas which concern a lot of patients and whose financial rentability is high. furthermore, only % of clinical trials concern new drugs and new targets. development of me-too and optimization of therapeutic strategies are the most frequent clinical trials. background and objective: the increasing use of performanceenhancing substances and methods in sport threatens not only the meaning and the ethical value of the sport itself, but also the health of the athletes. this study aims to assess the drug utilization profiles of the amateur football players, as well as their attitudes and knowledge on ''performance enhancing drugs''. design: this study was conducted on male players of the amateur football league. the players were asked to fill in a standard questionnaire, where information about their drug utilization profiles as well as their attitudes and knowledge on ''performance enhancing drugs'' were sought through various questions. setting: various amateur football clubs in turkey. main outcome measures: drug consumption rates of the players. answers to the pre-prepared questions. results: the age of the players ranged between and years. forty-four ( %) players thought that drugs have a positive impact on sports performance; while % did not share this idea. the resource of this idea was a team-member for . %, the trainer for . %, sports magazines for . % and a family-member for . %. the players were asked to name the drugs that could be used for performance enhancement and . % replied as vitamins, while . % replied as central nervous system (cns) stimulants and . % replied as anabolic steroids. thirty-six players ( . %) reported that they use various drugs with the aim of performance enhancement. the drug utilization profile was as follows: vitamins were consumed by . % of the players; where cns stimulants, anabolic steroids, diuretics and growth hormone were consumed by . %, . %, . % and . % of the players, respectively. the players reported the reasons referring them to drug-use as follows: . drugs always have a positive impact on sports performance ( . % of the drug-users); . drugs are necessary in case of inadequate training ( . % of the drug-users); . performance enhancement leads to individual success in the team and this brings prices and rewards in return ( . % of the drug-users). about % of the players thought that cns stimulants have the main effects of increasing the heart rate, endurance and strength. the main potential adverse effects of the cns stimulants were reported as gastrointestinal problems, dependency and tachycardia. about % of the players thought that anabolic steroids increase muscle volume, endurance and strength. the main potential adverse effects of the anabolic steroids were reported as hypertension, hepatotoxicity and dependency. conclusions: the results of the questionnaire suggests that drug-use with the aim of performance-enhancement was common among the amateur football players; and the players were not adequately and properly informed on the effects and adverse effects of the (so called performance-enhancing) drugs. this reality yields new responsibilities in this challenging area of practice, for the clinical pharmacist. background and objective: psychiatric disorders and opiate misuse are associated with chronic pain syndromes, but their incidence in fibromyalgia (fms) is unknown. the aim of this study was to identify if the incidence of affective disorders, and opioid misuse, was more common in patients with fms than those with other forms of nonmalignant chronic pain. design: a prospective, cohort study was carried out involving patients, who were internally referred to a chronic pain management program. subjects with a working diagnosis of fms were matched by age (mean = ) and sex ( % female) and compared to a control group of patients with alternative forms of non-malignant chronic pain. individuals were compared using urine toxicological screens, drugrelated criminal convictions, diagnoses of affective disorders, and responses to the following inventories: screener and opioid assessment for patients in pain, the pain disability index, the personal health questionnaire and the fibromyalgia impact questionnaire. patients also underwent a standardised physical examination using american college of rheumatology (acr) guidelines to diagnose fms. setting: this was a quantitative, hypothesis-testing cohort study, conducted in an academic general internal medicine practice. main outcome measures: diagnosis of fibromyalgia, clinician's awareness of acr guidelines, pain intensity and impact of fibromyalgia on physical and psychosocial activity. results: response rate was % (n = ; mean age = , median = , age range = - ; male = ); the most common background and objective: the increase in the frequency of the metabolic syndrome and its implication, in the development of ischemic cardiovascular disease and type ii diabetes mellitus, represent a real public health problem and of much interest in the medical field. otherwise, the cardiovascular pathologies are twice more frequent among chronics psychotics patients, for that reason we were interested in the prevention of these pathologys. this phenomena have been accentuated with the arrival of second generation antipsychotic drugs which were associated with weight gain, disorders of glycemia and lipidemia. in the aim to elaborate recommendations, at first we proceeded to the assessment of biological and clinical follow-up of the patients hospitalized in the saint-egreves hospital. design: literature review, months prospective study. setting: we asked doctors of each unit ( ) to answer questionaries corresponding to patient files. we worked out a general questionary to avoid revealing our objective. main outcome measures: blood pressure, body weight, height, abdominal perimeter, glycemia, total cholesterol, triglyceridemia, cholesterol's fractions(hdl and ldl). results: our results showed a good clinical follow-up but the frequency of biological control was not sufficient. the blood pressure and weight were evaluated respectively in and % of the patients, the total cholesterol, glycemia and triglyceridemia were in % of patients. on the other hand, cholesterol's fractions (hdl and ldl) were rarely evaluated. as for the abdominal perimeter, where the increase is predictive of cardiovascular disease, is never measured. conclusions: it seems difficult to evaluate the risk of either cardiovascular disease or metabolic syndrome of these patients or to determine if there is any possible relationship between the nombre of cardiovascular risk factors and the apparition of this syndrome. these findings imply to identify these high risk subjects and to define the optimal preventive, or curative, management strategy of metabolic syndrome and this, through simple measurements to realize in clinical practice. background and objective: availability of a medicine in western markets can be delayed either due to differential submission strategies of companies or differences in review process between countries. the aim of this study was to examine delays in patient access to medicines for compounds approved by two or more authorities (us fda, eu emea, australian tga, health canada and swissmedic), by characterising potential drivers for new active substances (nas) approved between and , from both a company and regulatory agency perspective. design: nass approved by fda since were compared to nass approved by the other agencies. this data was analysed comparing the difference between submission and approval dates and characterised by; type of approval route, company size, and therapy area. setting: data on nas's approved by the authorities was collected from agencies and from public domain sources. main outcome measures: the difference in patient access to new medicines in different countries and factors influencing such differences. results: nass have been approved by fda and one or more of the agencies studied. the median time ranged between submissions to fda and another authority from days at emea to days at tga. the difference between approval dates ranged from a median of days at emea to at health canada. however these differ depending on company size, therapy area and approval route. conclusions: availability of a new medicine is a mixture of company submission strategies and approval process, although therapeutic profile of submissions and company size are also influencing factors. in europe the main driver to patient access is review timelines rather than delay in submission by companies. main outcome measures: data on sex, age, origin of ppi prescription and indication were collected by a standardized questionnaire and were retrospectively analysed. results: men and women were reviewed. the median age was years (range, - ). % of patients received ppi therapy by pantoprazole (available in our hospital) when hospitalized. % of patients received daily mg of pantoprazole and % received mg a day. % of the prescriptions were validated. the main off-label indications were prevention of hemorrhagic risk of antiplatelet agent ( %), hemoglobin decrease( %), anticoagulant co-prescription( %), steroids co-prescription( %). conclusions: this prospective study confirms the large prescription of ppi therapy in a department of internal medicine. nevertheless, this study highlights the difficulties to interrupt this well tolerated therapy after the first prescription by family physicians. clinical pharmacist interventions in the department consist of explaining the difference of indications between pantoprazole mg and pantoprazole mg, he makes physicians aware of prescribing ppi therapy with a cautious reweighted cost/benefit consideration. background and objective: to develop and validate a system for regulatory authorities to provide feedback to companies on the quality of their submissions while companies report to authorities on the quality of the review. a standardised report format will allow performance comparisons within and across companies and agencies. design: draft scorecards were tested in a study on the same four compounds, each reviewed recently by the agencies in australia, canada and switzerland. the agencies provided feedback on the quality of submissions and sponsors, astrazeneca, gsk, novo nordisk and pfizer gave views on the conduct of reviews. background and objective: adherence with chronic medication such as inhaled corticosteroids (ics) has repeatedly been reported to be low. non-adherence could be related to inadequate knowledge of ics' actions and lack of ics' instructions on the use of inhalers. this has not been reported previously to our knowledge among new users of ics who discontinued ics treatment early. the aim of the study is therefore to describe, among new users of ics that discontinued, their knowledge of ics' actions and whether they were instructed on the use of their inhaler. design: a cross-sectional study among new users of ics that discontinued use. patients were interviewed by telephone and their gp received a mailed questionnaire. automated dispensing records of all patients were retrieved. setting: community pharmacies in the netherlands. main outcome measures: by use of conditional logistic regression the association between knowledge and study variables was assessed. results: from eligible patients, ( . %) were interviewed. the majority ( . %) of these new users of ics who discontinued ics early was not aware of the anti-inflammatory actions of ics. most patients ( . %) were instructed on the use of their inhaler, predominantly by the gp ( . %). after adjusting for symptom experience by acq, asthma diagnosis, having persistend asthma or use of medication only age (or . % ci . - . ) and male gender (or . % ci . - . ) were associated with unawareness of anti-inflammatory actions. conclusions: this study shows that a substantial number of new patients that did not refill their ics prescriptions, were unaware of ics' inflammatory actions. surprisingly only age and gender seemed associated with awareness of ics actions. most patients were instructed on the use on their inhaler by a health care provider. physicians and pharmacists could cooperate in identifying and motivating these patients to continue ics use. background and objective: to assess the quality of antibiotic therapeutic drug monitoring (tdm) in routine hospital practice and establish baseline status for rationally defining future actions aimed at improving it (by implementation of clinical pharmacy services). design: months prospective observational study with validated data collection form using predefined criteria for tdm quality assessment. descriptive statistics performed with spss . for windows Ò . setting: orthopaedic surgery, general surgery, neurosurgery, vascular surgery, haematology and pulmonary wards of a beds teaching hospital, using vancomycin twice daily and amikacin once daily administration schemes. main outcome measures: adherence to predefined criteria for sample timing, information transmission, and follow up of dose adjustment recommendations. criteria: (i) sampling time: less than ± min (amikacin) and ± min (vancomycin) deviation from preset time for peak levels; less than ± min for trough levels; (ii) information transmission: patient's full name, dose, schedule of administration, time of previous and current dose, actual time of peak and trough level sampling; (iii) quality of the analysis [internal and external controls]; (iv) acceptance of dose adjustment (more than %) recommendations. results: inclusion: patients ( vancomycin and amikacin courses). correct sampling times: (i) peak levels: % (n = ) for vancomycin and % (n = ) for amikacin, (ii) trough levels: % (n = ) for both antibiotics. correct information transmission: % (n = ). no issue noted for the quality of the laboratory analyses. implementation of recommendations: % (n = ) for vancomycin and % (n = ) for amikacin. conclusions: incorrect sampling times and deficiencies in communication between the ward and the laboratory are key factors affecting the quality of tdm, leading to dosage adjustment recommendations that are only infrequently implemented. the companion abstract examines the underlying reasons for such poor performance of the tdm process using a qualitative approach. corresponding values of d were: background and objective: to evaluate the relation between vancomycin and amikacin pharmacokinetic (pk) parameters in an intensive care unit population. design: data from intensive care unit patients were collected over a -month period, through a retrospective review of medical records and therapeutic drug monitoring (tdm) reports. patients were included only if at least two blood samples, at steady state conditions, had been drawn. data were first evaluated for completeness and consistency of recorded sampling and dosing times. individual pk parameters were estimated (bayesian analysis) using a one-compartmental pk model for amikacin and a two-compartmental pk model for vancomycin (pks Ò abbot). phase i of the study determined relationships between vancomycin and amikacin pharmacokinetic parameters, mainly clearance and volume of distribution. for that purpose, linear regression analysis of data from out of patients (analysis dataset) was performed. phase ii tested the predictability of the developed equations in an additional sample of patients (validation dataset) by comparing predicted pk parameters from equations (predeq) to those estimated by bayesian analysis (predbay) . t-test between predeq and predbay from each antimicrobial was performed. bias and precision were evaluated calculating the mean prediction error (mpe) and mean absolute error (mape), respectively (s-plus ). setting: intensive care units. tertiary university hospital. main outcome measures: patients demographics, clinical and tmd records, creatinine clearance by cockcroft-gault, vancomycin and amikacin blood levels and pk parameters. results: eighty-three critically ill patients ( females, males)were recruited for the study (mean values: age . yr, weight: . kg, cr: . mg/dl). a correlation between vancomycin and amikacin regarding their cl was found (clvancomycin = . clamikacin + . ; r = . ). however, no correlation was observed for vd (r = . ). concerning phase ii, differences in demographic data from both datasets were not statistically significant. no significant differences were observed when performing predeq versus predbay t-test. nevertheless, boxplot graphs for predeq and predbay residuals showed a wide variability of the values distribution and a lack of precision for both antimicrobials. conclusions: in our patient population this studied approach reveals an existing relation between amikacin and vancomycin pk parameters (or vice versa). however, the poor precision and large bias of residual values prevents us from recommending the use of these equations as pk parameters predictors (or regimen dose predictors) in intensive care patients. further studies with larger samples are definitely required in such an heterogeneous population. they were asked to identify which items of the ashp guidelines and gedefo they considered that must be filled in, in a prescription or in an identification label. consensus was defined as an agreement rate c %. prescriptions/labels evaluation: all breast or colon intravenous chemotherapy prescriptions, from a central hospital, have been evaluated from january to december (n = ), based on the parameters identified in the consensus document. a two month analysis of identification labels was performed. results: consensus document: a total of hospital pharmacists ( . %) completed the rounds of the delphi. consensus was obtained for . % of the prescription items and for . % of the labels items. prescriptions/labels evaluation: more than / of the analysed prescriptions were for breast cancer ( %) and the rest for colon. none of the analysed prescriptions had all the consensus items filledin. information that allowed the validation of the prescription by the pharmacist (ex: height, weight, body surface or number of cycle) was present in less then % of the prescriptions. no one had the prescriptor telephone, or the justification for dose reduction (when appropriate). only . % ( / ) of the labels mentioned the full identification of the solvent ( % miss the concentration) used and none of them stressed out the need for filter use when applicable. conclusions: consensus was obtained about a large number of items, which may constitute a difficulty in daily practice the evaluation of prescriptions highlights the lack of information that could allow confirmation by the pharmacist. labels do not seem to alert about special administration conditions. background and objective: background: the ministerial advisor on hepatitis c in catalonia has established a series of recommendations concerning hepatitis c treatment distinguishing between two groups: viral genotypes and , and viral genotypes and . in genotypes and it is necessary to evaluate treatment continuity after and weeks, depending on viral load and it is also necessary to prolong the treatment to weeks if there is viral response. in genotypes and it has not to be longer than weeks regardless of viral load. objective: to evaluate the adequacy of hepatitis c treatment and analytic monitoring, following the recommendations of the catalonian ministerial advisor. design: observational and retrospective study. setting: patients that started treatment with peg-interferon plus ribavirine in this hospital during . the information obtained was: viral genotype, the beginning and the end of treatment, quantitative basal rna (in all genotypes), quantitative rna ( weeks) and qualitative rna ( weeks) in genotypes and and at the end of the treatment in all genotypes. main outcome measures: the application of global advices ranges from % to % according to viral genotype and established recommendation. results: the compliance degree in genotypes and : % application of quantitative basal rna and % after weeks. % of treatments were discontinued with quantitative rna positive after weeks, % with qualitative rna positive after weeks and % the treatment was continued longer than weeks. in genotypes and : % application of quantitative basal rna, % application of qualitative rna after weeks and % the treatment was continued longer than weeks. conclusions: following the recommendations on viral response evaluation after and weeks allows the early suspension of therapy in non-responsive patients. this leads to and improvement in patients' quality of life, a reduction in adverse side-effects and savings in medical care costs. treatment monitoring by hospital pharmacist provides medical decision support. in consequence these patients constitute a target group to establish pharmacy care programs focused on hospital outpatients. topical application of mitomycin . % during two minutes and postoperative stenting for a period of four-ten weeks were used, no second application of mitomycin was used. the follow-up was years in bilateral case and six months for the unilateral case. setting: pharmacy and otorhinolaryngology service. hospital universitari joan xxiii de tarragona. spain. main outcome measures: the success of the repair was measured according to the following items: endoscopic evaluation of the patency of the choanae, respiratory distress and nasal drainage. results: bilateral membranous choanal atresia was surgical repaired five days after birth, using transnasal endoscopic approach and topical mitomycin . % during de proceeding. no operative complications occurred and stents were removed four weeks after repair. the choanae was inspected endoscopically to asses healing and no presence of re-estenosis was found. no clinical symptomatology. unilateral mixted atresia on the left side was diagnostic at six years old and surgical repaired because of association of respiratory distress and nasal mucus drainage symptomatology. no restenosis has appeared and syntomatology has improved. nome patient required surgical revision. conclusions: although, the exact role of the topical application of mitomycin . % must to be further investigated, the use of this drug as an adjunct to the surgical repair of choanal atresia may offer decreased need for revision surgery due to re-estenosis. keywords: mitomycin . %, choanal atresia pt- adherence to clinical guidelines for upper respiratory and ear infections in out-of-hours primary carea retrospective study sara claesson biofarmaci, uppsala university, täby, sweden background and objective: infections in the upper airways and ears are a frequently occurring reason for patients to visit primary care settings. prescribing adherence to local guidelines for handling infections of ears and upper airways and antibiotic prescribing is of both local and national concern. increasing antibiotic resistance is one reason, cost and patient quality of care are others. the objective of this study was to investigate physician adherence to clinical guidelines at the out-of-hours primary care clinic in täby. design: a retrospective study. clinical case notes were scrutinised for all patients seeking care for problems with ear, nose, throat, fever, cough or cold between january and march . data was analysed for patients who were diagnosed with specified ear or upper respiratory infections. laboratory tests taken and antibiotic prescriptions were anonymously documented. antibiotic prescription filling dates were investigated. setting: husläkarjouren, the out-of-hours primary care clinic in täby. main outcome measures: adherence to and fulfillment of local therapeutic guidelines and professional quality criteria was defined with respect to immediate, delayed or no prescription, drug choice, dose and duration and the use of diagnostic tests. adherence was defined as complete or not, and deviations from the guidelines were separately analysed. prescription filling was analysed with respect to time from clinic visit to pharmacy visit. results: data from patient visits were analysed. adherence to local guidelines was disappointingly low. general treatment of infections was only according to guidelines in % of the cases and only laboratory testing met the quality criteria. adherence to antibiotic prescribing guidelines was even lower, only % of antibiotic prescriptions were completely according to local guidelines. % of all antibiotic prescriptions were filled within one day from the visit to the clinic. conclusions: communicating guidelines to prescribers and continuous follow up of prescribing behaviour is essential for improving patient care and decreasing the risk of antibiotic resistance in the community. this study exposes gaps in the quality of care that may not be picked up by traditional follow up measurments. studies with a wider scope and in depth analysis of reasons for nonadherence to guidelines are warranted if antibiotic use is to be improved. . lines after other therapies. before bortezomib one patient received therapeutic with thalidomide/dexametasone, one vad, one cyclophosphamide (ctx), three followed by vad, one mp followed vad and followed ctx, and another with vad followed by ctx. the therapeutic selection followed the nccn guidelines in all patients. the average number of cycles with bortezomib has . . from the patients, stopped, one had a generalized face oedema bortezomib related, although the disease was in remission, and another died by sepsis not related with this drug. in relation to bortezomib's effectiveness, ( %) patients had a very good response, since the immunoglobulin decreased and three ( %) of these patients are actually at consolidation cycles ( th and th). conclusions: the use of bortezomib in our hospital was according the nccn guidelines and the experience was very positive. nevertheless, and considering also the high cost of this therapeutic, we consider very important to continue to follow up this group in order to evaluate the rate of response to bortezomib during time. pt- evaluation of the time interval between admission on the emergency department and administration of the first dose of antibiotics background and objective: when patients are admitted with a proven or suspected infection on the emergency department, adequate antibiotic treatment must be started as soon as possible. literature reveals that the time between admission and administration of the first dose of antibiotics can reach about hours and this can influence the prognosis of the patient. international guidelines for community acquired pneumonia (cap) and bacterial meningitis define this time interval of hours( ) and less than hours( ) respectively. to evaluate the practice on the emergency department of our hospital, with an average daily admission of patients, a study on this interval was carried out. . % of all administered antibiotics is in adherence to the local guidelines; however this was only evaluated by the clinical pharmacist. a possible explanation for the relatively short time intervals could be that the antibiotics which are frequently used are available at the emergency department and that the guidelines are at all time available on our hospital intranet. the time intervals of cap and bacterial meningitis are shorter than the defined international guidelines.( , ) conclusions: the time interval, found in our study, between admission on the emergency department and administration of the first dose of antibiotics is short, compared with similar conducted studies.( , ) pharmacy, otolaryngology, de octubre university hospital, madrid, spain background and objective: a woman diagnosed with laryngeal papillomatosis. treatment consisted of laser excision of the granulomatous lesions, followed by mitomycin c instillation over the surgical bed. description of the preparation and utilization of mitomycin c instillation over the surgical bed for the treatment of laryngeal papillomatosis. design: request by the ent service for compassionate use of mitomycin c. following authorization from the health authorities, a literature search was made. after determining the dose/day for the patient, elaboration was carried out according to the literature references. setting: vials of mg of mitomycin c were used as starting material, with -ml syringes to facilitate instillation in the operating room. dilution was made to double the standard in our centre ( ml). main outcome measures: two -ml syringes were prepared per cycle, with a mitomycin c solution of . mg/ml for instillation, though finally in all cases only one of them was used. stability was established as hours at room temperature, without special considerations regarding light exposure, as specified by the literature. the entire procedure was carried out in a laminar flow chamber, in compliance with the specifications for manipulating cytostatic agents. results: the patient received a total of three cycles of mitomycin c, the last two being maintenance cycles. the patient has experienced no papilloma relapse, and only right vocal cord hyperaemia is observed, probably secondary to surgery. conclusions: collaboration between the service of pharmacy and the ent service allowed adequate treatment and recovery of the patient, without apparent adverse effects. swollen and tender joints over joints evaluated, and the value of erythrocyte sedimentation rate, comparing the das from one patient on two different time points; eular response, that classifies patients in groups according to treatment response, and decrease in mhaq (modified health assessment questionnaire), that indicates patient's awareness of disability. data were recollected at beginning and at weeks of initiating treatment with rituximab. differences between b-lymphocyte count at beginning and at weeks was of initiating treatment was used as a secondary variable. results: we collected data of patients, all being females, was rf positive and were anti-ccp positive. at women) a . % of patients received adjusted prophylaxis to their degree of risk ( . % with heparin prescription and . % without it) from all . % of inadequate prescription ( % of the patients with lmwh prescription); patients lost follow up. patients submitted to cancer surgery ( surgeries, men, women), % received apropiate prophylaxis ( . % with lmwh and . % without it). same percentage of patients ( %) was not adapted to correct prophylaxis ( % patients with lmwh); patients lost follow up. conclusions: there is a greater percentage of choledoco surgery patients with lmhw prescription in compliance with published guidelines than oncology surgery patients, however it will be necessary carry out a better implementation among healthy professionals in order to increase this percentage of patientes. results: prescriptions of these broad-spectrum antibiotics were collected and analysed. % of the prescriptions were initiated by residents, % by senior physicians. vancomycin was the most prescribed antibiotic ( %), mainly in the orthopaedic surgery unit. the indications of antibiotics were osteomyelitis ( %), septic arthritis ( %), prosthetic joint infections ( %) and pneumonia ( %). only % of antibiotic doses were not correctly adapted to creatinine clearances or to plasmatic vancomycin rates. the initial choice of antibiotic was considered appropriate in % of cases. regarding bacteriological results (bacteria, antibiogram), the continuation of the treatement was acceptable in % of cases. however in % of prescriptions, an adjustment of therapy with a more narrow spectrum antibiotic could have been done. conclusions: these results have to be extended with further investigation (at least months). were systematically reviewed to retrieve prospective trials describing esa doses in dialysis pts who converted from rhuepo to da. search words included: ''epoetin, darbepoetin, esrd, ckd, and dialysis.'' the inclusion criteria required a study to have dose data available during the evaluation period for both rhuepo and da. study selection and data extraction were performed by independent reviewers and verified by a rd. relative doses and dose changes after conversion from rhuepo to da were estimated using an initial rhuepo:da : conversion ratio (aranesp Ò eu label). study quality assessment was performed using the downs-black checklist, a standard method used to assess the quality of a study using ebm principles. setting: meta-analysis. main outcome measures: dose efficiency. results: the search yielded studies meeting the inclusion criteria. upon further review, studies were excluded ( had unextractable data, were retrospective analyses, and had predialysis pts). the remaining studies were analyzed: rcts with parallel control groups, cross-over trials, and observational conversion studies (table) . the studies yielded data on , rhuepo and , da pts, with a mean treatment duration being weeks. we found the average study quality was %, with rcts (n = ) having a higher quality score ( %) than crossover ( %; n = ) or observational ( %; n = ) studies. there was a notable dose efficiency observed when pts were converted to da from rhuepo. this effect was greater in the rcts ( . %) than in crossover ( . %) or observational ( . %) studies. conclusions: we found a notable da dose efficiency (up to %) in pts who were converted from rhuepo to da using a : conversion ratio. additionally, studies with the highest quality scores (eg rcts) had the greatest observed dose efficiency while non controlled studies scored lowest in both quality and dose efficiency. background and objective: the purpose of this study is to evaluate the frequency of otitis media and the assessment of its relationship to patients' socio-demographic characteristics and determination of systemic and topical drug profiles in otitis media. design: this retrospective study included the assessment of patients diagnosed with otitis media, who admitted to the study hospital during a months period (march-may ). demographic, clinical and prescription data of these patients were collected and analyzed. setting: the ear-nose-throat out-patient clinic of a states hospital. main outcome measures: the socio-demographic data of patients; the frequency of otitis media; type and percentage of prescribed drugs; the most used treatment regimens. results: patients ( women and men), who were diagnosed with otitis media, were included in our study. patients were diagnosed as chronic otitis media or serous otitis media or acute otitis media or external otitis media at rates of . %, . %, . %, and . %; respectively. in children, the acute and serous otitis media were seen more often than in adults (p \ . ). however, chronic otitis media were seen more frequently in adults (p \ . ). all patients were administered drug therapy for their diseases. it was observed that antibiotics (oral and/or topical), analgesics, decongestants, and topical corticosteroids were prescribed at rates of . %, . %, . %, and . %; respectively. prescribed oral antibiotics were cephalosporins [cefixime ( . %), cefuroxime axetil ( . %) and cefaclor ( . %)], amoxicillin-clavulanate, and floroquinolones [levofloxacin ( . %) and ciprofloxacin ( . %)] at rates of . %, %, and . % respectively. rifampin and ciprofloxacin were prescribed as topical antibiotics at rates of . %, . % respectively. when the number of drug used by the patients was evaluated, were on quadri-therapy, on tri-therapy, on dualtherapy and on mono-therapy. patients were treated with the combination of antibiotic-analgesic-decongestants. conclusions: our study indicated that the most frequently prescribed drugs were antibiotics in otitis media. clinical pharmacists have a potential role in rational antibiotic use by providing clinical pharmacy services such as antibiotic selection, drug monitoring and patient education; so that they would reduce both antibiotic resistance and treatment costs. background and objective: the sampling method is crucial for the physical and chemical quality control of antineoplastic chemotherapies. this step acts upon the dosage correctness and may lead to risk of needle-stick or cytotoxic drug projection during its achievement. beyond, the sampling time must be as short as possible and the sample be directly placed on the analysis machine. this work evaluates a new and improved sampling method, specially worked out for this application. design: this study was designed to ensure the vial airtightness and the volume sampling. a cost and time study was also performed. setting: the vial is an hplc type, chromacol Ò ml, mm dp = hpa, (interchim Ò , montluçon, ). the vial is airtight thanks to a ptfe silicon septum set with an aluminium collar. the vials are vacuum-packed in a pvc bag (didop Ò , compiègne, ). a void test was led on unpacked vials up to months, to ensure a maximum conservation, and showed a filling volume of ± ll, very close of the ll target. main outcome measures: one week samples have been weighed to calculate the filling volume in real conditions of use. this volume is ± ll ranging from to ll. since this sampling method has been set up, the percentage of refusal for insufficient volume is lower than . %. this technique was compared to the previous method in terms of cost: vial, sampling adjuncts, handlingtime and waste. the vials are filled with a secured double-needle eclipse Ò , mm, / (becton-dickinson Ò , le-pont-de-claix, ). results: about , chemotherapies controls are made each year in the hospital. the vial and the sampling device costs are higher than the previous ( € versus . €, and . € versus . €). on the contrary, the handling-time for sampling was estimated minute lower (which corresponds to . € less per sample). furthermore, the waste weight is gram lighter with the new devices, which costs . € less in the waste disposal. the total cost difference is . € higher per sample. an estimation of a needle-stick accident has been carried out, with the human cost (pharmacist technician's compensation and medical consulting) and the equipment including gloves, sterilization devices and post-sterilization check; the lowest estimation cost of an accident is . €. conclusions: the secured needle makes the sampling operation easier for the workers and it lowers the risk of needle-stick. besides, this closed system avoids completely the antineoplastic contact for the manipulators during the confection and the control. moreover, this system allows to secure the sample library. background and objective: the prevalence of diabetes mellitus in kuwait ranks amongst the highest in world at about %. diabetes is a well recognized independent risk factor for cardiovascular disease (cvd). the increased prevalence of several other known risk factors for cvd in kuwaiti diabetics further increases the risk. since cvd is the leading cause of death in kuwait, the high incidence of diabetes has major social and economic impact. diabetics aged years or older have, in general, an increased -year risk of developing cvd compared to younger patients but there have been no audits involving this group of patients in kuwait. the objective of this study was therefore to audit achievement of cvd risk factor goals according to pt- pharmacotherapy of first-episode psychosis in the psychiatry clinics of the north estonia regional hospital (nerh) and the tartu university hospital (tuh) jana lass , agnes männik , simon j. bell pharmacy department, north estonia regional hospital, tallinn, institute of pharmacy, university of tartu, tartu, estonia, faculty of pharmacy, university of helsinki, helsinki, finland background and objective: treatment guidelines provide recommendations for the evidence-based treatment of schizophrenia. adherence to these guidelines is often sub-optimal. our aim was to compare and contrast the pharmacotherapy of first-episode psychosis at the nerh and tuhs, with respect to both treatment location and evidence-based guidelines. design: retrospective study. case notes for consecutive patients with schizophrenia, schizotypal or delusional disorders (icd- ) admitted to the nerh and tuh between september and september were retrospectively reviewed. setting: psychiatry clinics of two tertiary care hospitals -nerh and tuh. main outcome measures: outcome measures included the choice and daily dose of antipsychotics, incidence of antipsychotic polypharmacy and reasons for changes in therapy plan. results: patients form nerh and from tuh were included in the final analyses. median age (sd) of the patients was ( . ) in the nerh and ( . ) in the tuh patients were hospitalised for longer in the nerh than in the tuh, ( . ) vs. ( . ). the most frequently prescribed antipsychotic was risperidone at both study locations - % of prescriptions in the nerh and % in the tuh. conventional antipsychotics were administered twice often in the nerh than in the tuh. in the tuh olanzapine was administered in higher prescribed daily doses than in the nerh. the number of antipsychotics prescribed per patient was higher in the nerh than in the tuh - . vs . . the prevalence of antipsychotic polypharmacy was . % among the patients in the nerh, whereas only one patient was treated with antipsychotic polypharmacy in the tuh. conclusions: analyses revealed significant differences in the pharmacotherapy of first episode psychosis at the nerh and the tuh. mechanisms to facilitate improved adherence to the evidence-based treatment guidelines should be investigated. pharmacy, hospital universitario de getafe, getafe, spain background and objective: to analyze the use of tnf-alfa inhibitors in rheumatoid arthritis diagnosed patients in a bed hospital. design: retrospective study of patients with anti-tnf-alfa during year . the following data were compiled: age, sex and anti-tnfalfa prescription including dosage and duration of treatment. setting: hospital universitario de getafe. main outcome measures: dosage and duration of treatment. results: a total of patients were included. patients ( . %) received just one anti-tnf-alfa drug, ( . %) needed two lines of treatment and ( . %) of them needed three. patients who were treated with just one drug had a median age of . years and those who required two lines of treatment had a median of . years. the first line drug was etanercept in . %, infliximab in . % and adalimumab in . % of patients. second option was etanercept in . %, adalimumab in . % and infliximab in . % of patients. the average duration of treatment with etanercept as forward edge was days. the treatment was suspended in . % of patients. when infliximab was used as first line, average duration was days, and treatment was interrupted in . %. with adalimumab, average duration was days and treatment was interrupted in % of patients. conclusions: those of our patients who need an only one treatment line are younger than those who need or , due to the chronic and progressive course of the disease. etanercept is used as much in first option (followed by infliximab) as in second one (followed of adalimumab), although these differences are not statistically significant and it would be necessary to make a study including more patients. the duration of treatment with infliximab is the longest, as this was the first drug available. regarding treatment failure, etanercept shows the greatest percentage. this should be taken into account when establishing first line treatment. background and objective: plantar warts are hyperkeratotic lesions on the plantar surface caused by infection with human papillomavirus. lesions caused by warts are commonly refractory to therapy and may become large and painful in immunodeficient patients. cidofovir is a cytidine analogue with activity against a broad spectrum of dna viruses. it is indicated for the treatment of cytomegalovirus retinitis in patients with acquired immunodeficiency syndrome and without renal dysfunction. we describe a case of plantar warts that was treated with topical cidofovir in a highly immunodeficient patient. design: case report, evaluation and discussion based in clinical chart and literature review. setting: pharmacy department, general teaching hospital. main outcome measures plantar warts regression, which was evaluated on the basis of change in overall surface area of the treated lesions compared with baseline. to evaluate the organoleptics properties of the galenic formulation. results: a years old woman, who received kidney transplant in , presenting plantar warts refractory to conventional therapy since last four years. she was treated with topical % cidofovir cream twice daily. the treatment was authorised as compassionate use by the national regulatory agency on drugs. the glomerular filtration rate (gfr) was monitored in order to detect nephrotoxicity due to cidofovir. the % cidofovir ointment was compounded as follows: -cidofovir mg/ml ml vial .…….. ml -anhydrous lanolin ……………………….. g -beeler base …..sufficient to produce g it was packaged and labelled in a light-resistant containers and we assumed an expiration date of months based on the duration of treatment and published studies. the quality controls of organoléptics properties were made according to the good manufacturing practice (gmp) after weeks of therapy the patient did not show any improvement and developed severe local erosion, so treatment with cidofovir was withdrawn. two weeks later this local erosion disappeared spontaneously. no systemic side effects were observed. the colour, texture and smell organoleptics characters were complied with gmps. conclusions: there are not formal studies of optimal formulations or treatment regimens and further studies are needed to elucidate the role of cidofovir in treatment of plantar warts. the immunodeficiency of the patient and the large wart area could be related with the failure to the treatment. background and objective: hypersensitivity reactions (hr) to platinum salts can be serious and should lead to interrupt chemotherapy regimen. skin tests may be used to confirm diagnosis of hr. usually, these tests are prick tests and intradermal tests performed with diluted solutions of platinum salts. there is no standard solutions for these drugs, which local toxicity depends on concentration. the main objective of this study was to assess the safety of platinum salts skin tests performed in our hospital. the secondary objective was to assess their efficacy to verify the allergic nature of the reactions observed. design: pilot study of skin tests preparations between january and june . these preparations were dilutions ( / , / , / ) of a primary platinum salt solution, which concentration was roughly the one used in the chemotherapy regimen of most patients. the compatibility of platinum salts with dilution solvent was checked, and all solutions were prepared extemporaneously in a centralised cytotoxic drug preparation unit, in order to protect handlers. setting: allergology department and pharmacy department in a university hospital. main outcome measures: skin tests results: positive if a papule appeared, negative if there was no reaction, local toxicity if an irritative reaction happened. results: patients with clinical symptoms of hr with a chemotherapy regimen containing platinum salts were explored by skin tests. drugs assessed were: oxaliplatin ( patients), cisplatin ( ), and carboplatin ( ) . no patient developed local toxicity. tests results were positive in cases ( oxaliplatin and cisplatin), and negative in cases. patients received both cisplatin and oxaliplatin skin tests: patients had a single positive reaction with no cross reaction between the two drugs, and the third had no reaction. in cases, tests results and clinical history of hypersensitivity mismatched. conclusions: this study shows that these skin test solutions were safe. their efficacy was judged correct: positive reactions confirmed the diagnosis of hypersensibility for patients. the main limit of the results is the absence of control subjects. these tests allowed to explore patients' hr, and to help oncologists to choose the more appropriate treatment for them. stability studies are still needed to assess the pharmaceutical quality of these diluted solutions. these preparations have now been standardized in our hospital. background and objective: guidelines regarding appropriate use of prophylactic antibiotics have been implemented at university hospitals leuven. however, the degree of compliance with these guidelines is unknown. the aim of this study is to develop a method to quantify compliance with antibiotic prophylaxis guidelines and to apply this method to the clinical areas of appendectomy and heart valve surgery. design: a retrospective case series was carried out of all prophylaxis episodes related to appendectomy and heart valve surgery at university hospitals leuven between august and february . four grades of compliance with antibiotic prophylaxis guidelines were identified: grade compliance, reflecting administration of the antibiotic proposed by the guidelines in a dosage within - % of the recommended dosage; grade compliance, defined as the administration of the antibiotic proposed by the guidelines in a dosage outside - % of the recommended dosage; grade compliance, referring to the administration of an antibiotic equivalent to the antibiotic proposed, but not mentioned by the guidelines; and grade compliance, representing any other antibiotic prophylaxis scheme. setting: divisions of abdominal and cardiac surgery, university hospitals leuven. main outcome measures: the percentage of prophylaxis episodes that satisfy each grade of compliance with antibiotic guidelines. results: prophylaxis guidelines relating to appendectomy ( , episodes) recommend administration of three times cefazolin g and a single dose of metronidazol . g. the proportion of episodes that satisfied grade , , and of compliance with guidelines amounted to %, %, %, and %, respectively. cefazolin g and metronidazol . g was used in episodes. prophylaxis guidelines applying to heart valve surgery ( , episodes) recommend administration of cefazolin g. the proportion of episodes that satisfied grade , and of compliance amounted to %, % and %, respectively. grade does not apply to heart valve surgery as no equivalent antibiotics were identified. the difference in compliance with prophylaxis guidelines between both surgical procedures could be explained by differences in infectious pathology, the peri-operative adaptation of the antibiotic regimen by the abdominal surgeon, and the use of a second regimen related to the severity of the appendicitis. a case can be made for combining grade - compliance with respect to appendectomy, resulting in a higher compliance rate. conclusions: our proposed method to measure compliance needs to be validated by future research. the method can be applied to different surgical procedures, thereby stimulating surgeons to explain differences in compliance between procedures and promoting the development of instruments to enhance compliance. closer interaction with surgeons is required to further develop the measurement of compliance with antibiotic prophylaxis. keywords: antibiotic prophylaxis, compliance, guidelines pt- non-specific immunoglobulins for immune neonatal thrombopenia nuria ibañ ez , maria del pilar m. p. bautista , ana maria a. m. iglesias , roberto r. ortiz , javier j. sanchez-rubio , maria del carmen m. c. giron , marta m. arteta pharmacy, pediatry, hospital universitario de getafe, madrid, spain background and objective: non-specific human immunoglobulins are being used at the moment in neonatal population for treatment of immune thrombopenia. the dosis commonly used varies between mg/kg and g/kg from one to five days. corticoids and platelet transfusions can be used jointly. to study the effectiveness and safety of non-specific human immunoglobulins in a neonatal unit for treatment of immune thrombopenia. design: retrospective study of neonatal patients diagnosed with immune thrombopenia during and treated with non-specific human immunoglobulins. a revision of clinical histories is made and following data are collected: sex, gestational age, born weight, age at the moment of infusion, administered dose and duration of treatment, use of corticoids and platelet transfusions, number of platelets/ll before infusion, at , at hours of initiate the treatment and at discharge. possible adverse reactions is also considered. setting: hospital universitario de getafe. main outcome measures: the effectiveness and safety of nonspecific human immunoglobulins for treatment of immune neonatal thrombopenia. results: three children were included in the study, two of them were males. thrombopenia was diagnosed from probable alloimmune origin, including positive confirmation study in one of the cases. gestational ages ranged from + to + weeks. born weight ranged between . kg and . kg. immunoglobulin treatment was initiated between first and sixth day of life. administered dose varies between mg/kg/day and g/kg/day from two to five days. all children needed platelet transfusions, while only one of them was treated with corticoids. the number of platelets/ll before infusion of immunoglobulins, at hours, at hours and at discharge was: children : , , , , , and , platelets/ll. children : , , , , , and , . children : , and , platelets/ll hours after initiation of treatment, there were no analytical data at hours, but number of platelets at discharge was , . no adverse effects were observed in any children. conclusions: although eventually the three children recovered the number of platelets, it can not be concluded that this was due to immunoglobulin treatment, because it is overlapped with administration of platelet transfusions and corticoids. a higher number of patients is required to evaluate efficacy and safety of non-specific human immunoglobulins in treatment of neonatal thrombopenia. background and objective: pulmonary hypertension (ph) is one of the most difficult childhood disease to treat. in spain, oral sildenafil has recently been approved in adults to treat ph, but it s an off-label drug for children (its utilization must be derived to ''compassionate use'', which requires a prior national health authorities approval for every children), and an oral suspension must be formulated at the pharmacy department for them. the objective of this study is to analyse the use of oral sildenafil for ph in paediatric patients. design: years retrospective study. % paediatrics patients with oral sildenafil for ph. clinical data review. setting: paediatric cardiology unit and pharmacy department ( pharmacist) in a paediatric hospital ( beds), in a large general teaching hospital ( beds, pharmacists). main outcome measures: patient data (diagnosis, age, weight). treatment description (dose, length of treatment). treatment effectiveness: peripheral arterial oxygen saturation and six-minute walk test. treatment security: side effects registered. results: children ( girls). age: months to years, median . years. diagnosis: / ph secondary to surgery due to congenital heart disease and / primary ph. sildenafil doses ranged from . mg/kg/ h to mg/ h; median length of treatment was . months ( month- . years). children have used the oral suspension formulated and monthly dispensed at the pharmacy department. other treatments: spironolactone ( ), furosemide ( ) , captopril ( ), acenocoumarol ( ), aspirin ( ), ranitidine ( ) and propranolol ( ). patients have experimented clinical improvement and are on treatment. sildenafil was withdrawn in patients because it was indicated to ameliorate the effects of inhaled nitric oxide withdrawn. patients died. no data available in patient. only patient experimented occasional headache. mensual treatment cost range from - €/patient. conclusions: oral sildenafilo seems to be a safe and effective therapy for paediatric patients with pulmonary hypertensión. due to the lack of an oral formulation for paediatrics patients, it should be elaborated at the pharmacy department. background and objective: we will describe the case of a bi-pulmonary transplant women who developed an invasive aspergillosis located in the lungs and the brain. she received intravenous voriconazole during days. she was then diagnosed with an aspergillus endophthalmitis. even though a dual therapy consisting of caspofungin and posaconazole was initiated, the patient underwent a partial vitrectomy. this therapeutic failure could be explained by a late diagnosis and insufficient vitreous and aqueous humor penetration of the systemic drugs. design: a retrospective analysis of an endophthalmitis management. setting: clinical unit in a french teaching hospital main outcome measures: to secure a high ocular concentration, the ophtalmologist recommended voriconazole intravitreal injections. his prescription was based on several case reports. results: we found articles dealing with animal testing: one concluded that voriconazole was a safe intravitreal agent which may be injected in human eye. another study described the successfull use of intra-ocular voriconazole to treat a fungal endophthalmitis: it allowed a significant improvement in visual acuity and the patient's recovery. however, further studies are needed to assess the optimal dosage and frequency of administration. we prepared voriconazole syringes under a horizontal laminar air flow hood, as follows: -preparation of a mg/ml solution with ml of water for injection and dilution in ml of water for injection, to obtain a mg/ml solution pharm world sci ( ) : - -we sampled . ml of this solution in a ml syringe, which was closed with an occluder, labelled and refrigerated. since we had no data regarding stability, it was administrated extemporaneously. conclusions: intravitreal injections failed to prevent deterioration. had they been introduced precociously, they might have been more efficient. an early diagnosis and prompt management might improve the extremely poor visual prognosis of this devastating condition. were are currently studying the preparation stability. pharmacy service, hospital universitario de getafe, getafe, spain background and objective: in professionals were alerted about an elevated frequency of early virological failure in patients treated with tenofovir (tdf) and didanosine (ddi) associated to lamivudine. in similar results related to the administration of tdf and ddi, in association with a non-nucleoside reverse transcriptase inhibitor (nnrti) were notified. therefore similar events can be observed when tdf and ddi are co-administered in combination with other antiretroviral classes, such as protease inhibitors (pi). subsequent pharmacokinetic studies have shown that tdf when co-administered with ddi increases ddi plasma concentrations leves by up to - %, with a higher risk of didanosine-related adverse events, like pancreatitis and lactic acidosis. the administration of a reduced dose of didanosine ( mg) to avoid over-exposure to didanosine may also contribute to a higher rate of virological failure and emergence of resistance at early stage. the objective of the study is to asses the rate of virological failure in patients treated with tdf and ddi associated to a pi. results: during the study period patients had prescription for drotrecogin alfa for sepsis syndrome; . % were male and the mean age was . years (range - years). all patients had proven infection: . % had pneumonia (n = ), . % pyelonephritis(n = ), . % soft tissue infection(n = ) and . % abdominal infection(n = ). the main isolated micro-organisms were klebsiella pneumonia(n = ), escherichia coli(n = ), pseudomonas aeruginosa(n = ), enterococcus faecium(n = ), staphylococcus aureus(n = ), legionella pneumophila(n = ), proteus vulgaris(n = ), enterococcus faecium(n = ), klebsiella oxytoca(n = ). all patients started treatment within h of the onset of severe sepsis. the treatment was not completed in one patient due to adverse events. contraindications were present in patients: platelet count \ . /l (n = ), age under years (n = ) and major surgery (n = ). the mean organ failures was . (range - organs). adverse reactions were present in patients: thrombocytopenia (n = ), pancytopenia (n = ), bleeding (n = ) and elevation of activated partial thromboplastin time (n = ). mortality at days was found to be % (n = ). conclusions: despite the presence of some contraindications, in most patients drotrecogin alfa was used according to current guidelines. nevertheless, since apache ii score was not determined, the real risk of death is unknown and there can be no extrapolation to literature results. upon these findings, a systematic evaluation of apache ii score must be implemented in order to optimize patient selection and the risk-benefit ratio, improving the use of drotrecogin alfa. pharmacy, oncology, hôpital tenon aphp, paris, france background and objective: it is necessary to focus on side effects for pharmaceutical analysis. dosage reductions are commonly used in cancer chemotherapy. however, little is known concerning the way these reductions are performed in clinical practice. the objectives were to evaluate the incidence, the reason and the percentage of dosage reduction. design: prospective four-week study during which we analysed prescriptions with dosage reductions. setting: pharmacy and clinical oncology department in a paris university hospital. main outcome measures: we focused on prescriptions with dosage reduction and we recorded : -patient information -cancer localisation -chemotherapy regimen -dosage reduction characteristics (date, reduction percentage, reason) the toxicities were classified according to the nci-ctc criteria (grade to ). individual interviews were performed in order to assess how physicians decided the reduction ratio. results: patients ( % women; mean age years) have been treated during that period. diagnosis majority were breast ( %), colorectal ( %) and lung ( %) cancer. patients required a dosage reduction (incidence %). hematological toxicities were the main cause of reductions ( %). the hematological toxicities observed were thrombopenia ( %), neutropenia ( %) and neutropenia-thrombopenia associations ( %). the toxicities observed were grade ( %) or ( %). the other major causes of reductions were neurological ( %) and gastrointestinal ( %). the average percentage of reductions was between % and %. the individual interviews have shown that physicians didn't base the dosage reductions on literature results (established criteria) but on their own clinical practice (experience). conclusions: % of the prescriptions showed a decrease of the regimen. even if there is few literature, clinical trials recommend a decrease of % of the usual dosage of the drugs. the percentage in practice is lower than the one defined by clinical trials. the choices of reduction percentage were not standardized. recommendations for dosage reductions are still needed. keywords: dosage reduction, anticancer chemotherapy, toxicity pt- interdisciplinary approach to dose adjustment in patients with renal impairment in secondary care liekweg a, hinnerkort a, ebeling g, braband s, dreischulte t, heilenkötter k, sander s, schiffmann s, schrimpff u, siems m, wagner k, weiland t, zeigermann g, melzer s hospital pharmacy of the asklepios kliniken hamburg gmbh background and objective: as part of a unit dose dispensing system, patient medication profiles are routinely entered in an electronic database. medication profile and laboratory data are accessible online by clinical pharmacists. the project was conducted in order to optimise pharmacotherapy in patients with renal impairment and to integrate the clinical pharmacist in the therapeutic team. setting: unit-dose supplied wards (n = ) in four asklepios hopitals in hamburg with approximately patients per day. the project was conducted in cooperation with clinical pharmacists, physicians and the laboratory department over a period of . months ( / - / ). programm description: clinical pharmacists receive a list of all patients with an estimated glomerular filtration rate (egfr) \ ml/ min/ , m (mdrd) from the laboratory department on a daily basis. they screen medication profiles daily with regard to apparently inappropriate dosing of renally excreted drugs (q o \ . ). critical cases are reported to physicians by phone or entry in medical case notes. following an interdisciplinary discussion with the physician the drug dose or dosing interval is either adjusted, the medication is stopped or paused or an alternative is started. during the pilot phase the number of altered medications as a result of pharmacists' recommendations was documented. results: a prevalence of % of patients with a egfr \ ml/min/ . m was found in the examined setting. during the pilot phase of prescribed drugs ( %) were renally excreted or considered nephrotoxic. antibiotics ( %), antidiabetics ( %), diuretics ( %) or nsais ( %) were predominantly involved. overall, of pharmaceutical recommendations ( %) were accepted and acted upon by physicians. conclusion: the number of recommendations demonstrates the importance of this service in optimising pharmacotherapy. clinical pharmacists' contributions in matters of dose adjustment in patients with renal impairment is well received by physicians especially in non-nephrologic departments. the new service was found to be feasible in daily practice and has become part of the clinical routine. background and objective: the sources and availability of drug information for patients are growing, e.g. through the internet and official patient information leaflets (pils). however, the quality of the information on the internet might be questioned. furthermore, pils are not standardized, the layout is not reader friendly and the information covers all approved indications for the drug, some of them not relevant for rheumatic patients. also, over the years various information leaflets for drugs have been developed in the departments of rheumatology in norway. these are not standardized and the accessibility is limited. the objective was to develop a system for producing and maintaining reader friendly patient information leaflets about antirheumatic drugs, which takes the quality assurance aspect into account, and is easily accessible for the users. design: development project, consensus method. a national multidisciplinary project group was set up in december , with members from the social leagues (two members), pharmacists' organization (four) and rheumatologists' organization (two). mandate and regulations were approved by the organizations, as well as a legal disclaimer. the pharmacists make a draft for each drug which is e-mailed to all the members of the group. based on the comments a revision is made followed by another hearing until consensus is reached. the rheumatologists approve the leaflet. setting: national multidisciplinary consensus including patients associations main outcome measures: establishment of a dedicated website. number of leaflets published. results: a web address for publication of the leaflets is set up on the home page of the norwegian society for rheumatology: www.legeforeningen.no/nrf. there is a link to this address on the home pages of the social leagues and the norwegian association of hospital pharmacists. during the first year different drug leaflets have been developed and published on the web site. it is possible to search by trade name, generic name and groups of drugs such as ''antiinflammatory drugs'', so the numbers of hits adding up to . conclusions: this national multidisciplinary approach has made it possible to develop a system for making patient information leaflets about anti-rheumatic drugs, which are standardized and easily accessible. keywords: patient information leaflets, drugs in rheumatic diseases, multidisciplinary the need for pharmaceutical care in the prevention of coronary heart disease; an exploratory study in acute myocardial infarction patients the right medicine: a strategy for pharmaceutical care in scotland pharmacy for health: the way forward for pharmaceutical public health in scotland. glasgow: phis . . the sector skills council for health. skills for health: public health practice competences qualitative research: consensus methods for medical and health services research prescribing problems and pharmacist intervention in community practice a dictation system for reporting prescribing errors in community pharmacies a competency framework for the care of a person with diabetes pharmaceutical care of the patient with type diabetes mellitus: a consensus model for delivery of structured pharmaceutical care by community pharmacists in scotland development of a leadership course tailored for pharmacists in scotland pharmacy students attitudes and views about portfolio-based learning: a questionnaire survey using portfolios to learn about prescribing: qualitative insights into students experiences implementing clinical pharmacy services in an outpatient oncology clinic an evaluation of pharmacist contribution in oncology ward in a swedish hospital evaluation of clinical pharmacy services in a hematology/oncology outpatient setting rheumatic illness (ri): ( . %), multiple sclerosis (ms): ( . %), hepatitis c (hc): ( . %). : cfk: ( . %), others: ( . %), ri: ( . %), hiv: ( . %), ms: ( . %), hc: ( . %). : cfk: ( . %) total economic impact (tei): , € valorisation of clinical pharmacy activities: validation of a standard tool for routine interventions quotation in french hospitals keywords: drug related problems hiv-infected patients' perceived satisfaction with an outpatient pharmaceutical care unit (opcu) quality perceived by outpatients at the pharmaceutical care clinic antibiothérapie par voie générale en pratique courante au cours des infectons respiratoires basses de l'adulte et de l'enfant éme conférence de consensus en thérapeutique anti-references simultaneous determination of olanzapine, clozapine and demethylated metabolites in serum by on-line column-switching comparative study and optimisation of the administration mode of three proton pump inhibitors by nasogastric tube is the administration of esomeprazole through a nasogastric tube modified by concomitant delivery of a nutrition mixture? gerontology and geriatric medicine the clinical implications of platelet transfusions associated with abo or rh(d) incompatibility platelet transfusion: products, indications anti-d ig pc- pharmacist's interventions in three medical care units: analysis and impact on the physicians' prescriptions ventilator-associated pneumonia epidemiology and outcomes of health-care-associated pneumonia keywords: pseudomonas aeruginosa, antibiotherapy, vap (ventilator-associated pneumonia) folic acid metabolism and human embryopathy folic acid in general medicine and dermatology folic acid awareness and intake survey in the united arab emirates keywords: pregnancy, folic acid, neural tube defects pepi- pre-diabetes screening program: a proactive study in istanbul community pharmacies www.diabetes.org keywords: type-ii diabetes years later…where do we stand? over-the-counter medications in pregnancy north american association for the study of obesity. consensus development conference on antipsychotic drugs and obesity and diabetes second-generation (atypical) antipsychotics and metabolic effects. a comprehensive literature review keywords: metabolic syndrome, second-generation antipsychotic, biological and clinical follow-up references analysis of taurine in blood plasma of epileptic patients using an improved isocratic hplc method for amino acids cardiovascular actions of taurine pharmacokinetics and pharmacodynamics of the effects of taurine on human blood pressure and heart rate references criteris d'indicació en el tractament de les hepatitis víriques. direcció general de recursos sanitaris peginterferon-alpha a and ribavirin combination therapy in chronic hepatitis c prevalence, incidence and predictors of severe anaemia with zidovudine-containing regimens in african adults with hiv infection within the dart trial the anemia prevalence study group. prevalence of anemia and correlation with biomarkers and specific antiretroviral regimens in human-immunodeficiency-virus-infected patients: findings of the anemia prevalence study clinical pharmacy and medication safety keywords: clinical pharmacist, chronic kidney disease, treatment safety reference nccn clinical practice guidelines in oncoloy for multiple myeloma a phase study of bortezomib in relapsed keywords: antibiotics, emergency department the use of mitomycin-c for respiratory papillomas: clinical, histologic and biochemical correlation mitomycin c: prevention and treatment of anterior glottic synechia preliminary results of intraoperative mitomycin-c in the treatment and prevention of glottic and subglottic stenosis airway complications from topical mitomycin c. otolaryngol head neck surg keywords: mitomycin c, laryngeal papillomatosis, papillomas pt- effectiveness of rituximab in rheumatoid arthritis background and objective: to assess the response to rituximab in patients with rheumatoid arthritis (ra) who were refractory to anti-tnf treatment. design: observational, cross-sectional study. performed on patients diagnosed of ra according to acr criteria the main variables used to assess clinical evolution were: decrease in das -esr, considering the number of references noss eh et al. rituximab as therapy for refractory polymyositis and dermatomyositis treatment of early and refractory dermatomyositis with infliximab: a report of two cases keywords: dermatomyositis, rituximab, infliximab were included in the study. main outcome measures: percentage of patients achieving optimum and minimum audit standards for serum total cholesterol, ldl-c, glycosylated haemoglobin (hba c), bp and take up of aspirin of these, optimum treatment standards for total cholesterol (\ mmol/l) and ldl-c (\ mmol/l) were achieved by . % (n = ) and . % (n = ), respectively. patients within the minimum audit goal for total cholesterol (\ mmol/l) and ldl-c (\ mmol/l) were joint british societies' guidelines on prevention of cardiovascular disease in clinical practice pt- pediatric use of infliximab: retrospective study vanida brunie reference french guidelines for assumption of responsibility of candida sp. and aspergillus sp. invasive infections. french society of anesthesia and reanimation keywords: assessment, antifungal agents comparisons of psychotropic drug prescribing patterns in acute psychiatric wards across europe to score personal risks factors ( to : standard risk, to : high risk) design of a therapeutic scheme following asco's guidelines, as follow. day : aprepitant mg per os h before chemotherapy ondansetron mg iv min before chemotherapy methylprednisolone (mp) mg iv min before chemotherapy days and : aprepitant per os mg methylprednisolone per os mg b.i.d. for the beam strategy, this treatment is given on day (carmustine) and on day (melphalan). the course of corticosteroid was reduced on purpose for clinical oncology guideline for antiemetics in oncology: update . kris and coll keywords: aprepitant, emesis, hematology pt- use of anti-tnf-alfa in rheumatoid arthritis intravitreal voriconazole for the treatment of endogenous aspergillus endophthalmitis intravitreal voriconazole for drug-resistant fungal endophthalmitis: case series fungal endophthalmitis caused by aspergillus ustus in a patient following cataract surgery intravitreal voriconazole: an electroretinographic and histopathologic study management of endogenous fungal endophthalmitis with voriconazole andcaspofungin histological examination of an eye with endogenous aspergillus endophthalmitis treated with oral voriconazole: a case report aspergillus endophthalmitis: an unusual complication of disseminated infection in renal transplant patients maria eugenia martínez nú ñ ez , javier sánchez-rubio ferrández , noelia garrido peño , carolina apezteguía fernández background and objective: zidovudine (zdv) is the first drug that approved for treatment of hiv infected patients and now has wide use in haart regimens. this drug can cause hypoproliferative anemia bone marrow toxicity. the object of this study is evaluation of incidence of anemia in iranian hiv positive patients that received zdv in haart regimens. design: in a prospective study, hiv positive patients were referred to iranian hiv research center that start zdv in haart combination were entered the study and have followed for at least one year. baseline and monthly hematological parameters were recorded. setting: iranian hiv research center. main outcome measures: patients demographic parameters, route of infection exposure, stage of disease, cd counts, cbc, and hematological parameters. results: twenty nine ( ) patients were excluded from the study because of impossible follow-up. from patients, of them have anemia (hemoglobin less than g/dl for female and less than g/dl for male). thirty there ( ) patients have anemia before starting haart. thirty four ( ) patients have showed anemia following received zdv. twenty ( ) patients have improved anemia after were changed zdv to stavudine. conclusions: about % of hiv positive patients that were received zdv have experienced anemia.background and objective: in june , the use of infliximab has been approved by emea for the treatment of severe active crohn's disease in pediatric patients aged to years old, who have not responded to conventional therapy (corticosteroid, immunomodulator and nutrition therapy). however, pediatricians were already using infliximab for patients with inflammatory bowel syndrome (ibd) such as crohn's disease (cd), ulcerative colitis (uc) and indeterminate colitis (ic). the goal of the study was to analyze infliximab prescriptions for children and to evaluate changes in prescriptions of corticosteroid due to the introduction of infliximab. design: retrospective study in ibd patients in a pediatric teaching hospital. setting: gastroenterology unit and pharmacy department. main outcome measures: indications, infliximab dosage, anterior treatments, reason of therapeutic change (non-tolerance or inefficiency of anterior treatment and/or cortico-dependance), evolution of corticosteroid dosage and months after the introduction of infliximab. results: thirty-three children were treated by infliximab: cd, uc and ic. age for diagnosis was an average of years old ( . - . ) and . years old ( - ) for the beginning of infliximab. previous treatment to infliximab was immunomodulators, single therapy for patients (azathioprine n = , mercaptopurine n = , methotrexate n = ) or dual therapy (n = azathioprine + methotrexate), with corticosteroids (n = ) and/or mesalazine (n = ). various etiologies justified infliximab administration: corticodependance (n = ), corticoresistance (n = ), non compliance to corticotherapy (n = ), insufficient efficacy of previous treatment (n = ), non tolerance to previous treatment (n = ). at the beginning, dosage of infliximab was mg/kg. dosages were increased ( mg/kg) for patients due to insufficient clinical results. one patient also had to be switched for adalimumab because he developed human antichimeric antibody (haca). among corticodependant patients ( ), corticosteroids have been stopped after or months, ( %) and patients ( %) respectively. for patients, corticosteroids were continued without reduction of dosages, six months after the introduction of infliximab. conclusions: infliximab is the only therapeutic alternative for children who are non tolerant or non respondent to conventional treatment. moreover, this treatment permits the use of decreased dosage of corticosteroids, limiting their side effects, especially on children growth. however, haca occurrence could limit its use in a long-term disease. keywords: infliximab, inflammatory bowel syndrome, pediatrics chemotherapy indication was autologous bone marrow transplant (bmt) ( %), leukaemia chemotherapy induction or consolidation ( %), leukaemia intensive chemotherapy ( %), myeloblastic allogeneic bmt ( %) and mini allogeneic bmt ( %).treatments were prophylactic ( %), empirical ( %) or curative ( % for aspergillus sp but no for candida sp infections); % of the prescriptions related to local candidosis and % remained unknown.although % of prescriptions were in accordance with internal guidelines concerned antifungal drug indication, % had wrong dosages e.g. no loading dose for voriconazole. moreover, only % of the prescriptions were in accordance with french recommendations: neither voriconazole is approved in prophylaxis of aspergillosis in patients with autologous bmt nor antifungal drugs associations (ten prescriptions). nevertheless, it may be a good way of medical management as hopeful patients outcomes have been obtained.conclusions: hematology department guidelines should be reviewed in accordance with french recommendations, department's ecology and the state-of-the-art about treatment of fungal infections in patients with haematological malignancies. the accordance to further recommendations should be regularly assessed as well as resistance emergence. background and objective: to evaluate the efficacy and safety of rituximab (rtx) for the treatment of refractory autoimmune cytopenia, including autoimmune hemolytic anemia (aha) and immunemediated thrombocytopenia (idiopathic thrombocytopenic purpura itp and thrombotic thrombocytopenic purpura ttp) design: descriptive, retrospective study based on rituximab prescriptions analysis. patients were identified through medical reports delivered by compassionate use program. data collection was made through the pharmaco-therapeutic profile and medical chart review setting: general teaching hospital ( beds) main outcome measures: patients who received any course of rtx for refractory immune cytopenia from january to may were evaluated. data recorded included patients details, diagnosis, previous treatment, rtx schedule, number of courses and baseline hemoglobin (hb) and platelet count (pq) values. effectiveness and tolerance were also considered. response was evaluated according to criteria found in the literature: clinical symptoms resolution and a normal pq count of . /mm for itp/ttp or an hb level [ g/ dl achieved and maintained for at least months for aha. additional response criteria for aha was an hb increase [ . g/dl month after the last dose of rtx. results: patients ( men), doses rtx; average age years (range - ). diagnosis: aha ( cases cold agglutinin disease), itp and ttp. in patients cytopenia (aha) was associated with chronic lymphocytic leukemia. all patients had been previously treated with steroids and had received or more other treatment modalities ( splenectomy, immunosupressive agents, intravenous immunoglobulin). patients received - rtx infusions at a standard dose of mg/m once per week, in combination with steroids therapy in cases. no serious infusion-related effects occurred, but patients reported hematologic toxicity (fever and infection). all patients with aha ( / ) and patients with itp ( / ) responded to the first course of rtx. one patient aha had relapse after months and responded to retreatment. itp responders achieved durable response ( and months) and were offered second course of rtx after relapse ( patients did not respond to retreatment). after months follow-up, patient with ttp remained with acceptable pq counts. hb levels increased by a median of . g/dl (range - , ) among the aha responders. itp + ttp responders achieved a median increase in pq count of . /mm (range - ). only responders who reached a months follow-up were considered for response duration assessment: aha, ttp, itp ( retreatment). median response duration was months (range - ) for aha and months (range - ) for itp + ttp conclusions: most of the literature findings for rtx in this setting were related to small series or isolated case descriptions. despite the common limitation of the number of patients, our results showed that rtx appears to be a promising agent for the treatment of refractory autoimmune cytopenia. key: cord- -g qe fi authors: pullano, g.; valdano, e.; scarpa, n.; rubrichi, s.; colizza, v. title: population mobility reductions during covid- epidemic in france under lockdown date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: g qe fi on march , , french authorities implemented a nationwide lockdown to respond to covid- epidemic emergency and curb the surge of patients requiring critical care, similarly to other countries. evaluating the impact of lockdown on population mobility is important to help characterize the changes in social dynamics that affected viral diffusion. using travel flows reconstructed from mobile phone trajectories, we measured how lockdown altered mobility patterns at both local and country scales. lockdown caused a % reduction in countrywide number of displacements, and was particularly effective in reducing work-related short-range mobility, especially during rush hours, and recreational long-range trips. anomalous increases in long-range movements, localized in both time and space, emerged even before lockdown announcement. mobility drops were unevenly distributed across regions. they were strongly associated with active population, workers employed in sectors highly impacted by lockdown, and number of hospitalizations per region, and moderately associated with socio-economic level of the region. major cities largely shrank their pattern of connectivity, reducing it mainly to short-range commuting, despite the persistence of some long-range trips. our findings indicate that lockdown was very effective in reducing population mobility across scales. caution should be taken in the timing of policy announcements and implementation. individual response to policy announcements may generate unexpected anomalous behaviors increasing the risk of geographical diffusion. on the other hand, risk awareness may be beneficial in further decreasing mobility in largely affected regions. our findings help predicting how and where restrictions will be the most effective in reducing the mobility and mixing of the population, thus aiding tuning recommendations in the upcoming weeks, when phasing out lockdown. french authorities responded to the rapid growth of covid- cases by imposing heavy restrictions on mobility, as many other countries in europe and beyond . lockdown was enforced on march , , and helped slow down infection rates and limit the strain on the healthcare system . accurately measuring changes in human mobility under these restrictions is essential to (i) quantitatively determine how imposed measures and recommendations (e.g. regarding telework where possible, ban of leisure trips) translated into reduced mobility at specific scales and times, (ii) inform models estimating the effectiveness of the ongoing lockdown in reducing the epidemic spread , , (iii) help devising social distancing measures needed for the post-lockdown phase. accessing human mobility data to measure these changes is now possible at several spatial and time scales, and often in nearly real-time. these data have been proven useful in many epidemiological contexts -including for example the west africa ebola epidemic -and are being used now for covid- pandemic in many countries -namely, belgium , germany , india , italy , , poland , spain , uk , , usa [ ] [ ] [ ] . mobile phone records are one of the main sources of mobility data. they describe travel flows among the different locations of a country. these flows can be analyzed over time to study population patterns, with no information on individual users, safeguarding privacy , , . in this report, we used data provided by orange business service flux vision, and studied how mobility in france changed before and during lockdown. we broke down our results by trip distance, user age and residency, time of day, and analyzed regional data and spatial heterogeneities. we investigated behavioral responses to announcements of interventions, and to the epidemic burden, as well as associations of mobility reduction with demographic and socioeconomic indicators. considering the network of travel connections among french locations, we also identified the most vulnerable and most resilient connections to the mobility shock induced by lockdown, with a specific focus on main french cities. mobile phone data were provided by the orange business service flux vision. they comprised origin-destination travel volumes among ~ , geographic areas of mainland france, which group municipalities at the epci (Établissements publics de coopération intercommunale) level. the average distance between the centroids of two adjacent areas is km. travel volumes were computed on-the-fly from signals exchanged between mobile phones and the mobile network, which contain information about the identifiers of the mobile phone and of the antenna handling the communication. knowing the spatial localization of the antennas allows reconstructing the approximate position of the device in communication. this was then used to compute aggregated travel volumes among locations, with no residual information tracing back to the individual users. data provided the number of displacements (or trips) observed between any two consecutive locations where the user spent at least hour. for each pair of locations and any given day, data were provided stratified by age class. travel flows were adjusted by orange to be representative of the general population. regional hospitalization data were obtained from santé publique france . from them, we extracted as indicator the cumulated number of covid- -related hospitalizations per , inhabitants at a given date, for each region. on april , , grand-est . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . population data and regional socioeconomic indicators were obtained from the french national statistical institute (insee) . we used the following indicators: i) fraction of population in the age range - , corresponding with the peak of activity . Île-de-france had the highest value ( . %), bourgogne had the lowest value ( . %). the sample standard deviation across regions was . %. ii) th percentile of the regional standard of living (niveau de vie), defined by insee as the household's gross disposable income divided by the number of consumption units (measuring the size of the household -one unit for the first adult, . units for each additional person over years of age and . for each child under years of age). Île-de-france had the highest value ( , euros), hauts-de-france the lowest ( , euros). the sample standard deviation across regions was , euros. employment data were obtained from insee and from the report of the french ministry of labor on the impact of restrictions on economic activities . as indicator, we used the fraction of employees in the sectors mostly affected by lockdown. these are the sectors in which at least % of employees stopped working (hotels, hospitality, food services, and construction), or had been working remotely (finance, insurances, it). Île-de-france had the highest value ( . %), bourgogne the lowest ( . %). the sample standard deviation across regions was . %. ethics. mobile phone data were previously anonymized in compliance to strict privacy requirements, reviewed and approved by the french national commission for data protection (cnil, commission nationale de l'informatique et des libertés), ruling on all matters related to ethics, data, and privacy. timeline fit and prediction. to fit and forecast time series we used the forecasting procedure prophet by facebook open source . we enforced weekly seasonality, and used school holidays by region as additional (additive) regressors. trip analysis. our analyses were performed on all trips and on trips whose geodesic distance between location centroids is longer than km (long trips). the cutoff of km effectively discards commuting, as ~ % of daily work-related trips are shorter than km , . we distinguished between residents, i.e., users with french sim cards, and foreigners. we broke down data in three age classes: young (younger than y.o.), adults ( - y.o.), and seniors ( + y.o.). we classified tips by their time of day: daytime ( am- pm), nighttime ( pm- am), and distinguished between weekdays and weekends. during weekdays we also considered rush hours ( am- am, pm- pm). mobility reduction during lockdown. mobility reduction during lockdown was computed in a case-crossover framework by comparing the week starting monday april , ( weeks into lockdown), to the week starting monday february (control week). the latter was chosen as being before school holidays, and after strikes of public transport. all statistical analyses were performed in r, version . . . two-sided significance of pearson coefficients was determined at a level of . . network analysis. nodes in the networks represent the geographic locations in which we divided mainland france, and links represent trips between locations. links are directed (trips have origins and destinations), weighted (by the number of trips linking . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint two locations), and evolve in time. to handle and analyze networks we used standard python libraries, among which networkx. to smooth spatial data, we used a standard gaussian kernel with fixed characteristic distance, and adjusted locations by their population (see appendix). the radius containing % of outgoing traffic from a city was computed by considering all mobility links that start from that city, each with its geodesic distance. they were included incrementally from the shortest to the longest (in terms of geodesic distance), until the cumulative sum of the weights of the included links reached % of the total outgoing traffic. changes in circle radius capture changes in the geographic pattern of outgoing mobility. if mobility is reduced homogeneously across distances, the radius will remain constant. if reduction increases with the distance, the radius will decrease (and vice versa). three phases have marked the french response to covid- epidemic (figure ). phase started in early january and can be identified with the first publication of covid- case definition by santé publique france . its aim was to detect imported cases as quickly as possible and conduct case-contact epidemiological investigations to identify possible local transmissions and isolate cases. phase started on february , upon appearance of localized clusters, and featured the same measures of phase coupled with targeted social distancing interventions (e.g. school closure, gatherings and public transport bans) to stop possible transmission in the community. during this phase, two clusters were identified, in oise and haute-savoie. phase was declared on march when the virus was recognized to actively circulate in the territory. starting few days before phase , a set of announcements were made by french authorities that progressively led to the lockdown on march , ( . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . while no observable change in mobility occurred during phase and of the epidemic, the start of phase on march had a substantial impact on mobility in france (fig. a) . this transition occurred prior to the announcement (march ) and implementation (march ) of lockdown measures, and saw nationwide mobility go from ~ m trips per day down to ~ m trips after lockdown entered into effect. the shock in mobility spread out over a transition period lasting almost a week. to study in detail this transition, we quantified the deviation of measured traffic flows from the predicted evolution of traffic over time. predictions were obtained from fitting is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint mobility data from january , , to monday, march (training set, fig. ) , and assuming no perturbation due to covid- and associated interventions after march . total flow was significantly below predictions starting march , as a likely consequence of the start of phase . mobility further decreased on sunday, march , when local elections took place. instead, an anomalous rise in traffic took place on the day before lockdown enforcement, which had higher volume than the surrounding days, whereas still lower than the predicted baseline. long trips (> km) were also significantlyalbeit slightly -below the predicted baseline during the weekend (march , ). they however went back to seemingly normal values on march -i.e., in agreement with the unperturbed prediction -, and near-to-normal values on lockdown day. however, this country-level behavior hid anomalous deviations from the predicted mobility behavior in specific locations, as fig. b shows. spikes in outgoing traffic are distinctively visible in Île-de-france (the region of paris) and, at the same time, in incoming traffic in normandy and bretagne. they measure the pre-lockdown exodus out of paris occurring before lockdown took effect , . analyses at finer scales within Île-de-france revealed that anomalous outgoing traffic concentrated in the paris area, and western Île-de-france. similar spikes of outgoing and incoming traffic were also visible in the south east, close to the alps, as reported previously . the transition starting with phase reshaped weekly patterns compared to those measured in the unperturbed mobility. before the mobility shock, a stable weekly pattern was observed, with peaks on fridays and troughs on sundays for all trips, and peaks on both fridays and sundays for long trips. during the transition, no weekly pattern was recognizable, as mobility was perturbed in different ways across several days. following the transition, patterns no longer featured peaks in mobility on fridays (for all trips) or on sundays (for long trips). mobility patterns quickly entered a new equilibrium after lockdown enforcement, marking the end of the transition period. using a crossover framework (see methods), we found that lockdown decreased the overall number of trips by % (figure a) . reduction was stronger for trips made by foreigners (~ %), suggesting that the enforcement of lockdown disrupted tourism and impacted more the mobility of foreign nationals in the country . their number of trips was however very small even before lockdown compared to french residents ( %), therefore we excluded them from the rest of the analysis as their contribution is negligible. long-range traffic (> km) was disrupted more severely than average ( % reduction, fig. a) . this was likely associated with a disruption of long-range transportation (trains, flights), and the ban of leisure-related trips, also confirmed by the almost disappearance of long trips during the weekend (see below). . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint figure . mobility reduction during lockdown across user type, age and time of day. reduction is computed as the average over the week starting monday april , with respect to the average over the first week of february (starting monday february ). a), b) and c) show the relative reduction broken down by residents/foreigners, age classes, and times of the day. they also show statistics for all trips (orange) and long trips (green), defined as trips with geodesic distance longer than km. horizontal orange and green lines indicate relative reduction on all residents (all, long, respectively). mobility reduction in total trips was homogeneously distributed across age classes (fig. b) . when considering only long trips, reduction instead increased with age, as seniors reduced their trips above km by ~ %. drops in mobility were uneven across the time of the day (fig. c) . movements during rush hours were the most disrupted, indicating that the combined effect of school closure and telework led to a ~ % reduction. daytime movements during weekends also exhibited a higher-than-average decrease, hinting at a successful reduction of recreational activities. nighttime movements during weekdays instead recorded the lowest reduction, well below average. they might be related to unavoidable workrelated mobility, whose impact is however likely to be limited, as these movements make up for only ¼ of the total. long-range mobility almost completely stopped during weekends (around % decrease). regional heterogeneities in mobility reduction during lockdown. traffic reductions were not homogeneous across the regions of mainland france. reduction of internal traffic was above average in regions (Île-de-france, auvergne-rhône-alpes, grand est, provence-alpes-côte d'azur), whereas markedly below average in bourgogne-franche-comté, centre-val de loire, and normandy (figure ) . similar fluctuations were visible in outgoing traffic (coefficient of variation equal to . % compared to . % for internal traffic). Île-de-france, hauts-de-france and grand est all experienced above-average reductions in outgoing mobility, as high as % for Île-de-france. corse also exhibited a reduction comparable to Île-de-france, showing a clear disruption of the long-range connections linking the island to mainland france. similar reductions were obtained with incoming fluxes in the regions (not shown). . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . the impact of nationwide lockdown in the reduction of outgoing mobility per region was strongly associated with the fraction of the population in the most active age range ( - y.o.) (pearson r = . , p < . ) and the fraction of workers employed in sectors that substantially modified their organization during lockdown, due to telework, partial or full closure of activities (pearson r = . , p < . ) (table and figure ) . it was moderately associated with the standard of living of the region (pearson r = . , p = . ). regional drops in mobility in a given week (april - , ) were strongly associated with covid- hospitalization rates registered and communicated in the week before (april ) (pearson r = . , p < . ; figure ) . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . similar results were obtained for drops in mobility within the region, except for the association with the hospitalization rate per region, which however showed a similar, though non-significant, tendency (table s and fig. s in appendix). taking out the data point of Île-de-france as the region mostly affected by a departure of inhabitants for relocation in other regions led to similar results ( table s in appendix). disruption of mobility connections. shifting the focus from overall traffic reductions to mobility connections between locations, we found that some connections completely disappeared, as individuals stopped going from one location to another (figure ) . the probability that a mobility connection observed in the control week (week of february , ) was also observed when interventions were announced and after they entered into effect (persistence probability, fig. b ) decreased steadily during the transition period ( % of connections surviving in the week of school closure and nonessential activity closure announcements, march to ; % in the week of announcement and implementation of lockdown, march to ) to stabilize in the first . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint full week of lockdown ( % of connections surviving, march ) and beyond. long connections were less resilient than average, as only / of them survived lockdown. after lockdown effects stabilized (e.g. starting the second full week of lockdown, march ), connections usually characterized by small traffic prior to restrictions (weak connections) were the most likely to disappear, with % of them corresponding to trips per week (fig. c) . the traffic lost on these connections however barely contributed to total traffic reduction ( % contribution). restricting the analysis to long mobility connections (> km), the fraction of the weak connections disappearing slightly increased (from % to %), however with a reduction of % of the traffic. the disruption in connections occurred with a certain delay compared to reductions in traffic. for example, on monday march -the day before lockdown -traffic was reduced by % with respect to the previous monday, but the number of connections went down by % only. one week after (march ), traffic drop was % and the drop in the number of connections was %. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint lost on connections which used to have at most a certain weight in the baseline week. for all panels: orange: all links, green long links (longer than km). restrictions on mobility during lockdown had an uneven impact on the most populated french cities. the circle containing % of outgoing traffic from each city decreased after lockdown took effect for all cities, indicating that long-range mobility was disrupted more than short-range one (figure ) . but reductions varied from more than % (paris, bordeaux, nice) to % (strasbourg, lille), mainly due to different patterns of commuting and connectivity characterizing the mobility of each city. in normal circumstances, paris is connected to almost the rest of the country, whereas the other cities have a more localized pattern of mobility with fewer long-range connections. once lockdown was implemented, surviving mobility shrank around the cities. connections among main cities disappeared too. considering the connections per city with highest traffic that are compromised by the lockdown, we no longer detected mobility from bordeaux, montpellier, and nantes to lyon, or from montpellier to strasbourg (figure ), compared to pre-emergency situation. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . using travel flow data extracted from mobile phone trajectories, we documented a large drop in both short-range and long-range population mobility following lockdown enforcement in france. overall, trips were reduced by %, similarly to reductions found in belgium , spain , and italy during lockdown, albeit different data sources, spatial resolutions, and definitions of mobility proxies prevent direct numerical comparisons. the transition signaling the drop in mobility lasted almost a week, anticipating the enforcement of lockdown and creating opposite mobility behaviors. individuals started spontaneously reducing their mobility on saturday following the announcement of school closure, likely because of fear of the growing epidemic and heightened risk awareness - generated by the first governmental decision on nationwide interventions. the weekday-to-weekend pattern was disrupted, with overall mobility on monday following the closure of all non-essential activities similar to the preceding saturday. at the same time, fear of an imminent change in policy imposing stricter restrictions -as already implemented in italy, spain, austria pushed individuals to relocate themselves even to farther away regions where to spend the period of lockdown, if put in place. the exodus, largely covered by the press , , occurred already before the announcement of lockdown and led to anomalous increases in mobility flows out of certain regions (e.g. Île-de-france) and incoming in others (e.g. normandy). such behavior was similarly reported in china (from wuhan to other regions), in italy (from the north to the south) prior to the implementation of lockdown, and in india . it demonstrates that the timing at which a policy is announced might disrupt social dynamics as much as the direct effect of the policy, at least in the short term. increased caution should therefore be considered in the period from announcements to enforcement to avoid unpredictable behaviors that may result in unwanted seeding of the epidemic to other areas. no increase in viral circulation became then visible in the receiving regions in the following weeks, as lockdown strongly suppressed epidemic activity in all regions , , , . seeding events due to relocations may however become more important in phasing out the lockdown, as less strict social distancing measures may prevent such suppression. region-specific interventions may increase this risk by inducing similar behavioral responses. new york state reported for instance increased mobility in counties with no imposed lockdown . in this perspective, nationwide interventions and restrictions limiting displacements were adopted by several countries , to prevent compensation effects and reduce the possible geographical spread of the epidemic. once lockdown entered into effect, population mobility reductions were heterogeneous across regions. larger reductions were measured in regions more severely hit by the epidemic, with an estimated % decrease in regional mobility every additional hospitalizations (per , inhabitants). this suggests that individuals witnessing a larger covid- burden on the hospital system in their region may have further limited displacements compared to those living in less affected regions. media largely communicated on the epidemic, also providing early on region-specific information on hospitalizations and mounting pressure on the healthcare system. exposure to this information likely triggered a behavioral response increasing compliance to movement restrictions. a similar, though stronger, behavior was observed during a -day national lockdown enforced nationwide in sierra leone in march in an effort to control ebola epidemic . the correlation remains significant even taking out the region of Île-de-france, which experienced a reduction in population due to relocation of individuals. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint clearly, other factors may have come into play to differentiate drops in regional mobility. lockdown restrictions had a severe impact on jobs and the organization of work. regions with the higher proportion of activity sectors mostly impacted by the lockdown (due to telework, but also to complete or partial closure of sectors, such as tourism, entertainment, food services, and construction) also experienced larger drops on mobility. a smaller fraction of active individuals continued to go to work, while the others limited their displacements respecting lockdown mobility restrictions. indeed, regions with larger portions of the population in the most active age range ( - y) , were also the ones where lockdown had the largest effects. besides the displacements to go to work, active population is also highly mobile for leisure activities, which were completely banned by restrictions (with short exceptions to do sport once a day for at most hour). uneven mobility drops were also associated with socioeconomic disparities. increasing evidence points at different socioeconomic strata getting uneven shares of the covid- burden . higher income jobs can often be performed remotely, in confinement, whereas lower income jobs cannot. a survey in france reported that % of low income workers were still going to their workplace during lockdown, against only % of high income workers . also, wealthier population strata weather short-term financial losses better, making them more prone to stop working and stay at home if they are afraid or sick. at the same time, they can afford more leisure activities and have a more varied social network , , leading to a higher rate of leisure-related mobility in normal circumstances. wealthier populations then likely experienced a larger mobility reduction because of the possibility to work remotely or stop working, as well as for the imposed ban on leisure activities. a strong response was documented in the older age class, which is at highest risk of developing severe forms of covid- if infected. seniors almost stopped taking trips longer than km, likely to avoid leisure activities and family trips, as recommended by authorities. the most effective reduction in overall mobility occurred during rush hours, associated with a disruption of commuting patterns. this reduction alone likely boosted the role of mobility restrictions in suppressing viral diffusion, as mounting evidence shows that public transportation is a main risk factor for transmission , . lockdown had a different impact on mobility depending on distance, causing larger disruptions on long-range mobility, as also reported in belgium and italy . short-range and long-range mobility flows play different roles in the spread of an infectious disease epidemic. short-range connections are mainly responsible for local diffusion in the community within and around a metropolitan area, whereas long-range connections drive the spatial spread of the epidemic, acting as seeding events to otherwise unaffected or weakly affected areas . mobility flows out of the city of wuhan were shown to have seeded other prefectures in china in the early phase of the epidemic before travel restrictions and substantial control measures were implemented [ ] [ ] [ ] . a delayed response or less efficient lockdown would have likely led to a larger outbreak increasing its geographical range. coupled with social distancing interventions, longrange mobility restrictions are therefore critical to geographically contain the epidemic, especially when epidemic activity is largely heterogeneous at the spatial scale, showing a patchy geographical pattern observed in many affected countries including france. banning trips above km as announced by french authorities will continue breaking the spreading pathways along which the epidemic could spread and reducing the locations reachable by the virus, as observed during the lockdown. nonetheless, in the lockdown phase we documented that some long-range mobility connection, among the ones with highest traffic, survived the restrictions -namely, from paris to . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org / . / . / montpellier, and from the other most populated cities to paris (except toulouse and strasbourg). these movements should be carefully accompanied by strict hygienic and preventive measures to avoid re-seeding events from visitors or returning residents, as discussed above. the largest reduction of mobility across distance was reported for paris. before lockdown, % of outgoing traffic reached destinations within km from the city center, approximately the distance between paris and lille, close to the belgian border. after lockdown, this radius reduced to km, the distance from the city center to disneyland paris. achievable distances from large cities shrank during lockdown, even in absence of explicit limitations on distance, also reducing the number of reachable destinations. mobility became more localized and restructured around metropolitan areas, serving the needs of individuals who continued their daily displacements associated to work, e.g. in essential professional categories. a similar geographical fragmentation induced by restructured local job markets was also observed in italy . our analysis offered plausible interpretations on how the labor market, demographic and socio-economic indicators, and awareness of increased epidemic risk might have shaped the reduction in mobility, confirming evidence observed in previous , and current , outbreaks. being observational in nature, the study does not allow us to identify causal relationships; also, confounding effects among the covariates may be expected, but the available sample was too small to take this into account. focusing on the reduction in mobility during lockdown and its association to hospitalizations in the same time period, our study did not aim to assess the role of mobility in shaping the epidemic spread, but to investigate a behavioral response likely induced by risk awareness. associations were robust also removing the data point for Île-de-france, the region mostly affected by the exodus of individuals relocating in other locations. this suggests that associations are not biased by a change in population size of the region. regional variations in mobility may be induced by differential restrictions based on estimated epidemic activity in the region . however, this was not the case in france, where a nationwide lockdown was applied uniformly in the country. local authorities additionally imposed heavier restrictions in certain areas over time, like curfews in cities in hauts-de-france and in the south of france . we did not consider these additional restrictions as possible factors in our analysis. we expect them to result in smaller effects, likely not visible at the resolution scale under study here. using aggregated flow data extracted from mobile phone trajectories, we documented the large impact that lockdown had on reducing mobility in france. different effects were observed across scales, with larger disruptions on long-range connections leading to a localization of the mobility. factors related to demography, professional categories, and socio-economic level were all associated with the reduction in mobility. uneven drops in population movements by region may also be explained by a different behavioral response linked to the perceived risk of the epidemic in the region. our findings may help predicting how and where restrictions will be the most effective in reducing the mobility and mixing of the population, thus aiding tuning recommendations in the upcoming weeks, when phasing out lockdown. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint let ! be the value in location of the quantity we want to smooth. let ! be the population in , and ∆ !" the geodesic distance between locations , . then, the smoothed value is ! #$%%&'() = ∑ " " *+ ∆ !" -. where is the characteristic distance parameter. the sum runs over all the locations. table s . correlation coefficients. the table reports the correlation coefficients and their p-value for the four indicators considered and internal and outgoing regional mobility. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . table s . correlation coefficients without Île-de-france. the table reports the correlation coefficients and their p-value for the four indicators considered and internal and outgoing regional mobility, computed excluding Île-de-france. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint figure s . reduction in internal mobility for the week april - , vs. epidemic, socioeconomic, and demographic indicators. the following plot is the equivalent of figure for internal mobility. correlation is evaluated between outgoing traffic and the four considered indicators: a) the population in active age ( - years old), b) the fraction of employees in the sectors mostly affected by lockdown. c) the th percentile of the regional standard of living. pearson correlation coefficients and their p-values are reported, d) the cumulated number of covid- hospitalizations per , inhabitants on april , . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint how will country-based mitigation measures influence the course of the covid- epidemic? santepubliquefrance.fr. covid- : point épidémiologique du avril expected impact of reopening schools after lockdown on covid- epidemic in Île-de-france expected impact of lockdown in Île-de-france and possible exit strategies human mobility: models and applications population mobility reductions associated with travel restrictions during the ebola epidemic in sierra leone: use of mobile phone data mobile phone data and covid- : missing an opportunity? covid- : belgium analyses telecom data to measure the impact of confinement covid- mobility project in germany mapping the lockdown effects in india: how geographers can contribute to tackle covid- diffusion covid- outbreak response: first assessment of mobility changes in italy following lockdown mobile phone data analytics against the covid- epidemics in italy: flow diversity and local job markets during the national lockdown community mobility changes due to the coronavirus (covid- ) outbreak in poland informe sobre los cambios de movilidad en españa debido a las medidas de confinamiento contra la extensión del covid- oxford covid- impact monitor analysis of human mobility in the uk during the covid- pandemic assessing changes in commuting and individual mobility in major metropolitan areas in the united states during the covid- outbreak effect of social distancing measures in the new york city metropolitan area mapping county-level mobility pattern changes in the united states in response to covid- protect privacy of mobile data aggregated mobility data could help fight covid- indicateurs : cartes insee. bilans économiques des régions françaises activité et conditions d'emploi de la main-d'oeuvre pendant la crise sanitaire covid- coronavirus : le grand exode des citadins confinement : plus d'un million de franciliens ont quitté la région parisienne en une semaine cnews. coronavirus : quel impact sur le tourisme epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong what can we learn about the ebola outbreak from tweets? outpatients' behavior of seeking medical care after onset of severe acute respiratory syndrome and community control measures in beijing public perceptions, anxiety, and behaviour change in relation to the swine flu outbreak: cross sectional telephone survey survey on the likely behavioural changes of the general public in four european countries during the / pandemic covid- epi dashboard estimating the burden of sars-cov- in france covid- : one-month impact of the french lockdown on the epidemic burden stratégie de déconfinement fase american inequality meets covid- premiers de corvée et premiers de cordée, quel avenir pour le travail déconfiné network diversity and economic development using big data to study the link between human mobility and socio-economic development the subways seeded the massive coronavirus epidemic epidemiology and transmission of covid- in cases and of their close contacts in shenzhen, china: a retrospective cohort study multiscale mobility networks and the spatial spreading of infectious diseases population flow drives spatio-temporal distribution of covid- in china the effect of human mobility and control measures on the covid- epidemic in china effect of non-pharmaceutical interventions to contain covid- in china location data says it all: staying at home during coronavirus is a luxury this study is partially funded by: anr projects evalcovid- (anr- -covi- ), sphinx (anr- -ce - - ) and dataredux (anr- -ce - - ); eu h grants recover (h - ) and mood (h - ); reacting covid- modeling grant. we thank luca ferreri, sylvain bourgeois, erwan le quentrec, zbigniew smoreda, chiara poletto, pierre-yves boëlle for useful discussions. key: cord- -med c q authors: fovet, thomas; lancelevee, camille; eck, marion; scouflaire, tatiana; becache, eve; dandelot, dominique; giravalli, pascale; guillard, alexandre; horrach, pierre; lacambre, mathieu; lefebvre, tiphaine; moncany, anne-hélène; touitou, david; david, michel; thomas, pierre title: prisons confinées: quelles conséquences pour les soins psychiatriques et la santé mentale des personnes détenues en france? date: - - journal: encephale doi: . /j.encep. . . sha: doc_id: cord_uid: med c q résumé objectif. en france, les mesures de confinement ont été accompagnées de dispositions spécifiques pour les prisons: suspension des activités, parloirs et interventions extérieures. plus de dix mille personnes détenues ont en outre été libérées pour diminuer le taux d’occupation des établissements et limiter la propagation du virus. l’objectif de cet article est de décrire la réorganisation des soins psychiatriques en milieu pénitentiaire en contexte de pandémie de covid- et d’interroger les conséquences du confinement et des libérations anticipées sur la santé mentale des personnes détenues. méthode. ce travail s’appuie sur une enquête menée en avril auprès des soignants de unités sanitaires en milieu pénitentiaire et des unités hospitalières spécialement aménagées en france. une synthèse de la littérature internationale sur la question des soins psychiatriques en milieu pénitentiaire durant l’épidémie de covid- a également été réalisée. résultats. l’épidémie de covid- semble avoir été plutôt contenue dans les prisons françaises au cours de la période de confinement mais le poids des mesures mises en place sur la population carcérale est important. les niveaux de soins psychiatriques en milieu pénitentiaire ont instauré des mesures spécifiques pour assurer la continuité des soins, accompagner les personnes incarcérées et contenir l’épidémie. parmi les plus importantes, on note la restriction des consultations, la création de « secteurs covid », la déprogrammation des hospitalisations non urgentes, le renforcement des mesures d’hygiène et le remaniement des effectifs. actuellement, les soignants sont principalement confrontés à des sevrages forcés, des symptomatologies anxieuses et des décompensations de troubles psychiatriques chroniques. certaines libérations anticipées sont aussi très préoccupantes, pouvant entraîner des ruptures de soins, par manque de préparation des relais de prise en charge. discussion. les remaniements en lien avec le confinement donnent une visibilité accrue au fossé qui sépare la psychiatrie en milieu libre de la psychiatrie en milieu pénitentiaire. il nous apparaît important de rappeler la vulnérabilité des personnes incarcérées qui doivent impérativement être considérées dans les politiques de santé publique. abstract objective. the impact of the covid- pandemic on the million people currently incarcerated worldwide is the subject of many concerns. prisons and jails are filled with people suffering from many preexisting medical conditions increasing the risk of complications. detainees’ access to medical services is already limited and overcrowding poses a threat of massive contagion. beyond the health impact of the crisis, the tightening of prison conditions worries. on march , , in france, the lockdown measures have been accompanied by specific provisions for prisons: all facilities have suspended visitations, group activities and external interventions. over prisoners have been released to reduce the prison population and the risk of virus propagation. these adjustments had major consequences on the healthcare system in french prisons. the objectives of this article are to describe the reorganization of the three levels of psychiatric care for inmates in france in the context of covid- pandemic and to have a look at the impact of lockdown measures and early releases on mental health of prisoners. methods. this work is based on a survey conducted in april in france among psychiatric healthcare providers working in ambulatory units for inmates and in the full-time inpatient psychiatric wards exclusively for inmates called “uhsas” (which stands for “unités hospitalières spécialement aménagées”, and can be translated as “specially equipped hospital units”). a review of the international literature on mental healthcare system for inmates during the covid- epidemic has also been performed. results. the covid- epidemic has been rather contained during the period of confinement in french prisons but the impact of confinement measures on the prison population is significant. the three levels of psychiatric care for inmates have implemented specific measures to ensure continuity of care, to support detainees during coronavirus lockdown and to prevent an infection’s spread. among the most important are: limitation of medical consultations to serious and urgent cases, creation of “covid units”, cancellation of voluntary psychiatric hospitalizations, reinforcement of preventive hygiene measures and reshuffling of medical staff. prolonged confinement has consequences on mental health of detainees. currently, mental health workers are facing multiple clinical situations such as forced drug and substance withdrawal (linked to difficulties in supplying psychoactive substances), symptoms of anxiety (due to concerns for their own and their relatives’ wellbeing) and decompensation among patients with severe psychiatric conditions. early releases from prison may also raise some issues. people recently released from prison are identified as at high risk of death by suicide and drug overdose. the lack of time to provide the necessary link between health services within prisons and health structures outside, could have serious consequences, emphasizing the well-known “revolving prison doors” effect. discussion. the current lockdown measures applied in french jails and prisons point out the disparities between psychiatric care for inmates and psychiatric care for general population. giving the high vulnerability of prison population, public health authorities should pay more attention to health care in prisons. objectif. en france, les mesures de confinement ont été accompagnées de dispositions spécifiques pour les prisons : suspension des activités, parloirs et interventions extérieures. plus de dix mille personnes détenues ont en outre été libérées pour diminuer le taux d'occupation des établissements et limiter la propagation du virus. l'objectif de cet article est de décrire la réorganisation des soins psychiatriques en milieu pénitentiaire en contexte de pandémie de covid- et d'interroger les conséquences du confinement et des libérations anticipées sur la santé mentale des personnes détenues. méthode. ce travail s'appuie sur une enquête menée en avril auprès des soignants de unités sanitaires en milieu pénitentiaire et des unités hospitalières spécialement aménagées en france. une synthèse de la littérature internationale sur la question des soins psychiatriques en milieu pénitentiaire durant l'épidémie de covid- a également été réalisée. résultats. l'épidémie de covid- semble avoir été plutôt contenue dans les prisons françaises au cours de la période de confinement mais le poids des mesures mises en place sur la population carcérale est important. les niveaux de soins psychiatriques en milieu pénitentiaire ont instauré des mesures spécifiques pour assurer la continuité des soins, accompagner les personnes incarcérées et contenir l'épidémie. parmi les plus importantes, on note la restriction des consultations, la création de « secteurs covid », la déprogrammation des hospitalisations non urgentes, le renforcement des mesures d'hygiène et le remaniement des effectifs. actuellement, les soignants sont principalement confrontés à des sevrages forcés, des symptomatologies anxieuses et des décompensations de troubles psychiatriques chroniques. certaines libérations anticipées sont aussi très préoccupantes, pouvant entraîner des ruptures de soins, par manque de préparation des relais de prise en charge. discussion. les remaniements en lien avec le confinement donnent une visibilité accrue au fossé qui sépare la psychiatrie en milieu libre de la psychiatrie en milieu pénitentiaire. il nous apparaît important de rappeler la vulnérabilité des personnes incarcérées qui doivent impérativement être considérées dans les politiques de santé publique. mots-clés : coronavirus, covid- , sars-cov- , épidémie, pandémie, psychiatrie, milieu pénitentiaire, confinement abstract objective. the impact of the covid- pandemic on the million people currently incarcerated worldwide is the subject of many concerns. prisons and jails are filled with people suffering from many preexisting medical conditions increasing the risk of complications. detainees' access to medical services is already limited and overcrowding poses a threat of massive contagion. beyond the health impact of the crisis, the tightening of prison conditions worries. on march , , in france, the lockdown measures have been accompanied by specific provisions for prisons: all facilities have suspended visitations, group activities and external interventions. over prisoners have been released to reduce the prison population and the risk of virus propagation. these adjustments had major consequences on the healthcare system in french prisons. the objectives of this article are to describe the reorganization of the three levels of psychiatric care for inmates in france in the context of covid- pandemic and to have a look at the impact of lockdown measures and early releases on mental health of prisoners. methods. this work is based on a survey conducted in april in france among psychiatric healthcare providers working in ambulatory units for inmates and in the fulltime inpatient psychiatric wards exclusively for inmates called "uhsas" (which stands for "unités hospitalières spécialement aménagées", and can be translated as "specially equipped hospital units"). a review of the international literature on mental healthcare system for inmates during the covid- epidemic has also been performed. results. the covid- epidemic has been rather contained during the period of confinement in french prisons but the impact of confinement measures on the prison population is significant. the three levels of psychiatric care for inmates have implemented specific measures to ensure continuity of care, to support detainees during coronavirus lockdown and to prevent an infection's spread. among the most important are: limitation of medical consultations to serious and urgent cases, creation of "covid units", cancellation of voluntary psychiatric hospitalizations, reinforcement of preventive hygiene measures and reshuffling of medical staff. prolonged confinement has consequences on mental health of detainees. currently, mental health workers are facing multiple clinical situations such as forced drug and substance withdrawal (linked to difficulties in supplying psychoactive substances), symptoms of anxiety (due to concerns for their own and their relatives' wellbeing) and decompensation among patients with severe psychiatric conditions. early releases from prison may also raise some issues. people recently released from prison are identified as at high risk of death by suicide and drug overdose. the lack of time to provide the necessary link between health services within prisons and health structures outside, could have serious consequences, emphasizing the well-known "revolving prison doors" effect. discussion. the current comme dans tous les lieux d'enfermement, la contagion est une menace constante en prison et les exemples historiques ne manquent pas pour illustrer ce constat. dès le xviii e siècle, john howard dénonce les conditions d'incarcération dans les prisons anglaises ainsi que les conséquences dramatiques des épidémies de typhus (connu alors sous le nom de « fièvre des prisons ») [ ] . la pandémie grippale de dite « grippe espagnole » aurait quant à elle touché environ un quart de la population carcérale, une prévalence bien plus importante qu'en population générale [ ] . plus récemment, des épidémies de grippes ont été rapportées dans des établissements pénitentiaires de plusieurs pays [ , ] . par ailleurs, ces observations épidémiologiques ne s'accompagnent pas toujours des mesures de prévention et de prise en charge adaptées, comme en témoignent les faibles taux de vaccination de la population carcérale au cours de la pandémie de grippe h n [ ] . l'impact de la pandémie de covid- sur les onze millions de personnes actuellement incarcérées à travers le monde [ ] fait donc l'objet de nombreuses inquiétudes et interrogations [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . la population carcérale apparaît particulièrement fragile et potentiellement plus exposée aux formes sévères de la maladie. en effet, parmi les personnes détenues, la prévalence des maladies chroniques associées à une immunodépression est élevée [ , ] et un vieillissement de cette population est observé dans de nombreux pays ( personnes sont âgées de plus de ans dans les prisons françaises au er janvier ) [ , ] . de plus, plusieurs facteurs comme la surpopulation ou le cadre de fonctionnement sécuritaire, peuvent constituer des freins à un accès aux soins de qualité en milieu pénitentiaire [ ] . de nombreuses recommandations ont émergé ces derniers mois des organismes internationaux et des sociétés savantes pour limiter la propagation de l'infection à sars-cov- en population générale. toutefois, les conditions d'incarcération actuelles interrogent quant à la possibilité de mettre en place, en milieu carcéral, l'ensemble des mesures de distanciation sociale actuellement recommandées [ ] . au er janvier , personnes sont détenues en france pour places opérationnelles. plus de la moitié de ces personnes incarcérées se trouvent dans une structure sur-occupée à plus de % et l'administration pénitentiaire dénombre matelas au sol [ ] . c'est dans ce contexte que l'entrée en vigueur des mesures générales de confinement annoncées le mars a été accompagnée de dispositions spécifiques pour les prisons comme la suspension de toutes les activités considérées comme non essentielles (travail, formation, culte, etc.), la limitation des mouvements et la suppression des parloirs et interventions extérieures. le rôle décisif que joue le phénomène de surpopulation dans la transmission des infections en milieu pénitentiaire a très rapidement conduit de nombreux auteurs à proposer la libération massive des personnes incarcérées dans le contexte de la pandémie de covid- [ , , , ] . une revue de la littérature récente a en effet mis en évidence une association entre la transmission des maladies infectieuses et la surface disponible par personne détenue dans les cellules [ ] . ainsi, plus de prisonniers iraniens ont été libérés et cette politique de « décarcéralisation » a été adoptée par de nombreux autres pays [ ] . en france, plus de personnes détenues (en majorité des personnes qui présentaient un reliquat de peine inférieur à mois) ont été libérées (assignations à domicile, les données épidémiologiques actuellement disponibles sur le nombre de prisonniers atteints du covid- sont limitées [ ] . aux États-unis, sur les prisonniers fédéraux, cas de covid- (dont personnes décédées) ont été confirmés. parmi les personnels pénitentiaires, cas sont recensés (recherche effectuée le mai [ ] ). au royaume-uni, au moins personnes incarcérées sont décédées du covid- [ ] . en chine, l'impact de l'épidémie en détention aurait été largement minimisé selon certains auteurs [ ] . en france, les données communiquées par la direction [ ] . en france, des dispositions ont ainsi été mises en place le mars pour accompagner les restrictions liées au confinement (crédit téléphonique, gratuité de la télévision, etc.), mais elles ont été jugées insuffisantes par la contrôleure générale des lieux de privation de liberté [ ] . l'ensemble des aménagements pris pour limiter l'impact de l'épidémie de covid- a eu des conséquences majeures sur le système de soins psychiatriques en milieu pénitentiaire. celui-ci doit pourtant impérativement continuer à effectuer ses missions compte tenu de la prévalence élevée des troubles psychiatriques en détention [ , ] mais aussi des conséquences potentielles des mesures de confinement sur la population carcérale [ ] . cet article se propose de décrire la réorganisation des trois niveaux de soins psychiatriques en milieu pénitentiaire au cours de la pandémie de covid- et en france, les établissements autorisés en psychiatrie ont très rapidement créé des unités permettant de prendre en charge les patients souffrant de troubles psychiatriques et du covid- [ ] . toutefois, une nette reprise de l'activité est décrite depuis mi-avril . en ce qui concerne l'admission des personnes détenues sur les secteurs de psychiatrie générale en soins psychiatriques sur décision d'un représentant de l'État, aucune donnée nationale n'est disponible et l'hétérogénéité des pratiques ne permet pas d'établir un état des lieux. le poids du confinement en détention il est difficile de prédire quelles seront les conséquences du confinement en population carcérale. les rares données actuellement disponibles en population générale font état de phénomènes fréquents de peur de la contamination, d'inquiétude pour les proches, d'irritabilité ou de sentiments de frustration et d'impuissance [ ] . pour limiter ces réactions, de nombreuses recommandations insistent sur la nécessité de planifier des activités, de pratiquer des exercices physiques, de maintenir des liens sociaux ou d'entretenir une hygiène de sommeil, par exemple. l'accent est mis sur l'utilisation de réseaux sociaux, de sites internet ou d'applications mobiles pour un accès aux pratiques de relaxation et méditation [ ] . là encore, les mesures proposées apparaissent bien éloignées de la réalité du milieu carcéral et quasiment impossible à mettre en oeuvre pour les personnes incarcérées. les mesures de confinement actuelles exacerbent des difficultés bien connues en milieu pénitentiaire comme l'isolement ou l'inactivité contrainte. elles entraînent l'inquiétude des personnes détenues sur leur santé ou celle de leurs proches. des difficultés matérielles (problème d'approvisionnement en linge propre suite à la suspension des parloirs ou difficultés financières dues à l'arrêt du travail en détention par exemple) sont aussi rapportées. enfin, les reports d'audience et la en ce qui concerne le suicide, qui constitue une préoccupation majeure en milieu pénitentiaire [ ] , les chiffres semblent stables (la disp de lille a par exemple enregistré suicides entre le [ ] . on sait à quel point la période suivant la libération est associée à une mortalité élevée, principalement en raison du suicide et des overdoses [ ] . ce risque est d'autant plus important que la personne a connu, au cours de sa détention, une période d'isolement [ ] . la sortie de détention est aussi identifiée comme un risque majeur de rupture de prise en charge si elle n'a pu être anticipée [ ] . « la santé en prison, c'est de la santé publique » expliquent les épidémiologistes, insistant sur l'importance d'intégrer les mesures visant à limiter la diffusion du covid- en détention à la réponse globale de santé publique [ ] . mais cette formule s'applique également à la santé mentale et devrait nous interroger sur la place donnée actuellement à la psychiatrie en milieu pénitentiaire en france. car si les prisons françaises restent, pour le moment, peu impactées par la maladie, les remaniements en lien avec le confinement donnent une visibilité accrue au fossé qui sépare encore et toujours la psychiatrie en milieu libre et la psychiatrie en milieu pénitentiaire [ ] , bien illustré par les difficultés rencontrées pour le relais des prises en charge à la libération. les multiples recommandations relatives à la santé mentale publiées actuellement témoignent d'une préoccupation forte pour les conséquences psychiques du confinement. il nous apparaît particulièrement important de rappeler la vulnérabilité de certaines populations dont font partie les personnes incarcérées qui doivent être largement prises en compte dans les politiques de santé publique. ainsi, le déconfinement annoncé le avril et qui reste la source de multiples interrogations, devra s'accompagner d'un suivi rigoureux des indicateurs de santé mentale en détention mais surtout d'une réflexion globale sur l'organisation des soins psychiatriques en france intégrant les soins aux personnes détenues. les auteurs déclarent ne pas avoir de liens d'intérêts. the state of the prisons in england and wales | work by howard an analysis of influenza outbreaks in institutions and enclosed societies influenza outbreak in a correctional facility influenza outbreak in a canadian correctional facility distribution of a(h n )pdm influenza 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prevalence in the united states psychiatric effects of solitary confinement reforming punishment: psychological limits to the pains of imprisonment psychopathological effects of solitary confinement effects of sensory deprivation in psychiatric seclusion and solitary confinement the role of boredom proneness in self-reported anger and aggression release from prison -a high risk of death for former inmates solitary confinement placement and post-release mortality risk among formerly incarcerated individuals: a population-based study feuille de route sur trois ans pour la santé des personnes placées sous main de justice substance abuse and dependence in prisoners: a systematic review psychiatric disorders and repeat incarcerations: the revolving prison door psychotic disorders and repeat offending: systematic review and meta-analysis psychiatrie en milieu pénitentiaire : une sémiologie à part ? key: cord- - l pec z authors: terriau, a.; albertini, j.; poirier, a.; le bastard, q. title: impact of virus testing on covid- case fatality rate: estimate using a fixed-effects model date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: l pec z background in response to the sars-cov pandemic, governments have adopted a variety of public health measures. there are variations in how much testing has been done across countries. south korea, germany, and iceland take the bet of massive testing of their population. whereas tests were not performed widely in southern european countries. as the former undergo a lower case-fatality rate due to the covid- than the latter, the impact of the testing strategy must be investigated. in this study, we aimed to evaluate the impact of testing on the case fatality rate. methods we use data on inpatients across french geographic areas and propose a novel methodology that exploits policy discontinuities at region borders to estimate the effect of covid- tests on the case-fatality rate. in france, testing policies are determined locally. we compare all contiguous department pairs located on the opposite sides of a region border. the heterogeneity in testing rate between department pairs together with the similarities in other dimensions allow us to mimic the existence of treatment and control groups and to identify the impact of testing on mortality. results the increase of one percentage point in the test rate is associated with a decrease of . percentage point in the death rate. in other words, for each additional tests, one person would have remained alive. conclusion massive population testing could have a significant effect on mortality in different ways. mass testing may help decision-makers to implement healthcare measures to limit the spread of the disease. the increase of one percentage point in the test rate is associated with a decrease of . percentage point in the death rate. in other words, for each additional tests, one person would have remained alive. massive population testing could have a significant effect on mortality in different ways. mass testing may help decision-makers to implement healthcare measures to limit the spread of the disease. since it was reported in late december from hubei province in china, the severe acute respiratory syndrome coronavirus (sars-cov ) has now spread worldwide with more than million confirmed cases by the end of april . the outbreak reached europe via italy at the end of february and quickly affected the entire continent, making europe the epicenter by mid-march. the world health organization (who) declared the sars-cov to be a pandemic in mid-march . while research is still underway to find a curative treatment, the increasing number of severe cases admitted to hospital has raised fears of overburdening the health care systems. to prevent such a situation, governments have implemented various public health measures such as mobility restrictions, social distancing, or mass screening strategies. on march th, the head of the who pronounced in favor of massive population tests, because "you cannot fight a fire blindfolded". yet, there is a growing debate about the impact of mass testing on mortality rates. we have observed strong differences in testing rates between countries; for example, south-korea, germany, or iceland, have undertaken important screening policies and now report low casefatality rates. on the contrary, countries like spain or france have restricted access to diagnostic tests for inpatients or health care workers and now report higher mortality rates. unfortunately, cross-country comparisons are difficult due to the strong heterogeneity among countries. even in the united states of america, endowments for medical centers and lockdown strategies are very different from one state to another. by contrast, france kept a relatively centralized health system but as the epidemic was expanding, the health regional agencies (ars) were given autonomy in terms of screening strategies implementation; however, at the same time, a strict lockdown approach was instituted for all regions. , among french regions, the main difference in their strategies was the intensity of testing policies. screening policies and mortality rate might be related to the fact that testing allows authorities to detect and isolate infected people and to prevent them from transmitting the virus; and also enables early treatment, thus increasing the chances of cure. we propose a novel approach to assess the impact of focused screening strategies on mortality rates, which exploits policy discontinuities at region borders and contiguous department pairs that are located on opposite sides of a region border. this methodology has been used in an economic setting to evaluate the effects of the minimum wage on earnings and employment in the us. we conducted a retrospective study, with a prospective database, including the total of patients who were admitted to hospital and afterwards discharged, the total of casualties and the total of tests performed for screening covid- infection (rt-pcr) by out-of-hospital medical laboratories. the sample covers the period from / / to / / , which corresponds to a lockdown period in france. all the information was provided daily by the french public health agency (santé publique france; https://www.data.gouv.fr/). the data was gathered from different geographic areas within france and no other countries were included. we merged this dataset with . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . information on hospital occupancy rates for intensive care units published by the french ministry of health (https://www.sae-diffusion.sante.gouv.fr/). sociodemographic data were extracted from the national institute of statistics and economic studies (https://statistiques-locales.insee.fr/). our analysis took place at the department level. in our study, we exploit the fact that from / / , the french government has activated the third stage of the national plan for the prevention and the control of the epidemic, which translates into non-systematic testing of symptomatic individuals. from this date, testing policies were determined at the region level by the regional public health agencies. we used a fixed-effects model to assess the impact of the number of tests performed over time at a local geographical level (department) on fatality-cases. in fixed-effects models, subjects serve as their own controls, providing a means for controlling omitted-variable bias. otherwise stated, fixed-effects models allow controlling for time-invariant heterogeneity, i.e. all possible characteristics that do not change over time. we used two distinct samples: i) the all-department sample (ad sample) that includes departments distributed across regions; ii) the contiguous border department-pair sample (cbdp sample) that contains all the contiguous department pairs that straddle a region boundary. metropolitan france counts departments. we excluded two departments, haute-corse and corse-du-sud, that are part of a region, corsica, that does not share any direct border with others. among the departments, lie along a region border. as each department may belong to several department-pairs, we have a total of distinct department-pairs. our strategy consisted in comparing all contiguous department pairs sharing a region border (see figure for an example) to identify the effect of testing on the case fatality rate. tests rate and death rates were calculated using the number of rt-pcr tests and the number of deaths related to covid- divided by the number of patients admitted to the hospital, respectively. we first estimate the effect of testing on case-fatality rate using the canonical fixed-effects model and the ad sample (specification ( )): where denote the department, the time, is the case fatality rate in department at time , represents the percentage of people hospitalized that are tested in department at time , is a department fixed effect, and an error term. we now turn to our preferred identification strategy, which exploits policy discontinuities at region borders. to achieve identification, we estimate the following model using the cbdp sample (specification ( )): = + + + + . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . where denote the department, the department-pair, the time, is the case fatality rate in department in department-pair at date , represents the percentage of people hospitalized that are tested in department in department-pair at date , represents a department fixed effect and a department-pair fixed effect. standard errors are clustered on the region and border segment separately to account for possible correlation in the residuals. although fixed-effects models control for all characteristics which do not change over time, we report some time-invariant variables in table s for information. the average number of tested individuals was . per department with a share of positive cases of approximatively percent. we count a total of hospitalizations. the observed share of the population above age was roughly percent. as shown by figure , the number of deaths increased quickly, from on march th to on april th . over the same period, the number of tests increased from to . the path of mortality and testing was not homogenous over the territory. the autonomy given to regional public heaths agencies generated unprecedented differences in testing rate across regions and strong discontinuities at region borders (see figure ) . the department fixed effect captures time-invariant heterogeneity across departments. this includes sociodemographic variables (such as the structure of age, race, or gender in the population), but also many variables related to health facilities (number of hospitals, medical density or medical devices). we add time-varying confounding factors in specifications (b) and (c). specification (b) includes the occupancy rate of the resuscitation units, while specification (c) also controls for the rate of positive tests. the first variable controls for the capacity of hospitals to treat patients at different stages of the covid- epidemic while the second controls for selection bias. table reports the estimates provided by specifications (a)-(c). our baseline estimates reveal that a percentage point (pp) increase in the tests/hospitalizations ratio leads to a statistically significant decrease in the case mortality rate by slightly less than . pp. finally, table displays the results for specification (d)-(f). our estimates reveal that a pp increase in the tests/hospitalizations ratio leads to a statistically significant drop of case mortality rate by . pp. putting these numbers into perspective involves that for each additional tests, one person would have remained alive. sars-cov- outbreak is one of the major public health emergencies of international concern for decades. countries have implemented various measures mostly based on mobility restriction, social distancing, and regional or national lockdown. all of these public health measures are aimed at "flattening the curve" of the infected cases to limit avoidable mortality due to overburdened health care systems. we evaluate the effect of mass screening covid- on mortality rate in france during the first month of the lockdown. we take advantage of the difference in screening . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . intensity among french regions. we first estimate the effect of testing on case-fatality rate using the canonical fixed-effects model and the ad sample and find that the increase of screening rate of pp allows mortality rate to decrease of nearly . pp. we confirmed our results by estimating the fixed-effects model using the cbdp sample which compares contiguous french departments sharing region borders. to the best of our knowledge, no large randomized controlled trial (rct) has been implemented to investigate the effect of tests on the case-fatality rate, probably due to time, budget, or ethical constraints. when rct are difficult to implement or unethical, natural experiments (ne) are one of the best alternatives. the principle of ne is to mimic the existence of treatment and control groups using an instrumental variable that induces a change in the explanatory variable but has any direct effect on the outcome. however, in the case of the covid- epidemics, finding a suitable instrument remains a hard task. in the absence of rct or ne, many researchers try to approximate using standard methods such as linear regression, logistic regression, or propensity scores. however, such methods are subject to the well-known omitted-variable bias, leading to severe bias in estimating the effects of the variables that are included. consequently, causal inference via statistical adjustment represents a poor alternative to randomized experiments. in such a context, panel data models represent the best way to control for heterogeneity and to improve causal estimation. we use a fixed-effects model because it represents a powerful tool for longitudinal data analysis. however, such a model requires substantial differences between treatment intensities across entities and time to get precise estimates. our data meets these conditions: i) no region has the same test rate path than other regions over the period considered; ii) the test rate varies greatly across regions and time. methods based on regional controls and policy discontinuities have several advantages: i) contiguous border departments are relatively similar, in particular with regard to health trends, which are of major importance in the context of an epidemic; ii) the testing policy is determined at the region level and is largely exogenous from the point of view of a department, which rules out potential reverse causality. until a vaccine is developed, the only way to prevent an unrestrained scenario is to control the spread of sars-cov- . this is a challenging task because some asymptomatic infected patients could potentially spread the virus. literature reports an alarming proportion of asymptomatic infected cases. epidemiological data from the diamond princess cruise sheep revealed only % of positive cases reported no symptoms. two hospitals in new york implemented universal testing for sars-cov- with nasopharyngeal swabs in women who were admitted for delivery, and revealed that nearly % of patients who were positive for sars-cov- at admission reported no symptoms. overall population screening in iceland revealed that only % of participants with positive tests reported symptoms of covid- . this proportion could be even higher, because of false negative results of tests to detect sars-cov- . testing is part of a strategy to limit the transmission of the virus and who recommends a rapid diagnosis and isolation of cases in combination with a rigorous tracking and precautionary self-isolation of close contacts. several authors support the implementation of mass screening policies. , in our opinion, mass screening may positively impact the fatality case rate in different ways. first, unfocused testing, i.e. not limited to symptomatic subjects, could improve the monitoring of the progress of the epidemic and facilitate decision-making by the health authorities. the use of "case definition", given the limited knowledge of the new disease, probably resulted in a low sensitivity to detect infected subjects, resulting in a delayed perception of the progression of the epidemic. , screening strategies are subject to the availability of tests which indirectly shapes epidemic curves. while the usa increased their screening capacities between late-february to early-march, the country experienced a rapid increase of total infected cases. second, mass screening may also allow early identification of infected subjects and rapid implementation of isolation measures. early reports from wuhan suggest that public health interventions combining universal symptoms survey, traffic restriction and home quarantine resulted were temporarily associated with an increased control of the outbreak. , a modeling from singapore suggests that quarantining of infected individuals and their family members, school closure and workplace distancing could reduce the progression of the epidemic but is associated to a significant economic cost. review from the cochrane database concludes that quarantine is important in reducing the number of covid- cases but is dependent on screening strategies. also, a us survey on the impact of school closure on mortality reports that the transmission prevention by school closure needs to be weighted with the potential loss of health-care workers. this supports that public health decisions should be as focused as possible in order to limit the negative impact on the economy and the society. importance of rapid diagnosis and case identification and isolation will become of utmost importance with the end of lockdowns. our study far supports a significant impact of screening strategies on the case-fatality rate in france. notwithstanding, there are some limitations to our results. first, they belong to france and it would be very hazardous to pretend that they apply to other countries because their exposition to covid- is different, they adopted different strategies, and have different health structures. second, to provide further evidence on this relation, it would be worth applying this methodology to other countries for which such data are available and in which testing policies are sufficiently heterogeneous across geographical areas. in addition, the data on tests collected by the french public health agency are those made by private laboratories and do not include those made in public hospitals. this represents an important share of tests (between half and two thirds) and we cannot rule out the possibility that this unobservable amount of screening activity may affect our results. lastly, our study cannot quantify the respective contribution of the treatment delivered to screened and infected individuals or the lower dissemination of the virus that results from quarantining policies. covid- intensive screening policies were significantly associated with a decrease in the fatality-case rate in france. these results support the implementation of mass screening strategies and could provide important information for decision-makers in the fight against sars-cov pandemic. the optimal testing strategy might also concern economic issues. indeed, the bank of france estimated that each fortnight of lockdown costs to france . % of annual gdp (nearly usd billions). , from a costs/benefits perspective one might naturally wonder what is the optimal policy capable of containing the outbreak and lowering the fatality rate. this is in our research agenda. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . we thank dr natalia lucia gomez, from the hospital italiano de buenos aires, for it's attentive revision of the manuscript and pr emmanuel montassier, from the university of nantes for his expert advices. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . standard errors between parentheses. *** p< . , ** p< . , * p< . . source: santé publique france and authors calculations. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . number of regions standard errors between parentheses. *** p< . , ** p< . , * p< . . source: santé publique france and authors calculations. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . example with "nouvelle aquitaine" and "occitanie" regions. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . test rate: number of rt-pcr tests divided by the number of patients admitted to the hospital for covid- . death rate: number of deaths in hospital due to covid- divided by the number of patients admitted to the hospital. we use shapefiles for regions and departement to construct the maps and compute the contiguity matrix (https://www.data.gouv.fr/fr/datasets/contours-desregions-francaises-sur-openstreetmap/; https://www.data.gouv.fr/fr/datasets/contours-desdepartements-francais-issus-d-openstreetmap/ ). reading: paca belongs to the top % of regions that test more and to the bottom % of regions that have the lowest fatality ratio. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . note: sample means are reported for all departments in france and for all contiguous border department-pairs with a full balanced panel of observations. source: santé publique france . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. 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health-care workforce and net mortality: a modelling study tackling covid- : are the costs worth the benefits? update on business conditions in france at the end of gdp and spending -gross domestic product (gdp) -oecd data key: cord- - xnjj h authors: cécile, couchoud; florian, bayer; carole, ayav; clémence, béchade; philippe, brunet; françois, chantrel; luc, frimat; roula, galland; maryvonne, hourmant; emmanuelle, laurain; thierry, lobbedez; lucile, mercadal; olivier, moranne title: low incidence of sars-cov- , risk factors of mortality and the course of illness in the french national cohort of dialysis patients. date: - - journal: kidney int doi: . /j.kint. . . sha: doc_id: cord_uid: xnjj h the aim of this study was to estimate the incidence of covid- disease in the french national population of dialysis patients, their course of illness and to identify the risk factors associated with mortality. our study included all patients on dialysis recorded in the french rein registry in april . clinical characteristics at last follow-up and the evolution of covid- illness severity over time were recorded for diagnosed cases (either suspicious clinical symptoms, characteristic signs on the chest scan or a positive reverse transcription polymerase chain reaction) for sars-cov- . a total of , infected patients were reported on the rein registry from march th, to may th, . of these, died. the prevalence of covid- patients varied from less than % to % between regions. the probability of being a case was higher in males, patients with diabetes, those in need of assistance for transfer or treated at a self-care unit. dialysis at home was associated with a lower probability of being infected as was being a smoker, a former smoker, having an active malignancy, or peripheral vascular disease. mortality in diagnosed cases ( %) was associated with the same causes as in the general population. higher age, hypoalbuminemia and the presence of an ischemic heart disease were statistically independently associated with a higher risk of death. being treated at a selfcare unit was associated with a lower risk. thus, our study showed a relatively low frequency of covid- among dialysis patients contrary to what might have been assumed. -rein registry, agence de la biomédecine, saint-denis la plaine, france -chru-nancy, inserm, cic, epidémiologie clinique, nancy, france -nephrology department, caen university hospital, france -nephrology department, aphm university hospital, marseille, france -nephrology department, ghr mulhouse sud-alsace, france -university of lorraine, chru-nancy, vandoeuvre, france -calydial, vienne, france -nephrology department, nantes university hospital, france -nephrology department, ap-hp pitié-salpêtrière hospital, paris, france -nephrology-dialysis-apheresis department, nîmes university hospital, france introduction due to their frequent contact with hospitals and their comorbid condition, dialysis patients are identified as high-risk patients for severe forms of infection from sars-cov- . guidelines to mitigating risks have been published ( ) ( ) ( ) ( ) ( ) ( ) ( ) . however, few studies including case reports or the experience of centres have included sufficient numbers of patients to have a complete overview of their real risk and course of illness ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in those studies, case fatality varied from % to %. on march th , , the french national end-stage kidney disease rein registry began to record all patients on dialysis in france who were diagnosed with covid- . the aim of this first report from the french rein registry is to describe the population of infected dialysis patients and their course of illness, estimate the incidence and lethality of covid- disease and identify the risk factors associated with the probability of death. from march th , to may th , , patients were declared as being infected with sars-cov- on the rein registry. this represents . % of all dialysis patients treated in dialysis units in metropolitan france and overseas territories. the clinical and care situation at the first report in the registry was "hospitalized -moderate disease" for %, "mild disease treated at home" for %, "severe disease in an intensive care unit" for %, "death" for % and asymptomatic for % of cases. the first diagnosis was made in % of cases with a pcr on a nasopharyngeal swab, % on characteristic signs on the ct scan and % on suspicious clinical symptoms. finally, a positive pcr was available for patients ( %). in all, % were treated at home. outpatients were younger (median age . , iqr . - . , vs . , iqr . - . ), more often non-smokers and had less dysrhythmia and incapacity for transfer (suppl table ). their mortality was lower ( . %) compared to patient who were hospitalised ( . %). in all, % of patients were admitted to an icu unit. those patients were younger than the others (median age . , iqr . - . , vs . , iqr . - . ), less often had cerebrovascular disease, had a higher bmi and were less often treated by hospital-based hd (suppl table ). among the patients for whom information was available, % received invasive mechanical ventilation (suppl figure ). the mortality of icu patients was higher ( %) compared to patients who were not admitted to icus ( . %). the clinical situation at the last report in the registry for patients who were still alive, was "hospitalized -moderate disease" for %, "mild disease treated at home" for %, "in intensive j o u r n a l p r e -p r o o f care" for % and "recovered" for % and asymptomatic %, with a median follow-up of days (iqr - ). not all parts of france were affected in the same way. the prevalence of covid- patients varied from less than % in the overseas territories and metropolitan regions to over % in northeastern regions (especially in alsace, %, one of the first french clusters) and in the Île-de-france, %, the most densely-populated region ( figure ). these variations were not explained by age and were parallel to those of the general population (figure ). at that time, the percentage of infected persons in the french population was . % and the mortality among confirmed cases was % (no systematic screening). the cumulative incidence of new cases after an exponential increase has now stabilized itself ( figure the clinical characteristics of infected dialysis and control populations are represented in table . compared to the selected controls (treated in centres where at least one patient was infected), the probability of being a case was higher in males (or . among the infected patients, died due to a cause related to sars-cov- after a median time of days (iqr - ). the lethality in diagnosed cases was %. in the univariate analysis, higher age, being a former smoker, having a chronic respiratory disease, cardiovascular comorbidities ( e.g. peripheral vascular disease, ischemic heart disease, congestive heart failure or dysrhythmia) and frailty (hypoalbuminemia or inability to walk) were associated with a higher risk of death in sars-cov- infected dialysis patients. dialysis in self-care units or out-centres or being a current smoker were associated with a lower risk of death. in fact, most of these clinical characteristics and care j o u r n a l p r e -p r o o f were associated with older age. in the multivariate model, only older age, hypoalbuminemia and the presence of an ischemic heart disease were statistically independently associated with a higher risk of death (table ). being treated in a self-care unit was associated with a lower risk of death. neither chronic respiratory disease, obesity, diabetes nor smoking status were associated with a higher risk of death. the sensitivity analysis including the region of treatment gave similar results. the trajectory of care is represented in figure for the deceased patients for whom at least different clinical situations were reported in the registry. for severe cases hospitalized in intensive care units, the median time until death was days (iqr - ), whereas the median time for hospitalized patients until death was days (iqr - ) and, for patients at home, days (iqr - ). the trajectory of care is represented in figure for the patients who recovered (clinical situation coded as recovery or asymptomatic). the median time in hospital until recovery was days (iqr - ), similar to that for patients who were at home ( days, iqr - ). so far, more than dialysis patients have been diagnosed with sars-cov- infection in france. our study shows that the prevalence of sars-cov- infection in dialysis patients varied throughout the country from to %. mortality in this population of diagnosed cases is high at % and is mainly associated with a higher age ( % mortality in patients aged under compared with % of patients aged over ). the trend of the sars-cov- epidemic in patients on dialysis shows a parallel development as in the general french population, with north eastern regions and the ile-de-france being more affected. our global prevalence is % of dialysis patients but this reaches % in the most affected regions. in the absence of other population-based data, it can only be compared with the % of the haemodialysis centre in wuhan at the epicentre of the chinese epidemic ( ) . however, the nonsystematic detection of asymptomatic patients in france may lead to an underestimation of the true dissemination of sars-cov- in the french dialysis population. although the lockdown seemed to have significantly reduced the amount of contact among the general population, dialysis patients have to leave confinement to go to their dialysis units and, consequently, are still in contact with a large number of people. the risk of contamination may occur during transport, at the dialysis unit or during hospitalisation, but also at home with the family or caregivers. home dialysis was associated with a lower probability of being infected suggesting a protective effect of staying at home. dialysis centres affected later learned from units contaminated early on in the epidemic's progression and reorganized their patients' circulation and care ( , ). indeed, since the beginning of the epidemic, protective strategies have been broadcast by the sfndt (société francophone de nephrology dialyse transplantation) with weekly covid- webinars inviting all french nephrologists to discuss the overall covid- themes and topics available on the sfndt website (https//www.sfndt.org˃actualites) . thanks to this collaboration, the worst may have been avoided. however, we must now remain vigilant and protect our healthcare workers. the initial incidence of the disease in some dialysis units seemed very high, especially in the initial regions. the incidence in dialysis units is now decreasing, mirroring the decrease in the general population. this can also be associated with the implementation of all the necessary preventive actions prone by the sfndt, including /wearing a mask during transport and for the entire period of care, /systematic tracking of patients and screening at the entrance to dialysis units based on fever and symptoms or contact with an infected person and /restricting areas for covid- cases ( ),. as in the general population, male gender, diabetes and frailty, but not age, were associated with a higher risk of being infected. a selection bias, due to the fact that these patients may have a more severe form of the disease and are therefore more easily diagnosed, cannot be ruled out. as in the cohort of haemodialysis patients at the haemodialysis centre in wuhan, diabetes was associated with a higher risk of infection. this result was still significant when introducing regions in the model to take into account the fact that the epidemic was mainly located in the north east of france where the prevalence of diabetes is higher. smoking, even after taking comorbidities into account, was associated with a lower risk of infection, as discussed in the general population ( ) . surprisingly, being treated in a self-care unit was associated with a higher risk of being infected. at self-care units, care is provided without supervision by an on-site nephrologist ( ) . the presence of a nurse is mandatory and patients are helped with the hd process. all these units collaborate with a hospitalbased dialysis unit. moreover these units treat younger patients who may have had more contact at risk than elderly. despite the lockdown. these small units, with fewer caregivers on site, could have tarried in implementing protection strategies as proposed by others ( ) . further analyses are required to evaluate the impact of other risk factors, such as living in an institution or in a deprived neighbourhood area associated with overcrowded housing. international comparison of case fatalities should be made with caution given the case-mix, the various healthcare arrangements and the different dynamics of the epidemic. our mortality among diagnosed cases, % so far, is higher than the % reported for the dialysis center in wuhan ( ). the older age and more frequent comorbidities of french dialysis patients may explain a higher mortality than in china ( , ) . furthermore, the non-systematic detection of asymptomatic patients favors more seriously ill patients. in france, case fatality was lower than the % reported in j o u r n a l p r e -p r o o f outpatient dialysis facilities in italy ( ), the % in a single center in madrid ( ) , or % in a single center in new york ( ) . higher mortality in these studies may be explained by a selection bias for more severely ill hospitalized patients. compared to the general population, the dialysis lethality observed in our cohort was similar to the % case fatality rate observed with patients aged over in italy ( ) . it is also similar to the mortality rate for confirmed cases in the french general population, where at least % of the people who died had a comorbid condition and % were aged or older. apart from age, which seems to be the major factor in the general population ( ), nutritional status, indirectly assessed by albumin levels and the presence of ischemic heart disease, seem to be the main risk factors. further in-depth analyses are planned in order to better estimate the excess mortality in dialysis patients at this period, taking into account the underlying mortality risk. being treated in a self-care units was associated with lower mortality, even after taking into account age and comorbidities. after adjustment, home dialysis mortality did not differ from the mortality rate for hospital-based haemodialysis. however, the small number of patients with home dialysis has not allowed us to make an in-depth analysis so far. other factors (such as living conditions, delay in alerting and other home-based care) which are not available in our registry, need to be explored. although incomplete, the illness trajectory seems to show rapid worsening and a slow healing process. the short lapse of time before death could corroborate the physiopathology with the delay in host inflammatory response phase reported to days after the initial infection ( ). this rapid negative development raises the question of reinforced surveillance at home, during dialysis sessions and, why not, preventive hospitalization in a safe environment. our definition of recovery should be taken with precaution since the definition of recovery is still under debate. some patients were maintained in hospital under isolation for days. very soon after the start of the epidemic, the french-speaking society of nephrology, helped by infectious disease specialists made recommendation that for each dialysis patient with fever, a viral syndrome, pulmonary symptoms or diarrhoea, a ct scan should be prescribed as well as a pcr on a nasopharyngeal swab. contact subjects were also tested in the later period explaining the occurrence of few asymptomatic patients. these recommendations applied to all hospitalized patients and outpatients as well throughout the whole country. however, due to possible variations in diagnosis strategies, day for each patient may vary from one unit to another. access to intensive care units was a concern for nephrologists in certain areas. some tensions could be noted in highly affected regions but, in general, dialysis patients could be transferred to intensive care as required depending on their age and comorbidities. the strength of this study is its national scale, including the whole population of french dialysis patients. however, these results must be interpreted bearing the following limitations in mind. various screening strategies may influence the detection of the disease. this could be the case especially for patients treated at home or asymptomatic patients or sudden death, but mild cases and hospitalized patients can be considered as being exhaustive. non-systematic screening favours the collection of more severe cases and leads to an overestimation of lethality. the second limitation is the lack of granular data on clinical presentation, laboratory results and treatment and the precise protective strategy implemented in the units. our study is based on a registry, which gives an exhaustive national overview but with a limited dataset -not on medical records, which could give more detailed data on treatment and clinical presentation but on a limited number of patients with a risk of selection bias. third, due to the confinement of registry research assistants, the data quality control procedure was limited. post hoc controls will be taking place to complete the data. fourth, the total number of patients tested and not considered as covid positive is unknown. as in the general population, the true lethality of covid- in infected dialysis patients needs to be confirmed by a longer follow-up and deployment of screening methods. despite the difficulty to have a "true" estimation, this preliminary report of the french registry shows a relatively low frequency of covid- among dialysis patients contrary to what might have been feared but, as in the general population, the epidemic did not evenly affect the whole territory. mortality in diagnosed cases ( %) is, associated with the same causes as in the general population, namely, high age, frailty and comorbid conditions. the french rein registry is intended to include all end-stage renal disease (esrd) patients on renal replacement therapy (rrt) living in france, including overseas territories. patients with a diagnosis of acute renal failure were excluded, i.e. those who recovered all or some renal function within days or were considered by experts to have acute failure when they died before days. the details of organizational principles and quality control are described elsewhere ( ) . the rein network includes nephrologists, nurses, patients, public health representatives and epidemiologists coordinated within regional and national steering committees. the national coordination center is based at the agence de la biomédecine, a public health agency that oversees the activity of organ and tissue procurement and transplantation. the clinical characteristics at last follow-up included age, gender, comorbidities, mobility status (walks without help, needs assistance for transfers, or is totally dependent for transfers), body mass index (bmi), tobacco use, haemoglobin and serum albumin, dialysis technique (haemodialysis or peritoneal dialysis) and location (hospital-based, out-centre, self-care unit, home). this study analysed comorbidities: diabetes, congestive heart failure, ischemic heart disease, peripheral vascular disease, aortic aneurysm, cerebrovascular disease, dysrhythmia, active malignancy, cirrhosis, and severe behavioural disorders (defined as including dementia, psychosis, or severe neurosis that may have affected the functional status or adherence to treatment). the last residence and last dialysis unit before february , were taken into account to avoid misclassification of patients transferred to another dialysis centre due to their infection status. the clinical characteristics of patients were expressed as frequencies and percentages for qualitative variables and medians with interquartile ranges for quantitative variables. the percentage of infected patients in the dialysis units of each region was adjusted on age (indirect standardization) to take into account the underlying age distribution of the dialysed patients. the crude ratio and the standardised ratio are presented on a map, according to the patients' area of residence. to give an overview of the epidemic in france, hospital mortality due to covid- on april , extracted from the platform of the national public health agency, santé publique france: https://geodes.santepubliquefrance.fr/#c=indicator was reported. we also presented the cumulative number of infected patients on a day-to-day graph for the whole country. to describe the characteristics of infected patients, we compared this population with two control groups. the first one included all the dialysis patients in france who were not infected. the second, to take into account the heterogeneity of the distribution of the epidemic in the country, included only patients treated in the dialysis units where at least one infected patient had been declared. risk factors associated with being a case in those units were analysed by logistics regression with a stepwise selection of variables. the final model is based on complete data (no imputation). a p-value of < . (two-sided) was considered statistically significant. results are reported as odds-ratios (ors) with their % confidence interval. lethality was estimated from the proportion of deceased patients among the diagnosed cases. to identify the risk factors associated with death in sars-cov- dialysis patients, a logistics regression with stepwise selection of variables was used. interactions between age and other factors were explored. a p-value of < . (two-sided) was considered statistically significant. results are reported as odds-ratios (ors) with their % confidence interval. sensitivity analyses were made including the region of treatment, either as a fixed effect or with a random intercept. finally, when available, the course of illness was represented on a graph to describe the process of care for patients who died and for those who recovered. for each transition between the various care statuses, the number of patients and the median duration before transfer were calculated. j o u r n a l p r e -p r o o f recommendations for the prevention, mitigation and containment of the emerging mitigating risk of covid- in dialysis facilities covid- and the inpatient dialysis unit: managing resources during contingency planning pre-crisis consensus recommendations for the care of children receiving chronic dialysis in association with the covid- epidemic covid- from the nephrologist's point of view pandemic uncertainty: considerations for nephrology nurses how we mitigate and contain covid- outbreak in hemodialysis center (hd): lessons and experiences a case of novel coronavirus disease in a chronic hemodialysis patient presenting with gastroenteritis and developing severe pulmonary disease coronavirus disease (covid- ) pneumonia in a hemodialysis patient first reported nosocomial outbreak of severe acute respiratory syndrome coronavirus (sars-cov- ) in a pediatric dialysis unit peritoneal dialysis during the coronavirus (covid- ) pandemic: acute inpatient and maintenance outpatient experiences an analysis on the clinical features of mhd patients with coronavirus disease : a single center study deisolation of covid- -positive hemodialysis patients in the outpatient setting: a singlecenter experience management of patients on dialysis and with kidney transplant during sars-cov- (covid- ) pandemic in brescia covid- : clinical course and outcomes of hemodialysis patients in spain presentation and outcomes of patients with eskd and covid- lessons from the experience in wuhan to reduce risk of covid- infection in patients undergoing long-term hemodialysis hemodialysis with cohort isolation to prevent secondary transmission during a covid- outbreak in korea covid- and smoking: a systematic review of the evidence spatial analysis of case-mix and dialysis modality associations case-fatality rate and characteristics of patients dying in relation to covid- in italy opensafely: factors associated with covid- -related hospital death in the linked electronic health records of million adult nhs patients. | medrxiv siddiqi hk, mehra mr. covid- illness in native and immunosuppressed states: a clinical-therapeutic staging proposal the renal epidemiology and information network (rein): a new registry for end-stage renal disease in france covid- therapeutic trial synopsis we gratefully acknowledge all participants of the rein registry, nephrologists and research assistants alike, especially at this very particular time. the centres participating in the registry are listed in the rein annual report: http://www.agence-biomedecine.fr/le-programme-rein.we also thank teresa sawyers, medical writer at nîmes university hospital for her help in editing the text. the authors of this manuscript declare that they have no competing financial interest and no conflict of interests to disclose. the registry is supported by the agence de la biomedicine, france. key: cord- -abxv mkz authors: izopet, jacques; dubois, martine; bertagnoli, stéphane; lhomme, sébastien; marchandeau, stéphane; boucher, samuel; kamar, nassim; abravanel, florence; guérin, jean-luc title: hepatitis e virus strains in rabbits and evidence of a closely related strain in humans, france date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: abxv mkz hepatitis e virus (hev) strains from rabbits indicate that these mammals may be a reservoir for hevs that cause infection in humans. to determine hev prevalence in rabbits and the strains’ genetic characteristics, we tested bile, liver, and additional samples from farmed and wild rabbits in france. we detected hev rna in % ( / ) of bile samples from farmed rabbits (in ) and in % ( / ) of liver samples from wild rabbits (in – ). full-length genomic sequences indicated that all rabbit strains belonged to the same clade (nucleotide sequences . %– . % identical to hev genotypes – ). comparison with hev sequences of human strains and reference sequences identified a human strain closely related to rabbit strain hev. we found a -nt insertion in the x domain of open reading frame of the human strain and all rabbit hev strains. these findings indicate that the host range of hev in europe is expanding and that zoonotic transmission of hev from rabbits is possible. h epatitis e virus (hev) is a major cause of acute hepatitis in many developing countries in asia and africa, where it is transmitted by the fecal-oral route because of poor sanitation practices ( ) . acute hepatitis e is also increasingly reported in industrialized countries, where the transmission is mainly zoonotic ( ) . the initial discovery of hev transmission from domestic pigs ( ) has been followed by evidence that other mammals, such as wild boars and deer, are also potential reservoirs of hev ( ) . although the course of hev infection is generally self-limiting and asymptomatic (or symptomatic with acute hepatitis), fulminant hepatitis can occur in pregnant women and in persons with underlying liver disease ( ) ( ) ( ) . hev infections can also become chronic in immunocompromised patients, such as recipients of solid-organ transplants ( ) ( ) ( ) , those with hematologic diseases ( , ) , and patients infected with hiv ( ) ( ) ( ) . hev, genus hepevirus, family hepeviridae, is a positive-sense, single-stranded, nonenveloped rna virus ( ) . the hev genome is ≈ . kb long and contains open reading frames (orfs) as well as ′ and ′ untranslated regions: orf encodes nonstructural proteins, orf encodes the capsid protein, and orf encodes a small phosphoprotein. phylogenetic analysis of hev sequences has led to the recognition of major genotypes that infect mammals from a variety of species. hev and hev are restricted to humans and transmitted through contaminated water in developing countries. hev and hev infect humans, pigs, and other mammals and are responsible for sporadic cases of hepatitis e in developing and industrialized countries ( ) . hev is distributed worldwide, whereas hev largely is found in asia. although hev and hev infections have been linked to the consumption of raw or undercooked meats, such as pig liver sausages or game meats ( , ) , the full spectrum of animals that are reservoirs of hev is still unknown. recent studies have characterized new hev genotypes in isolates from rats in germany ( ) , wild boars in japan ( ) , and farmed rabbits in the people's republic of china ( , ) . because the potential risk for zoonotic transmission strain in humans, france of hev from rabbits in france is unknown, and cases of autochthonous hepatitis e are commonly reported in this country ( , ) , we investigated the prevalence of hev in farmed and wild rabbits. we also looked for a genetic link between hev strains circulating in rabbits and hev strains circulating in humans in france. bile specimens (n = ) were collected in september from rabbits raised on farms in western france, in the departments of maine et loire (n = ), vendée (n = ), deux-sèvres (n = ), calvados (n = ), and loire atlantique (n = ), the main geographic areas of rabbit farming in france. we sampled rabbits from each farm when they were slaughtered at - days of age (table ) . all rabbits were healthy and intended for human consumption. all samples were immediately stored at − °c. liver specimens (n = ) were collected during september -november from populations of wild rabbits, established in warrens; each population was considered epidemiologically independent. the populations were located in several departments of mainland france: dordogne (n = ), finistère (n = ), deux-sèvres (n = ), loire-atlantique (n = ), haute-garonne (n = ), charentes (n = ), morbihan (n = ), and pyrénées-orientales (n = ) ( table ). the number of rabbits sampled in a given warren ranged from to . they were > months of age, apparently healthy, and intended for human consumption. each rabbit was eviscerated within a few hours of its death, and a sample of its liver was taken and immediately frozen at − °c. necropsies were performed on a group of rabbits from the same warren in haute-garonne (w ), and samples of their intestine and cecum were taken, in addition to samples from the liver. serum specimens were collected from immunocompetent and immunocompromised patients who had received a diagnosis of hepatitis e from the department of virology at toulouse university hospital. all samples were stored at − °c ( , ) . samples ( μl of rabbit bile and mg of liver, intestine, and cecum) were disrupted with trizol (invitrogen, saint aubin, france). rna was extracted with qiaamp viral rna mini kits (qiagen, courtaboeuf, france). we used -step real-time reverse transcription pcr on the light cycler instrument (roche diagnostics, meylan, france) to amplify a -bp fragment. the primers and probes targeted the orf region: forward primer hevorf -s: ′-ggtggtttctggggtgac- ′, reverse primer hevorf -as: ′aggggttggttggatgaa - ′, and probe ′-fam-tgattctcagcccttcgc-tamra- ′ ( ) . each -μl reaction mix contained μl of superscript iii platinum one-step quantitative rt-pcr system (invitrogen), μl of rna, primers ( nmol/l) and probes ( nmol/l), and u of rnase out (invitrogen). reverse transcription was carried out at °c for min, followed by denaturation at °c for min. dna was amplifi ed with pcr cycles at °c ( s) and °c ( s). hev rna was quantifi ed by using a transcribed rna standard constructed from a genotype f hev strain (genbank accession no. eu ). the limit of detection was copies/ml. two fragments, one within orf ( bp) and the other within orf , encompassing the hypervariable region and x domain (≈ , bp), were amplifi ed and sequenced in both directions by the dideoxy chain termination method (prism ready reaction ampli taq fs and dye deoxy primers; applied biosystems, paris, france) on an abi xl capillary dna analyzer (applied biosystems, foster city, ca, usa). the primers used for the orf fragment were the following: forward primer hevorf -s: ′-gacagaattratttcgtcggctgg- ′ and reverse primer hevorf -as: ′-tgytggttrtcataatcc tg- ′. the primers used for the orf fragment were the following: forward primer hevorf -s: ′-tgacggcyacygtkgarcttg- ′ and reverse primer hevorf -as: ′-acatcracatccccctgy tgtatrga- ′. the whole genomes of rabbit strains (w - and w - ) and human strain (tls- -human) were amplifi ed by overlapping rt-pcr. the primers are listed in table . the genotype was determined by using reference strains as previously described ( ) . phylogenetic analyses were performed with genotype information on reference sequences based on the hev classifi cation proposed by lu et al. ( ) . sequences were aligned by using clustalw (mega , www.megasoftware.net; bioedit version . , www.mbio.ncsu.edu/bioedit/bioedit). phylogenetic trees were created by the neighbor-joining (kimura -parameter) method with a bootstrap of , replicates. the partial sequences of orf and the full-length sequences reported in this study have been deposited in genbank. the accession numbers are jq and jq for orf , and jq to jq for the full-length sequences of w - , w - , tls- -human, tr (genotype c), and tr (genotype e), respectively. all bile specimens from the farmed rabbits and the liver specimens from the wild rabbits were tested for hev rna (table ) . samples from farms ( %) and warrens ( %) tested positive for hev rna. hev rna was found in a bile samples ( %) from farmed rabbits. the median hev rna concentration in the bile samples was . × copies/ml (range copies/ml- copies/ml). a total of liver samples ( %) from wild rabbits were positive for hev rna; median hev rna concentration was . × copies/g (range , copies/g- . × copies/g). we tested the liver, intestine, and cecum samples from wild rabbits from the same warren (w ) in triplicate to obtain a clear picture of the tissue distribution of hev in infected rabbits. hev rna was detected in all the tissues from rabbits (nos. , , , ) , in the liver and intestine of rabbit (no. ), and in the liver only of rabbit (no. ) ( table ) . the virus loads in the liver (mean . log copies/g), intestine (mean . log copies/g), and cecum (mean . log copies/g) were not signifi cantly different. phylogenetic analyses, conducted on the basis of a -nt fragment within orf of the hev strains from rabbits, hev strains from humans circulating in france, and hev reference sequences (hev , hev , hev , hev , rabbit hev, rat hev, wild-boar hev) indicated that the new orf sequences from rabbit hevs were clustered. one cluster contained orf sequences from previously characterized hev from farmed rabbits from china, orf sequences from hevs from farmed rabbits in france, and orf sequences from hevs from wild rabbits in france (figure ). this cluster also contained an orf sequence from a strain from a person in france (tls- -human) (figure ). this strain was found in a serum sample from a -year-old man with an elevated alanine aminotransferase level ( iu/l, reference < iu/l). phylogenetic analysis based on a , -nt fragment within orf , indicated that the orf sequences from hev strains from rabbits in france (n = ) or china (n = ) and the orf sequence from the human strain tls- human formed a distinct genetic group among sequences of hev genotypes - (data not shown). the cluster of rabbit hev sequences was also distinct from the hev sequences from wild-boar and rat hev genotypes that were characterized recently. comparison of the orf sequences from rabbit hev strains with reference orf sequences from hev genotypes - showed an insertion of nt in the x domain of the orf of all the rabbit hev strains. this insertion was also found in the tls- -human strain. the deduced amino acid sequences corresponding to this insertion, located between amino acids and (burmese strain, m ), were not very similar, except for conserved amino acids at the c-terminal end. we obtained the full-length genomic sequences of hev strains from wild rabbits in france and the tls- -human strain. the phylogenetic tree, constructed by the neighbor-joining method using the full-length genomic sequences (including the sequences of genotypes f, e, and c, which were circulating in france), revealed that the hev genomes from the rabbit strains and the tls- -human strain belonged to the same clade. this clade was clearly separated from genotypes - , found in other mammals and from the new hev genotypes found in wild boars and rats (figure ) . the length of the rabbit strain w - genome, excluding the poly(a) tract at the ′ terminus, was , nt. the length of the rabbit strain w - was , nt, and that of the tls- -human strain was , nt. the nucleotide sequences of the rabbit strains and the tls- -human strain were . %- % identical ( table ). the nucleotide sequences of the rabbit or tls- human strains were . % to . % identical to those of genotype , . % to . % identical to those of genotype , . % to . % identical to those of genotype , and . % to . % identical to those of genotype . these comparisons therefore indicate that the sequences of the rabbit hev strains and the tls- -human strain are distinct from all known strains of hev genotypes - and from the newly described hev genotypes from wild boars and rats. we found that farmed and wild rabbits in france are naturally infected with hev. we also characterized a human hev strain that is closely related to rabbit hev strains; this fi nding thus supports the potential of zoonotic transmission from rabbits to humans. the hevs found in farmed rabbits in several geographic areas of china have been identifi ed ( , ) . hev was also recently found in farmed rabbits in virginia, usa ( ) . our study results show that rabbits in europe are infected with hev and that some farmed rabbits and wild rabbits in france are infected. we found hev rna in % of the farmed rabbits and in % of the wild rabbits. however, the ages of the rabbits and the tissues tested (bile samples from farmed rabbits and liver samples from wild rabbits) may explain the observed difference in hev prevalence. nevertheless, previous studies have shown that the virus loads in liver and bile samples from swine infected with hev are similar ( , ) . although the greater prevalence of hev in wild rabbits could be linked to their older age, we could not test for a relationship between the prevalence of hev and rabbit age because we did not know the rabbits' precise ages. our analysis of the distribution of hev in the tissues of infected wild rabbits showed hev rna not only in the liver, but also in the intestine and cecum; our analysis also showed that the virus loads from these organs were not signifi cantly different. this fi nding suggests that emerging infectious diseases • www.cdc.gov/eid • vol. extrahepatic sites of hev replication exist in rabbits, as has been demonstrated for hev in pigs ( ) . however, because the intestine and cecum samples may have been contaminated with blood, our results need to be confi rmed in future studies using methods that ensure that tissues other than the liver are not contaminated with blood. to determine whether rabbits could be a reservoir for viruses that cause human infection, we analyzed partial and complete nucleotide sequences of the rabbit hev strains and compared these sequences with those of human hev strains circulating in france. analysis of orf showed that the sequences from rabbit hev strains formed clusters, one of which included the sequences of hev genotypes and . the bootstrap values were very low because the fragments analyzed were small. in contrast, phylogenetic analyses based on orf and the full-length genome indicated that all the rabbit strains from china and france belong to the same clade. one human strain, tls- -human, clustered with the rabbit strains and appeared to be somewhat different from the major hev genotypes found in mammals and the newly described hev genotypes from rats and wild boars. although the full-length sequences of the genomes of the rabbit strains and the tls- -human strain are more similar to that of hev than to those of hev , hev , and hev , they do not seem to belong to the established hev genotype found in humans and swine, as recently suggested ( , ) . differences in the classifi cation of rabbit hev could be because the full-length genomic sequences were used as the reference for phylogenetic analyses. genotype is highly diverse, with identifi ed subtypes ( ) . we included in our analysis the full-length genomes of subtypes f, c, and e, which account for ≈ %, %, and % of the human and swine hev strains circulating in france ( , ) . we also included the other full-length genomes representative of hev subtypes, but subtypes d, h, and i are not yet available in genbank. our data indicate that the genomes of rabbit hev strains or tls- -human were < % identical with hev , regardless of which method was used to align the sequences. this fi nding is compatible with the defi nition of a new genotype, as previously proposed ( , ) . we found a -nt insertion in the x domain of the orf of the human strain tls- -human and of all the rabbit hev strains. this insertion, also found in the rabbit hev strains from china ( ), is not present in any known strain of hev genotypes - or in the new hev genotypes from rats and wild boars. the x domain corresponds to a macro domain found in the nonstructural polyproteins of several positive-stranded viruses such as togaviruses and coronaviruses ( ) ( ) ( ) . this domain can bind polyadenosine diphosphate-ribose regions and could play a role in the replication or transcription of virus rna. whether the insertion in the x domain infl uences the function of the hev macro domain warrants further investigation. several determinants, including this insertion, could be essential for specifying the host range, zoonotic transmission, and pathogenesis of rabbit hev strains ( ) . what rabbit hev strains contribute to the epidemiology of hepatitis e in humans is not clear. hev is endemic to southwestern france, and the annual incidence of locally acquired hev infections has been estimated as . % ( , ) . a case-control study found that the only factor independently associated with hev infection was the consumption of game meat, mostly wild boar, deer, and wild rabbit ( ) . however, molecular data from various studies in france indicate that most hev strains identifi ed belong to genotypes f, c, or e, which are prevalent in pigs and wild boars ( , , ) . a recent study showed the same proportions of genotypes f, c, and e in human and pig populations ( ) . although this fi nding could indicate that rabbit hev strains are less readily transmitted to humans than hev genotype strains, the primers used for pcr amplifi cation were not specifi cally designed for rabbit hev strains. therefore, the true prevalence of hev rna among rabbits and humans may have been underestimated. in addition, genotyping rabbit hev may have been diffi cult because reference sequences have become available only recently. the immunocompetent or immunocompromised status of the patient that became infected with a rabbit hev strain, as well as the source of his contamination, is unknown because of the lack of medical follow-up. molecular and epidemiologic studies are needed to determine the prevalence of rabbit hev strains among immunocompetent and immunocompromised patients. in conclusion, we have shown that in france, farmed and wild rabbits can be infected with hev. phylogenetic analysis, based on full-length genomes and a molecular signature in the x domain of orf , indicates that rabbit hev strains could be a new genotype. our identifi cation of *hev, hepatitis e virus; hev (m - a-burma, d - b-japan, x - c-india, ay - e-chad, ay - d-morocco); hev (m - a-mexico) ; hev (af - a-usa, ay - j-canada-sw, ab - b-japan, tls-tr - c, tls-tr - e, ab - e-japan-sw,eu - f-france,af - g-kyrgyzstan-sw); hev (ab - c-japan, ab - g-china, ay - d-china-sw). a human hev strain that is closely related to rabbit hev strains reinforces the potential zoonotic risk for infection with this virus. further studies are needed to demonstrate cross-species transmission directly and to evaluate the contribution of the rabbit reservoir to human hev infection and disease. hepatitis e: an emerging awareness of an old disease hepatitis e: an emerging infection in developed countries a novel virus in swine is closely related to the human hepatitis e virus hepatitis e virus: animal reservoirs and zoonotic risk locally acquired hepatitis e in chronic liver disease clinical course and outcome of sporadic acute viral hepatitis in pregnancy fulminant 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at slaughterhouse determined by real-time rt-pcr evidence of extrahepatic sites of replication of the hepatitis e virus in a swine model zoonotic hepatitis e: animal reservoirs and emerging risks close similarity between sequences of hepatitis e virus recovered from humans and swine phylogenetic analysis of the full genome of rabbit hepatitis e virus (rbhev) and molecular biologic study on the possibility of cross species transmission of rbhev structural and functional basis for adp-ribose and poly(adpribose) binding by viral macro domains computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis e virus: delineation of an additional group of positive-strand rna plant and animal viruses differential activities of cellular and viral macro domain proteins in binding of adp-ribose metabolites hepatitis e virus infection without reactivation in solid-organ transplant recipients hepatitis e virus antibodies in blood donors a national survey of acute hepatitis e in france we thank pascal bihannic, thierry delhorme, christian bernard, olivier galaup, francis berger, and jacky aubineau for their help with obtaining samples from wild rabbits.dr izopet is chief of the virology laboratory of toulouse university hospital. his research interests include the molecular virology of hev and its pathogenesis. key: cord- -jqm hxps authors: nan title: abstract date: - - journal: eur biophys j doi: . /s - - - sha: doc_id: cord_uid: jqm hxps nan standard proteomics techniques are unable to describe the stoichiometry, subunit interactions and organisation of assemblies since many are heterogeneous, present at low cellular abundance and frequently difficult to isolate. we have combined two existing methodologies to tackle these challenges: tandem affinity purification (tap) and nanoflow esi-ms. we use methods designed to maintain non-covalent complexes within the mass spectrometer to provide definitive evidence of interacting subunits based on the masses of complexes and subcomplexes generated by perturbation both in solution and gas phases. structural models will be presented for three oligomeric protein complexes of unknown structure: the yeast exosome and the human u snrnp and eif complexes. these models will then be examined within the context of their function. recent developments in mass spectrometry have added a further dimension to our studies of protein complexes: that of their collision cross-section. using ion mobility mass spectrometry we have been able to add spatial restraints to our models validating our models with measurements of collision cross-sections. very recently we have had a considerable breakthrough which has enabled us to preserve intact membrane complexes in the gas phase . this enables us to establish lipid and nucleotide binding and to define the stoichiometry and post translational modifications within the intact transmembrane regions of a number of complexes. in vivo molecular sensing: fluorescence beyond labeling f. beltram scuola normale superiore, i- pisa, italy fluorescent molecules are powerful reporter tools that have much extended the impact of optical microscopy, particularly thanks to the flexibility of genetically encoded tags. detection can now target single molecules even in the complex environment of intact live cells offering unprecedented insight on biological processes in real time in live cells and tissues. fluorescent labels, however, can do more that this. our increased ability to tailor molecule and, more in general, nanosystem properties allows us to design, produce and exploit intelligent tags that can actually analyze the cellular environment. today multifunctional nanosystems can be produced that provide a signal dependent on the value of a specific biochemical parameter. importantly these nanosystems can target specific subcellular domains and have the ability to be used also in the case of live organisms. recent results will be discussed that highlight the impact of nanobiotechnology in this context with a particular emphasis given to methods suitable for in vivo studies that can be transferred to the biomedical world. far-field optical nanoscopy s. w. hell max planck institute, göttingen, germany the resolution of a far-field optical microscope is usually limited to d = λ/ ( n sin α) > nm, with n sin α denoting the numerical aperture of the lens and λ the wavelength of light. we will discuss lens-based fluorescence microscopy concepts that feature a resolving power on the nanoscale. all these concepts share a common basis: exploiting selected (pairs of) states and transitions of the fluorescent marker to neutralize the limiting role of diffraction. specifically, the fluorophore is switched on and off, that is, between a bright and a dark state, to detect the emission of adjacent features sequentially in time. the first viable concept of this kind was stimulated emission depletion (sted) microscopy in which the fluorescence ability of the dye is switched off by stimulated emission. in the sted microscope, the extent of the region in which the molecule is able to fluoresce follows d ≈ λ/ n sin α + i/i s , meaning that fluorophores that are further away than d can be separated. i is the intensity that drives a fluorophore from the bright fluorescent state to the dark ground state by stimulated emission. i s depends (inversely) on the lifetime of the states. for i/i s → ∞, it follows that d → , meaning that the resolution can be molecular). altogether, far-field optical 'nanoscopy' is a fascinating development in optics with high relevance to the many areas of sciences, in particular the life sciences. since it has already been a key to answering important questions in biology, and owing to its simplicity and commercial availability, we expect far-field fluorescence 'nanoscopes' to enter most cell biology and many nanoscience laboratories in the near future. grabbing the cat by the tail: discrete steps by a dna packaging motor and the inter-subunit coordination in a ring-atpase c. bustamante, j. moffitt university of california, berkeley, california, u.s.a. as part of their infection cycle, many viruses must package their newly replicated genomes inside a protein capsid. bacteriophage ϕ packages its . mm long double-stranded dna into a nm dia. x nm high capsid using a multimeric ring motor that belongs to the asce (additional strand, conserved e) superfamily of atpases. a number of fundamental questions remain as to the coordination of the various subunits in these multimeric rings. the portal motor in bacteriophage phi is ideal to investigate these questions and is a remarkable machine that must overcome entropic, electrostatic, and dna bending energies to package its genome to near-crystalline density inside the capsid. using optical tweezers, we find that this motor can work against loads of up to ∼ piconewtons on average, making it one of the strongest molecular motors ever reported. we establish the force-velocity relationship of the motor. interestingly, the packaging rate decreases as the prohead fills, indicating that an internal pressure builds up due to dna compression attaining the value of ∼ megapascals at the end of the packaging. we show that the chemical energy of atp is converted into mechanical work during phosphate release. using ultra-high resolution optical tweezers, we determined the step size of the motor and established the coordination of the polymerases around the ring. we propose a comprehensive model of the operation of this motor. watching proteins function in real time via timeresolved x-ray diffraction and solution scattering p. a. anfinrud laboratory of chemical physics/niddk, nih, bethesda, maryland, u.s.a. to generate a deeper understanding into the relations between protein structure, dynamics, and function, we have developed x-ray methods capable of probing changes in protein structure on time scales as short as ps. this infrastructure was first developed on the id b time-resolved x-ray beamline at the european synchrotron and radiation facility, and more recently at the id b biocars beamline at the advanced photon source. in studies of ligand-binding heme proteins, a picosecond laser pulse first photolyzes co from the heme, then a suitably delayed picosecond x-ray pulse passes through the protein and the scattered x-rays are imaged on a d detector. when the sample is a protein crystal, this "pump-probe" approach recovers time-resolved diffraction "snapshots" whose corresponding electron density maps can be stitched together into movies that unveil the correlated protein motions that accompany and/or mediate ligand migration within the hydrophobic interior of the protein. when the sample is a protein solution, we recover timeresolved small-and wide-angle x-ray scattering patterns that are sensitive to changes in the size, shape, and structure of the protein. scattering studies of proteins in solution unveil structural dynamics without the constraints imposed by crystal contacts; thus, these scattering "fingerprints" complement results obtained from diffraction studies. this research was supported in part by the intramural research program of the nih, niddk dc-sign is a trans-membrane protein expressed on antigen presenting cells and recognizes pathogens like hiv- , hepatitis c virus and ebola. by electron microscopy and near-field optical nanoscopy, we demonstrated that at the plasma membrane dc-sign is organized in well-defined nanodomains of - nm in diameter. intensity-size correlation analysis revealed remarkable heterogeneity in the nanodomains molecular packing density. we constructed and characterized several dc-sign mutated forms lacking specific molecular domains. by immunogold labeling and spatial point pattern analysis, we show that the extracellular neck domain is essential for dc-sign nanoclustering. finally, we present a model that describes the probability of a cell to have a certain number of receptors joining the contact site in the initial encounter with an external object. monte carlo simulations subsequently define the parameters that are determinant in the object-cell encounter. our results show that receptor nanoclustering is of particular importance for binding objects of sizes comparable to the nanocluster size, indicating that the nanoscale spatial organization of dc-sign is optimized for binding to virus-sized objects. imaging of mobile stable lipid rafts in the live cell plasma membrane m. brameshuber , j. weghuber , v. ruprecht , h. stockinger , g. j. schuetz johannes kepler university linz, austria, medical university of vienna, austria the organization of the cellular plasma membrane at a nanoscopic length scale is believed to affect the association of distinct sets of membrane proteins for the regulation of multiple signaling pathways. based on in vitro results, conflicting models have been proposed which postulate the existence of stable or highly dynamic platforms of membrane lipids and proteins. here we directly imaged and further characterized lipid rafts in the plasma membrane of living cho cells by single molecule tirf microscopy. using a novel recording scheme for "thinning out clusters while conserving stoichiometry of labeling" , molecular homo-association of gpi-anchored mgfp was detected at • c and ascribed to specific enrichment in lipid platforms. the mobile mgfp-gpi homo-associates were found to be stable on a seconds timescale and dissolved after cholesterol depletion. having confirmed the association of mgfp-gpi to stable membrane rafts, we attempted to use an externally applied marker to test this hypothesis. we used bodipy-gm , a probe that was recently reported to be enriched in the liquid-ordered phase of plasma membrane vesicles. when applied to cho cells at different surface staining, we found that also bodipy-gm homo-associated in a cholesterol-dependent manner, thus providing further evidence for the existence of membrane rafts. [ ] appl phys lett , ( ) . synapsin knock-out mice as an in vitro model of human epilepsy studied with multi-electrode arrays d. f. boido, p. farisello, p. baldelli, f. benfenati department of neuroscience and brain technologies, italian institute of technology, genova, italy mutant mice lacking synapsins (syn), a family of synaptic vesicles (sv) proteins implicated in the regulation of neurotransmitter release and synapse formation, are epileptic. the attacks appear after the third month of age and severity increases with age. several mutations of syn genes have been found in families of patients with epilepsy. we used micro-electrode arrays (meas) to study spontaneous and chemically evoked epileptiform activities in cortico-hippocampal brain slices obtained from wild-type (wt) and synko mice. -months old synko mice show sporadic ictal (ic) events in the entorhinal cortex. a potassium channel blocker, aminopyridine ( ap), elicits ic and inter-ictal (i-ic) events in both wt and synko slices. in the hippocampus of young synko ( -days old) mice, ap induces i-ic events at higher frequencies than in wt mice. also the frequency of ic events, mainly observed in the cortex, is higher in synko. the analysis of adult ( -year old) mice, revealed a clear age-related aggravation, which paralleled the increase in the severity of the epileptic phenotype observed in vivo. many slices from adult synko mice showed an ic event, while wt slices were refractory at this age to experience ic activity. synko mice are useful to study how neuronal network hyperexcitabilty due to mutations in sv proteins leads to the development of epileptiform activity. meas proved themselves to be useful tools to characterize the epileptic signals foci and patterns of propagation. high electron mobility transistor (hemt) structures were used to bridge the gap between the analysis of biological reactions and biophysical characterization. the combination of nanotechnological measurement approaches with biological reactions provides new possibilities for living cell examinations after exposure to ionizing radiation and basically during the irradiation experiments itself. in this transdisciplinary approach experimental data and handling of biological material enables the identification and specification of systems properties of biological responses to ionizing radiation at different hierarchical levels. gan/algan-heterostructures form a hemt with a gate very sensitive to ph-value changes and potential changes in general. to record cell membrane potentials and ion fluxes during and after irradiation experiments living cells are cultivated on the functionalised biocompatible chip surface. here, we present results of x-ray stimulated cell responses grown on gan-chip surfaces. we recorded transistor signal changes of . µa within s caused by an irradiated cell monolayer. to measure cell potentials, not only after irradiation experiments but also during the irradiation itself expands the examination restrictions in an enormous way. measuring diffusion by spatial-cross-correlation e. gratton, m. a. digman laboratory for fluorescence dynamics, university of california, irvine, u.s.a. fluorescence correlation spectroscopy (fcs) has emerged as a very powerful method to study the motions of proteins both in the interior and exterior of the cell. it provides information at the single molecular level by averaging the behavior of many molecules thus achieving very good statistics. single particle tracking (spt) is also a highly sensitive technique to measure particle movement. however, the fcs method suffers in spatial resolution while the spt technique only allows for the tracking of isolated molecules. here we propose a change of paradigm in which using spatial pair cross-correlation functions we can overcome this limitation. our method measures the time a particle takes to go from one location to another by correlating the intensity fluctuations at specific points on a grid independently on how many particles are in the imaging field. therefore we can trace the average path of the particles. for example, our method could be used to detect when a protein passes the nuclear barrier and the location of the passage. this information cannot be obtained with the frap (fluorescence recovery after photobleaching) technique or the image correlation spectroscopy method. the interaction of the bax c-terminal domain with membranes j. c. gomez-fernandez, s. sanchez-bautista, a. perez-lara, s. corbalan-garcía departamento de bioquimica y biologia molecular. universidad de murcia, murcia, spain the c-terminal domain of the pro-apoptotic protein bax (bax-c) acts as a membrane anchor during the translocation to the membrane of this protein leading to programmed cell death. we have used static and mas-nmr techniques to show that the interaction of bax-c with membranes is modulated by the presence of a negatively charged phosphatidylglycerol. the width of the resonance peaks were considerably more increased by bax-c, in the presence of phosphatidylglycerol. bax-c substantially decreased the t relaxation times of phosphatidylglycerol and those of phosphatidylcholine when mixtured with phosphatidylglycerol but they were not decreased when phosphatidylcholine was the only phospholipid present in the membrane. c-mas-nmr showed that t values were decreased when bax-c was incorporated and, when phosphatidylglycerol was also present, the decrease in t affected considerably more to some carbons in the polar region. these results indicate that bax-c interacts differently with the polar part of the membrane depending on whether phosphatidylglycerol is present or not, suggesting that an electrostatic interaction of bax-c with the membrane determines the membrane disposition of this domain. fluorescence spectroscopy showed that the trp residues of bax-c were located in a microenvironment more hydrophobic when phosphatidylglycerol was present. h. g. franquelim , l. m. s. loura , n. santos , m. castanho instituto de medicina molecular, univ. lisboa, portugal, faculdade de farmácia, univ. coimbra, portugal since the efficacy of hiv fusion inhibitors was previously reported to be related to an ability to interact with membranes, we studied the interaction of the hiv fusion inhibitor sifuvirtide, a aa negatively charged peptide, with lipid vesicles. since this peptide has aromatic residues, fluorescence spectroscopy techniques were used with no need for attached probes. results showed no significant interaction with both zwitterionic fluid phase and cholesterol-enriched membranes; however extensive partition to fluid phase cationic membranes were observed. in the dppc gel phase, however, an adsorption at the surface of these membranes was detected by using a differential quenching approach with lipophilic probes, as well as by fret. moreover, the interaction with gel phase membranes seems to be specific towards pc vesicles, since no significant interaction was retrieved for membranes composed by shingomyelin and ceramide. besides fluorescence, atomic force microscopy and zeta-potential were used to further investigate this issue. our results show a selectivity and specificity of the peptide towards rigid domains, where most of the receptors are found, and help explain the importance of the interaction with membranes in the improved efficacy of sifuvirtide compared to other fusion inhibitors, by providing a local increased concentration of the peptide near the fusion site on both cellular and viral membranes. the study of molecular dynamics at the single-molecule level with fluorescence far-field optics offers new detailed insights into scientific problems, especially in living cells. unfortunately, the resolution of common far-field techniques is limited to about nm in the lateral direction by diffraction. in recent years, several concepts such as stimulated emission depletion microscopy (sted) have been successfully applied to overcome the diffraction barrier by exploiting the photophysical properties of fluorescent labels. we present the combination of high resolution sted microscopy with different fluorescence fluctuation techniques providing the unique ability to study molecular dynamics with high spatial (< nm) and temporal resolution (< ms) in living cells. using fluorescence correlation spectroscopy (fcs) and general single-molecule analysis, we were able to explore single-molecule dynamics in up to -fold reduced focal volumes on two-dimensional samples such as lipid membranes with excellent signal-to-noise ratios. special attention is drawn to inhomogeneous lipid diffusion on the plasma membrane of living cells . by extending the available spatial scale of standard single-molecule fluorescence far-field spectroscopy techniques, our experiments outline a new way of approaching scientific problems. modulation of the properties of membrane microdomains as a control mechanism in cellular physiology p. o'shea cell biophysics group, institute of biophysics, imaging & optical science, school of biology, university of nottingham, nottingham ng rd, u.k. this presentation will outline the molecular-physical rationale of how membrane microdomains may modulate the behaviour of membrane receptor systems as a controlling mechanism in cell signaling. a number of external factors that modulate these properties will be indicated that have a bearing on controlling cellular behaviour. some of key questions will be considered such as the factors that control the assembly and disassembly of microdomains, the size and numberdensity of the microdomains and the lifetime that they exist within the membrane. the technical challenges that these questions identify will also be outlined with some possible solutions. throughout this presentation, correlations will be made between theory and experiment as well as between model membrane systems and real cellular systems. structural and dynamic properties of caveolin- and - fragments at the membrane interface c. le lan , j. gallay , m. vincent , j.-m. neumann , b. de foresta , n. jamin cea, ibitecs, sb sm, & ura cnrs , gif-sur-yvette, france, ibbmc, université paris-sud, umr -cnrs, ifr , orsay, france caveolins are major protein components of caveolae, microdomains of the plasma membrane involved in a large number of biological functions, including signal transduction, cholesterol homeostasis and transport. the consensus topological model of caveolin- includes a small central intramembrane region ( - ) flanked by two cytosolic amphiphilic domains ( - and - ) which probably constitute in-plane membrane anchors. we investigated the interaction of the cav- ( - ) juxta-membrane segment with various membrane mimics, using fluorescence, cd and nmr. this segment partitioned better in dpc and in dm/anionic lipids micelles than in dm micelles and this partitioning was coupled with the formation of an amphipathic α-helix. this amphipathic helix was located in an average shallow position, in the polar head group region of the dpc micelle, as shown by fluorescence data and intermolecular noes, with the aromatic doublet w -f probably pointing towards the inside of the micelle on average. the peptide encompassing the homologous sequence of cav- was also localized to the dpc micelle polar head group region, in which it adopted a more stable helical conformation than cav- ( - ) . these data brings experimental support for the role of this segment as an interfacial membrane anchor. resveratrol (trans- , ', -trihydroxystilbene) , a phytoalexin present in grapes and its analogue piceatannol (trans- , , ', '-tetrahydroxystilbene) are biologically active compounds and possess potential chemopreventive and anticancer properties. the activity of resveratrol and piceatannol can be mediated by membrane effects since structure of lipid membrane domains may play an important role in cell signalling pathways. drugs interactions with dmpc bilayers was investigated using a combination of esr spectroscopy and differential scanning calorimetry. spin probes used in epr experiment were located in different part of lipid bilayer. study was performed at temperatures below and above phase transition temperature (t m ). epr spectra were simulated and displayed with ghost condensation method. the values of ϑ and ϕ (the main and asymmetry cone angles of wobbling spin probe, respectively) were taken for free rotational space parameter (Ω) calculation. the decrease of Ω values was observed in the presence of both compounds and the effect was more pronounced in lipid gel phase. order parameter and correlation time were also determined and presented in form of ghost patterns. using this approach the differential influence of studied compounds on membrane heterogenity was revealed. separating hydrostatic pressure from cellular strain: development of an in vitro model system r. sulley , j. whatmore , c. p. winlove , a. shore , j. tooke , r. ellis , k. gooding peninsula medical school, school of physics, university of exeter, uk endothelial cells (ecs) line blood vessels & are constantly subjected to haemodynamic and mechanical stresses and strains. these stimuli are known to influence ecs, modifying their morphology, intracellular signalling & gene expression. most reported systems exposing ec to mechanical forces in vitro alter pressure & strain simultaneously, making it impossible to distinguish the two potentially independent stimuli. this distinction is particularly relevant when examining the interaction of haemodynamic forces on microvascular ecs, which are exposed to low hydrostatic pressure but significant strains. this research aims to create an in vitro system that can independently examine the effects of pressure and strain, over a range experienced by ecs in the microvasculature. human ecs are seeded ( x ˆ cells/cmˆ ) on to the inner surface of compliant mm diameter tubing. which is mounted on a perfusion rig inside a sealed, fluid-filled chamber. a continuous sinusoidal cyclical strain of - % is created by a pump attached to the external chamber. lumenal pressure is generated using two hydrostatic pressure heads. validation experiments show that pressure & substrate strain can be independently varied & controlled over a physiological range. the system is now being used to investigate the effects of pathophysiological haemodynamic abnormalities on ec function. membrane potential dynamics of living cells in response to femtosecond laser irradiation n. i. smith , j. ando , k. fujita , s. kawata photonics advanced research center, osaka university, suita, osaka - , japan, dept of applied physics, osaka university, suita, osaka - , japan the ultrashort pulsed near-infrared femtosecond laser has had a large impact in biomedical research fields and in microscopy, where it has enabled new imaging methodologies. at high intensities, the focused beam of a femtosecond laser has been used to irradiate specific locations inside a cell, often beneath the cell membrane, exploiting the inherent penetration and localized absorption that comes from the multiphoton absorption physics. this has been applied in photobleaching, photouncaging, laser surgery and other experiments where the light is used not merely for observation but is instead an integral tool to interact with the dynamics of cells, to probe and perturb the cell condition. in this talk i will discuss biological and mechanical effects that can be generated by short exposures to femtosecond laser irradiation, such as calcium waves, membrane hyperpolarization, and cell contraction. this talk will concentrate on the changes in membrane potential that can occur when the cell is subjected to focused femtosecond laser beams. both depolarization and hyperpolarization of the membrane potential could be evoked, depending on the laser parameters and on the position of the laser focus. these results have implications for the use of laser beams in microscopy, optical gene transfection, and laser nanosurgery. in a recent study we showed that the melting behavior of supported lipid bilayers (slbs) on mica can be influenced by the solution ionic strength and by the slb preparation temperature [ ] . by changing these parameters we could control the coupling between the two bilayer leaflets obtaining a coupled or decoupled melting behavior. thus, we could provide evidence that the slb model system is also suited for the study of lipid/protein interactions which had been questioned in the past. then we investigated the mutual interactions between the membrane lipids (pope:popg : ) and the kcsa potassium ion channel by studying kcsa proteins reconstituted in slbs. in particular, we studied the melting behavior of the slb and the ion channel distribution relative to the different membrane phases by temperature controlled atomic force microscopy (afm). by decreasing the temperature we found that the proteins underwent diffusion so to be excluded from the growing solid ordered regions. further, the ion channels tended to accumulate at the domain boundaries or they aggregated in the liquid disordered phase. both effects have been suggested to affect protein function. when we started from a low temperature at which the membrane was mainly in the solid ordered phase the membrane melting processes started in the vicinity of the included ion channels. we report fluorescence recovery after photobleaching (frap) measurements performed at variable spot radius for t -egfp-hmop receptors on sh-sy y neuroblastoma cells in the presence of ligands. two different agonists, damgo and morphine, caused markedly different changes to receptor diffusion as compared to the basal state. like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joint semi-permeable domains, but with smaller domain size and diffusion coefficient. this effect was inhibited by pertussis toxin, suggesting that this dynamic behaviour is associated with early steps of signaling. in the presence of damgo, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. hypertonic sucrose buffer suppressed this effect which we attribute to receptor entry into clathrin-coated pits. it is likely that the observation of distinct receptor dynamics in the presence of damgo and morphine involves the agonist-selective phosphorylation of the receptor. the alteration of the physiological transcriptional program is one of the constant features of cancer cells. however, the characterization of the chromatin changes at single-gene level requires going beyond the diffraction limit affecting conventional fluorescence microscopy. the ability of molecular biology techniques to obtain a detailed view of the chromatin status at a sub-promoter resolution has to pay instead the price of averaging over a cell population. we report here the application of an approach based on high-resolution cytometry, chromatin immuno-precipitation and transcriptional profiling (dna microarray) for the characterization of the transcriptional and chromatin changes induced by the oncogenic transcription factor pml/rarα. the presentation will focus on the imaging protocol employed to observe the effects on the chromatin status and the extent of the deregulation induced on transcriptional activity in acute promyelocytic leukemia cells. this multiple-approach examination provides a further step towards the comprehension of the hierarchy of chromatin modifications leading to the establishment of a malignant transcriptional program. dna is rigid negatively charged polymer and in solution exists in extended conformation. i n vivo, volume occupied by dna must be reduced to fit to tiny space of cell nucleus. to condense dna, dna-dna electrostatic repulsion must be cut off that is achieved by interaction with cationic ligands. in binding to dna, oligocations compete with salt cations (k + , na + , mg + ). description of salt dependence of oligocation-induced dna condensation is still lacking. we studied dna condensation by model oligocations, ε-oligo(l-lysines), with variation of charge from + to + . combination of light scattering, uv-monitored precipitation assay and isothermal titration calorimetry allowed covering wide range of dna (c dna ) and salt (c kcl ) concentrations. salt dependence of dna condensation efficiency of the ligand, ec (ligand concentration at the transition midpoint) displays two regimes: salt-independent at low c kcl and salt-dependent at higher c kcl (steep increase of ec with c kcl ). simple formula describing ec as function of ligand charge, c dna and dissociation constant of ligand-dna complex (k d ), was proposed. in the salt-independent regime ec is defined by c dna . salt-dependence of ec is rooted in the variation of k d with c kcl earlier described in ligand-dna binding studies. importance of our findings for description of chromatin is discussed. interaction between proteins from linker region of nucleosome in presence/absence of dna in solution i. b. kipenko , e. v. chikhirzhina , a. m. polyanichko faculty of physics, saint-petersburg state university, russia, laboratory of cell biochemistry, institute of cytology, ras, saint-petersburg, russia interactions in the linker region of the nucleosome play a key role in the structural organization of the chromatin. the most fascinating and least understood is the interplay between non-histone chromatin protein hmgb and a linker histone h . it is known that both h and hmgb bind the linker region of the dna in vivo. however it is still a matter of debate as to whether these proteins assist each other or compete upon binding. the main attention in this work is paid to the investigation of the interactions between the hmgb and h proteins in physiological environment. using circular dichroism (cd) spectroscopy we have studied the interactions between hmgb and h at various hmgb /h ratios (r). it has been shown that there is a cddetectable interaction between the proteins h and hmgb at r < . we have demonstrated that the interaction between these proteins results in changes of their secondary structure. cd indicates that the structural impact of the unordered fragments decreases while the net α-helicity of the proteins increases upon the interaction. we have also shown that large higher order structures are formed in solution. in this work we have also discussed the dna-binding properties of the hmgb and h proteins. the work was supported by a rfbr grant ( - - ) and the government of staint-petersburg. t. u. rodionova , a. m. polyanichko , v. i. vorob'ev faculty of physics, saint-petersburg state university, russia, institute of cytology of the russian academy of sciences, saint-petersburg, russia hmgb is a nonhistone chromosomal protein. data regarding the structure of the hmgb-proteins obtained so far are rather different. thermodynamic experiments reveal predominant α-helical structure of the proteins only at temperatures below + • c, i.e. under physiological conditions they are mainly disordered. despite a lot of experimental data biological role of hmgb still remains unclear. it is believed that the proteins perform structural functions in chromatin and participate in various regulatory processes in cell. using circular dichroism spectroscopy and dna melting analysis we have shown that hmgb changes its structure upon binding to dna. it was shown that at room temperature only about % of amino acid residues form α-helices, while in dna-hmgb complex the degree of the α-helicity of the protein increases to approximately %. based on the data obtained we estimate the size of hmgb binding site as - b.p. we have also demonstrated that despite of strong dna-bending properties of hmgb its binding to dna results in increase of the double helix termostability. the authors are grateful for the financial support from the russian foundation for basic research (grants - - , - - ) and the government of saint-petersburg. structural organization of supramolecular complexes of dna with chromosomal proteins hmgb and h a. m. polyanichko , h. wieser faculty of physics, saint-petersburg state university, russia, dept. of chemistry, university of calgary, canada a combination of uv and ir absorption and circular dichroism spectroscopy together with atomic force microscopy was applied to investigate the structure and formation of large supramolecular dna-protein complexes. this combination of techniques was used to overcome limitations of uv-cd spectroscopy due to considerable light scattering in such solutions. based on the analysis of ftir and uv circular dichroism spectra and afm data the interaction of dna with highmobility group non-histone chromatin protein hmgb and linker histone h was studied. it is believed, that hmgb-domain proteins perform both structural and regulatory functions in chromatin. however, the particular mechanisms of it functioning remain unclear/ our data show that histone h facilitated binding of hmgb to dna by interacting with the sugar-phosphate backbone and binding of asp\glu amino acid residues of hmgb . acting together, hmgb and h stimulated the assemblage of supramolecular dna-protein structures. the organization of the ternary complexes is modulated by the interactions between hmgb and h molecules. the dna-proteins interactions in the presence of metal ions were different, causing prominent dna compaction and formation of large intermolecular complexes. the work was supported by rfbr (grant - - ). biophysical properties and mechanisms of phage dna ejection t. mdzinarashvili , m. khvedelidze , a. ivanova , t. partskhaladze , n. shengelia i. javakhishvili tbilisi state university, tbilisi, georgia, institute of molecular biology and biophysics, tbilisi, georgia to determine the requirements for phage adsorption on bacterial cell and for the realizing resources of following dna ejection thermodynamic and hydrodynamic methods were employed. the temperature, bacterial membrane fragments and receptors had been chosen as such external factors. the phages with short and long tail, both contractile and noncontractile have been studied. our viscometric studies of the phage dna ejection induced by receptor by the example of t phage and its receptor fhua have shown that the minimum protein-to-phage ratio necessary for complete dna release is to . the viscometric study of ddvi phage dna ejection induced by membrane fragments obtained from its host cells has shown that the environmental conditions play significant role in ejection process. both methods show that the thermally induced phage dna ejection for all investigated by us phages have shown that this process is nonenthalpic. finally from our experimental results we conclude that the start of the dna ejection process from the phage particle occurs without additional energy from either a physical or chemical source. we thank gnsf for the financial support. nitroxides induce apoptosis through caspase- activation and collapse of mitochondrial potential k. matczak , a. koceva-chyla , k. gwozdzinski , z. jozwiak department of thermobiology, department of molecular biophysics, university of lodz, lodz, poland nitroxides are new class of antioxidants that have been proved to show high reactivity toward free radicals. they act as superoxide dismutase mimics dismutating superoxide anions, but can also exert pro-oxidative properties. in view of their possible dual activity nitroxides could be of great importance in medicine. we have investigated pro-apoptotic activity of pyrroline and pyrrolidine nitroxides pirolid (pd) and pirolin (pl) in human breast cancer cells. in cancer, it is the failure of malignant cells to undergo apoptosis that is crucial. using microplate fluorescence methods, we estimated kinetics of changes in mitochondrial transmembrane potential and caspase- activity in breast cancer cells mcf- treated with pirolin or pirolid. these features are connected with induction of apoptosis in some type of cancer cells. we observed steady-state increase in caspase- activity up to h of postincubation that was followed by a decrease in the enzyme activity at h. caspase- activation was considerably greater in cells treated with pirolid. both nitroxides also caused notable decrease in mitochondrial transmembrane potential, which suggest that they can induce apoptosis in breast cancer cells through mitochondrial pathway. o. chernyavskiy , l. vannucci , p. bianchini , f. difato , l. kubínová dept. of biomathematics, inst. of physiology, as cr, prague, czech republic, dept. of immunology, inst. of microbiology, as cr, prague, czech republic, lambs, dept. of physics, university of genoa, italy, dept. of neuroscience and brain technologies, the italian institute of technology, genoa, italy the second harmonic generation (shg) imaging along with confocal laser scanning microscopy in reflectance mode can be applied to imaging unstained tissues in vivo, so it can be considered as a fast and non-invasive tool for in vivo studies. murine b f melanoma cells after subcutaneous inoculation in syngeneic mice were let to develop into tumor up to - mm in diameter. microscopic images were taken before and after microwave hyperthermia treatment (mwht). the microscopic images were acquired by -photon imaging in reflectance mode, shg imaging and -photon imaging of tissue autofluorescence. the evaluation of changes in the images after mwht of the tumor demonstrated changes in the architecture and organization in both the tumor capsule and tumor mass. the presented study was supported by the academy of sciences of the czech republic (grant iaa , institutional research concepts no.av z , av z ), and ministry of education, youth and sports of the czech republic (research program lc ). intracellular delivery and fate of peptide-capped gold nanoparticles: towards cellular biosensors y. cesbron , v. sée , p. free , p. nativo , d. g. spiller , m. r. h. white , m. brust , b. lounis , r. lévy liverpool institute for nanoscale science, engineering and technology, liverpool, uk, université bordeaux i / cnrs, bordeaux, france gold nanoparticles (nps) have extraordinary optical properties that make them very attractive single molecule labels. although understanding their dynamic interactions with biomolecules, living cells and organisms is a prerequisite for their use as in situ sensors or actuators. while recent research has provided indications on the effect of size, shape, and surface properties of nps on their internalization by living cells, the biochemical fate of nps after internalization has been essentially unknown. here we show that peptide-capped gold nps enter mammalian cells by endocytosis. we demonstrate that the peptide layer is subsequently degraded within the endosomal compartments through peptide cleavage by the ubiquitous endosomal protease cathepsin l. preservation of the peptide layer integrity and cytosolic delivery of nps can be achieved by a combination of cathepsin inhibition and endosome disruption. this is demonstrated using a combination of distance-dependant fluorescence unquenching and photothermal heterodyne imaging. these results prove the potential of peptide-capped gold nps as cellular biosensors. current efforts focus on in-vivo labeling of nps, nanoparticle-based real-time sensing of enzyme activity in living cells, and the development of photothermal microscopy for single nanoparticle imaging in living cells. towards intravital two photon microscopy study of lymphocytes mobility in lymphonodes m. caccia , l. sironi , m. collini , i. zanoni , t. gorletta , m. di gioia , g. francesca dipartimento di fisica, università di milano bicocca, italy, dipartimento di biotecnologie e bioscienze, università di milano bicocca, italy during the last years the edge between optical fluorescence based microscopy and the world of bio-medical research has become thinner and thinner and two photon laser scanning microscopy (tplsm) is one of the most powerful tool for immunological and medical research. one of the most limiting step in intravital microscopy is the preparation of the animal model and the number of animals to be sacrificed to get good statistics. alternative routes must be searched. we employ tplsm to explanted lymphnodes. two photon excitation and a non descanned detection mode allow to increase, respectively, the excitation and detection efficiency while the explanted organs are kept very close to the condition they experience in live animals by means of an home-made temperature controlled box surrounding the microscope and a system for the flux of physiological fluids. experiments performed on explanted lymphonodes, kept under constant flux of co -o saturated buffers and at o c, agree with literature for what concern t-cell homing and motility. this seems to confirm that t-cells behavior in explanted organs mantained in physiological conditions is very similar to that observed in live animals. we then believe that our tplsm microscope would allow to study cell behaviors in in-vivo with high efficiency and a little technical effort. a generalized quantitative frap method with no restriction on the size of the photobleached area heterochromatin protein in dna damage response -recruitment or dissociation from repair sites? j. w. dobrucki division of cell biophysics, faculty of biochem. biophys. and biotechnol., jagiellonian university, kraków, poland we studied recruitment of dna repair proteins to damage sites in live cells, by microscopy approaches, using a new method of inflicting local, sublethal damage in nuclei of live cells. oxidative damage, which was inflicted by exciting dna-intercalated ethidium with focused green light, triggered recruitment of base excision repair enzymes. surprisingly, an epigenetic regulator, heterochromatin protein (hp ) (zarebski et al., ) was recruited to damage as well. hp is a constitutive component of heterochromatin, and plays an important role in transcriptional repression and regulation of euchromatic genes, however it was not known to be required for repair of oxidative damage. the finding of hp recruitment is particularly puzzling, since in another study hp was shown to dissociate from chromatin as a result of dna damage (ayoub et al. ) . technical aspects of live cell imaging that may explain these contradictory results will be discussed. in this presentation, we report a method for determining both the presence and the stoichiometry of protein complexes at pixel resolution and apply it to disassembling focal adhesions. the method is derived from fluorescence fluctuation methods that have single molecule sensitivity and is based on our previously described n&b (number and brightness) method that measures the number and brightness (aggregation state) of fluorescent molecules in every pixel of a confocal microscope image. the new method exploits the correlation of fluorescence amplitude fluctuations for two colors and detects the presence of molecular complexes and their stoichiometry. while the original n&b method was developed for one color, i.e., a single molecular species, the new method, ccn&b, extends the analysis to two colors and introduces the concept of cross-variance. this method is similar in concept to the two-color pch analysis. however, the covariancebased ccn&b method also generates pixel resolution maps of protein complexes and can be used on commercial confocal microscopes. the method is highly sensitive and has relatively high temporal resolution. we have applied this method to adhesion complexes in cells. in addition of their structural role, to link the extracellular substratum to actin filaments, they also serve as signaling centers that regulate many cellular processes including their own assembly and turnover, migration, gene expression, apoptosis, and proliferation. spatio-temporal analysis of membrane lipid remodeling during phagocytosis s. de keijzer , d. kilic , c. g. figdor , s. grinstein , a. cambi department of tumor immunology, radboud university of nijmegen, nijmegen, the netherlands, department of biochemistry, university of toronto, toronto, canada the constant threat posed by pathogens and cell debris is tackled by phagocytosis, the process through which cells engulf and destroy dangerous material. the signaling, targeting and trafficking during phagocytosis is dependent on cytoskeleton rearrangements and membrane remodeling. it is becoming increasingly evident that lipids play an important role and can affect the phagocytic response. they assemble microdomains which can act as signaling platforms and confer charge and curvature to the membrane surface promoting electrostatic attraction and retention of proteins. little is known about the mechanism(s) regulating membrane lipid remodeling during phagocytosis. here we used fluorescently labeled biosensors based on k-ras and h-ras proteins to obtain spatio-temporal information on phagosomal membrane lipid remodeling during fcreceptormediated phagocytosis. the results show that cell activation by cytokines modulates the kinetics of anionic lipids thus affecting the membrane charge and the recruitment of cytosolic proteins to the phagosomal membrane. our data emphasize the fundamental role of lipids in the generation and transduction of signals in phagocytosis, and we believe this can be extrapolated to many important processes in a cell. fluorescence correlation spectroscopy (fcs) is a useful technique for characterizing the mobility and concentration of fluorescent molecules both in vitro and in vivo. we utilize two-photon fcs to characterize the concentration and mobility of fluorescent molecules within living cells of bacillus subtilis. autocorrelation functions were measured in bacteria expressing green fluorescent protein (gfp) under the lac promoter in both nutrient rich and nutrient poor culture medium. although considerable heterogeneity was evident from cell to cell, on average, both intracellular concentration and mobility were found to be dependent upon culture medium. we also investigated bacteria expressing gfp under control of native promoters for involved in the regulation of the carbon metabolic cycle in bacillus subtilis. the gfp concentration, which should be related to promoter activity, was investigated for single cells and cell populations under different metabolic conditions. some photobleaching was observed during the course of the measurements as a decrease in the average fluorescence intensity. this is due to the small size of the bacteria (∼ fl) and low basal expression levels of gfp (∼ nm) in the absence of iptg. methods to take this into account during data analysis are discussed. a. fassio , s. congia , p. baldelli , f. benfenati dimes, university of genoa, genoa, italy, dnbt , iit, genoa, italy several mutations have been discovered in the synapsin i (syn) gene in families with epilepsy, but the mechanism inducing the epileptic phenotype is unknown. syn is a protein associated with synaptic vesicles (svs) that control sv trafficking and neurotransmitter release. the syn mutations subject of this study are a non sense (ns- ) and of two missense (ms- , ms- ) . syn knockout (ko) hippocampal neurons were transfected with the either wild type (wt) or mutated syns. all syns presented a common punctate pattern of expression at the level of axonal arborizations but, while ns- syn targeted to synapses as wt-syn, ms- and ms- syns reached the presynaptic terminal less efficiently. we next set up a live cell imaging experiment using synaptophysin-phluorin and evaluated the effects of ns- , ms- and ms- syns on the size of sv pools. restoring wt-syn in syn i ko terminals led to a increase of all sv pools. restoring ms- and ms- syns resulted in a phenotype not significatively different from syn i ko background whereas restoring ns- syn caused a decrease of all sv pools as compared either with wt-syn or syn i ko background. these data suggest an alteration in the subcellular distribution and function of syn in patients carrying ms- e ms- mutation and a more severe effect on synaptic activity in patient carrying ns- mutation. imaging dynamics of dc-sign at the plasma membrane of hiv- -stimulated dendritic cells o understanding how viruses interact with host receptors is essential for developing new antiviral strategies. dendritic cells (dcs) can efficiently capture and take up hiv- through multiple attachment factors, such as the c-type lectin dc-sign. however, the initial interactions between hiv- and this receptor on dcs are not fully understood yet. in this work, we have used single-molecule epi-tirf microscopy in combination with fluorescently labelled hiv- virus like particles (vlps) and dc-sign-specific antibodies to image the dynamic interaction between hiv- and dc-sign nanoclusters at the plasma membrane of dcs. by tracking individual trajectories of dc-sign on the cell surface of living dcs we have found heterogeneity in the modes of motion of dc-sign: some clusters are immobile whereas others move very quickly, with a few ones showing a directed motion on the cell membrane. to investigate how such motion might be correlated to dc-sign function as virus attachment factor, we have developed glass platforms functionalized with hiv- vlps to locally stimulate living dcs. these virus platforms have allowed us to measure dc-sign diffusion rates and motion modes over the cell surface of stimulated dcs and represent a powerful tool for studying the dynamic interactions between hiv- and the dc membrane. a. esposito , t. tiffert , j. mauritz , s. schlachter , j. n. skepper , v. l. lew , c. f. kaminski dept. of chemical engineering and biotechnology, univ. of cambridge, u.k., dept. of physiology, development and neuroscience, univ. of cambridge, u.k. plasmodium falciparum (pf ) causes the most lethal form of malaria in humans. early research exposed two paradoxes: ) during its intraerythrocytic cycle pf permeabilizes the host cell so much that a comparably permeabilized healthy red blood cell (rbc) would lyse prematurely, and ) pf digests far more hemoglobin than needed for its metabolism. a model of the homeostasis of a pf infected rbc suggested a common explanation of both puzzles: excess hemoglobin digestion is required to reduce the colloidosmotic pressure within the host cell thus ensuring its osmotic stability to the end of the pf asexual cycle. we investigated these predictions with direct measurements of [hb] and volumes of parasitized rbcs. reliable volume and morphological data was obtained by confocal microscopy and quantitative surface reconstruction. furthermore, we developed a new fret-based method to measure hemoglobin molecular crowding by exploiting the reduction in fluorescence lifetime of a donor fluorophore loaded in the rbc cytosol. fret imaging techniques are powerful tools for probing the biophysics of living cells. these tools provided a first validation of the colloidosmotic hypothesis and a deeper understanding of the homeostasis of the intraerythrocytic stage of pf. hiv- assembly and release occur at the plasma membrane of infected cells and are driven by the gag polyprotein. using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated assembly of fluorescently labeled hiv- at the plasma membrane of living cells with high time resolution. gag assembled into discrete clusters corresponding to single virions. after their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over - hours. using a photoconvertible fluorescent protein, we determined that assembly was nucleated by membrane bound gag molecules, while both membrane-bound and cytosol derived gag polyproteins contributed to the growing bud. assembly kinetics were rapid and three phases are observed. in phase i, the number of gag molecules at a budding site increases following a saturating exponential with a rate constant of ∼ x − s − . hence, gag assembly is complete in ∼ s. in phase ii, a plateau in fluorescence intensity is observed with no exchange of gag protein. the fluorescence intensity decays in phase iii. this decay, in some cases, corresponds to the release of a virion. the time scale from the onset of assembly to release of extracellular particles was measured to be ∼ , +/- s. fast d chromatin dynamics studied in living yeasts using a novel lab on chip technology h. hajjoul , m. dilhan , i. lassadi , k. bystricky , a. bancaud laas-cnrs, université de toulouse, france, lbme, cnrs umr , toulouse, france we present a novel lab-on-chip technology for d particle tracking yeast abased on v-shaped mirrors, which are used to observe fluorescent specimens from multiple vantage points, providing stereo-images that can be recombined for d reconstruction. our technology is based on v-shaped mirrors, which are fabricated by wet etching of silicon wafers, and used as optical and fluidic components. after rigorous optical optimization of the device in vitro, the lab-on-chip is applied to study chromatin dynamics in vivo using budding yeasts as a model system. yeasts cells are visualized using gfp fused to the associated repressor protein. we confirm earlier observations that telomeric sequences, i.e. located close to chromosome ends, accumulate at the nuclear periphery, whereas genes found midway along chromosome arms are mostly present in the nuclear lumen. the dynamics of these sequences is followed in d with an unpreceded temporal resolution of ms, showing that chromosome dynamics is non linear in the short time regime and switches to a linear regime at larger timescales. notably, this behavior is reminiscent of universal responses observed in polymer solutions, and is related to the confinement and the structure of chromosomes. this technique shows a great potential for studying dynamic processes in small living organisms. retrovirus induced remodeling of the host cell actin architecture m. gladnikoff, i. rousso department of structural biology, weizmann institute of science, rehovot, israel retrovirus budding is a key step in the virus replication cycle. yet, despite substantial progress in the structural and biochemical characterization of retroviral budding, the underlying physical mechanism remains poorly understood, primarily due to technical limitations preventing visualization of bud formation in real time. using atomic force-, fluorescence-and transmission electron microscopy we find that both hiv and moloney murine leukemia virus (mlv) remodel the actin cytoskeleton of their host cells and utilize the forces it generates to drive their assembly and budding. highly dynamic actin-filamentous structures which varied in size over the duration of budding appeared to emanate from the assembled virion. these actin structures assemble simultaneously or immediately after the beginning of budding, and disappear as soon as the nascent virus is released from the cell membrane. analysis of sections of cryo-preserved virus infected cells by tem reveals similar actin filaments structures emerging from every nascent virus. substitution of the nucleocapsid domain implicated in actin binding by a leucine-zipper domain resulted in budding of virus-like particles that was not accompanied by remodeling of the cell's cytoskeleton. notably, budding of viruses carrying the modified nucleocapsid domains was an order of magnitude slower than that of the wild type. the results of this study show that retroviruses utilize the cell cytoskeleton to expedite their assembly and budding. t. gensch institute of structural biology and biophysics (isb- ) cellular biophysics, research centre jülich, germany the determination of ion concentrations in cells -in particular in neurons -is very important for understanding cell function and life. ca + is an ubiquitous messenger in almost all cell types, cl − has several roles, e.g. plays an important role in some neuronal signaling pathways including olfaction, nociception and vision. fluorescence lifetime imaging (flim) is of advantage over intensity based fluorescence microscopy, when comparisons between micro-domains of one cell or between different cells of one cell type are performed. several (organic chromophores and fluorescent protein based) ca + -and cl − -sensors have been tested in culture cells with respect to their applicability in flim studies. the ca + -and cl − concentration in rodent olfactory sensory neurons, dorsal root ganglion neurons and neurons of the retina is investigated by time-resolved flim with two-photon excitation. viscosity is one of the main factors which influence diffusion in condensed media. in a cell viscosity can play a role in several diffusion mediated processes, such as drug delivery and signalling. previously, alterations in viscosity in cells and organs have been linked to malfunction; however, mapping viscosity on a single-cell scale remains a challenge. we have imaged viscosity inside individual cells using novel fluorescent probes, called molecular rotors, in which the speed of rotation about a sterically hindered bond is viscosity-dependent. , this approach enabled us to demonstrate that viscosity distribution in a cell is highly heterogeneous and that the local microviscosity in hydrophobic cell domains can be up to × higher than that of water. we have also shown that the intracellular viscosity increases dramatically during light activated cancer treatment, called photodynamic therapy (pdt). we have demonstrated the effect of such viscosity increase on intracellular reactions by directly monitoring the rates of formation and decay of a short lived toxic intermediate, crucial in pdt, called singlet molecular oxygen, in light perturbed cells. characean algae, close relatives of higher plants, represent a convenient model for studying the effect of propagating electrical signals, action potentials (ap) on photosynthesis. illuminated chara corallina cells produce coordinated spatial patterns of chlorophyll fluorescence (chl fl) and extracellular ph. photosynthesis is higher in the cell regions adjacent to acid zones compared to alkaline zones. under physiological conditions, the electrically induced ap differentially and reversibly suppresses photosynthesis in the alkaline and acid regions. in this work we examined the effects of an artificial psi acceptor, methyl viologen (mv), on chl fl with imaging-pam technique. mv is a divalent cation and poorly permeates through biological membranes. the presence in the medium of mv had no effect on fl as well as on p + absorbance signals until the application of a single excitatory stimulus. once an ap was generated in the presence of mv, it induced irreversible inhibition of native (nadpdependent) electron flow and a strong non-photochemical fl quenching all over the cell. this indicates that ap redirects electron flow from the main pathway to the artificial acceptor. we concluded that ap generation opens access for permeation of mv from the medium to the chloroplast stroma across two membrane barriers, plasmalemma and chloroplast inner membrane. we suggested that mv might enter chara cell via plasmalemma ca + -channels activated during ap. evaluation of cell damage after photodynamic and sonodynamic treatment h. kolarova, k. tomankova, s. binder, p. kolar, r. bajgar, m. strnad department of medical biophysics, faculty of medicine and dentistry, palacky university, hnevotinska , olomouc, czech republic photodynamic therapy (pdt) and sonodynamic therapy (sdt) is a new, combined therapy for treating cancer. the basis of the therapy is to administer a small amount of photosensitizer and sonosensitizer, which are selectively taken up by cancer cells, and then expose the body to light and ultrasound to activate these sensitizers. when the sensitizers absorb light of an appropriate wavelength, it may cause their excitation with subsequent energy transfer to oxygen; the oxygen then becomes highly reactive in cancer cells and produces reactive oxygen species (ros). the resulting damage to organelles within malignant cells leads to tumor ablation. sdt uses an agent that is sensitive to ultrasound, allowing deeper penetration and destruction of abnormal cells. changes in human melanoma cells were evaluated using fluorescence microscope and atomic force microscopy.we focused on obtaining pictures of the topography and pictures involving elastic properties of cell surface. the production of ros was investigated with the molecular probe cm-h dcfda, and morphological changes in cells were evaluated using fluorescence microscope. the quantitative ros production changes in relation to phthalocyanine concentration, irradiation doses and ultrasound intensity were measured by a fluororeader. activity correlation imaging: visualizing function and structure of neuronal populations s. junek , t.-w. chen , m. alevra , d. schild department of neurophysiology and cellular biophysics, university of göttingen, germany, dfg research center for molecular physiology of the brain (cmpb), university of göttingen, germany understanding a neuronal network relies on knowledge about both the function and the structure of its neurons. while he simultaneous observation of hundreds of neurons is possible by densely staining brain tissue with functional dyes, the low contrast of these stainings does not allow the identification of neuronal processes from the raw fluorescent images. however, as neurons are known to exhibit complex temporal patterns of activity, fluctuations in signal intensity over time could be exploited to generate contrast in densely stained tissue. we demonstrate that the uniform calcium signals within individual neurons of the olfactory bulb can be exploited to visualize the morphology and projection patterns of these cells in tissue slices. as different neurons exhibit distinct time patterns, it is possible to generate a highcontrast multi-color visualization of the network's active neurons, solely based on the specificity of temporal fluctuations of a single-color calcium dye. it is thus possible to use other spectral channels for additional labelling, for example using cell-type or protein specific markers. the ability to map function and structure of neuronal populations online opens up a number of intriguing applications, such as selecting cells or cell pairs with certain functional or projection profiles for targeted recordings, ablation or stimulation. -live cell imaging - investigation of the dynamics of redox elements in live cells by using fluorescence ratio microscopy g. maulucci , g. pani , v. labate , m. mele , e. panieri , m. papi , g. arcovito , t. galeotti , m. de spirito istituto di fisica, università cattolica s. cuore, roma, italy, istituto di patologia generale, università cattolica s. cuore, roma, italy oxidative stress or signaling events can affect cellular redox environment, which act simultaneously as regulator and indicator of key cellular functions in both physiological and pathological settings. by using a redox-sensitive protein (rxyfp), employed ratiometrically, it is possible to generate high resolution redox maps of cells. the spatial distributions of oxidized and reduced elements have been discriminated in human embryonic kidney cells by the deconvolution of the histograms of redox maps. by transfecting cell with glutaredoxin v (grx-v), a significant shift towards more reduced state with respect to that recovered from non-transfected cells is observed. despite such large differences, a common behaviour in the spatial distribution of reduced and oxidized couples can still be observed: oxidized population shows a pronounced localization on the cell borders, near the plasma membrane, while reduced population appears as a collection of well separated spots homogeneuosly distributed thoroughly the inner part of the cell with a mean dimension of um. furthermore we observe that the role of grx-v consists in causing a shift towards reduced values of the highly reduced region, while leaving unaltered the redox-balance of the intracellular side of the plasma membrane. fluorescence resonant energy transfer (fret) is a popular approach to studying molecular interactions. fluorescence lifetime imaging (flim) provides a robust read-out of fret but has largely been restricted to fixed cells owing to relatively long acquisition times. we present a highspeed wide-field multidimensional fluorescence microscope for flim fret in live cells on second timescales for high content analysis and studying fast dynamics. the system uses a nipkow disc for optical sectioning and a time-gated image intensifier for wide-field flim. a spectrally selected supercontinuum excitation source facilitates versatile realtime flim of live cells transfected with fluorescent proteins. for cell signalling studies we have developed a multiplexed fret approach. ras activation at the plasma membrane is demonstrated using flim to read out tagrfp-raf rbd interaction with mplum-h-ras. simultaneously, a spectral ratiometric read-out is used for a second fret (cfp/yfp cameleon) probe to monitor the downstream calcium flux. to further develop multiplexed fret as a tool for imaging multiple components of cell signalling networks, we are working to include polarisation-resolved imaging for steady-state and time resolved fluorescence anisotropy measurements. monitoring gene expression via novel nucleic acid and delivery methods k. lymperopoulos , c. spassova , a. seefeld , h. s. parekh , d. p. herten bioquant institute,heidelberg university, germany, school of pharmacy, university of queensland, australia application of single-molecule and high-resolution fluorescence methods to monitor gene expression in living cells increase the demand on novel probes and delivery methods.they require fluorophores with high photostability and quantum yield and highly-efficient delivery methods that ensure the minimum interference with cell processes such as metabolism and signal transduction. we used a novel class of dendrimers with varying generations and branching factors and different number of positive charges due to different moieties and functional groups.these different properties were tested for their efficiency to transfect eukaryotic cells with fluorescent oligonucleotides (odns).different parameters (temperature,concentration of dendrimers, ratio of dendrimers and odns) were evaluated and optimised.we utilised these established optimal conditions to deliver a modified concept of smartprobes to mammalian cells targeting mrnas involved in signal pathways.we tested different mrna targets and we optimised the fluorescence signal by varying a range of parameters,namely the fluorescent label and the intrinsic properties of the smartprobe (length of the loop and stem, conformation and number of guanosines).in the near future,we plan to use these probes for monitoring gene expression levels using diffusion imaging microscopy. we present a novel near-field scanning microwave microscope (smm) capable of providing surface impedance measurements of samples with nanometric resolution. the instrument is the integration of a microwave vector network analyzer (vna) and a scanning probe microscope (afm/stm). a key point is that our software, controlling and synchronizing both the instruments, creates simultaneously images of the sample at several frequency points. this can be used to extract several features of the sample depending on the frequency. moreover, close frequencies show the same features, added to random noise. exploiting this redundancy of information, we have achieved remarkable results. we have been working on the optimization of this system for biological applications, to detect functional characteristics of cells generating a variation of their dielectric properties. this instrument offers the possibility of performing local impedance measurements on a single live cell and, if correctly calibrated, it provides also quantitative information (e.g. absolute measurements of membrane permittivity). the system was demonstrated to work on saccharomyces cerevisiae. a better model for a full test of the potentialities of the new technique is given by excitable cells, characterized by a greater variability of dielectric properties. a challenging objective could be directly imaging ion channels. hiv- to efficiently complete a replication cycle has to integrate its genome into the host cellular dna.after hiv- enters target cells,neosynthesized viral dna forms along with other proteins the pre-integration complex (pic).pics are then transported into the nucleus where integration,catalyzed by the viral integrase,takes place.hiv- viral particles engineered to incorporate integrase fused to egfp have proven effective to study pics within nuclei of infected cells.in this study we report the live imaging analysis of nuclear pic dynamics obtained by time-lapse microscopy.intranuclear trajectories of in-egfp-labeled pic were collected in three dimensions and examined by both mean squared displacement (msd) and cage diameter (cd) analysis.in cd the maximum distances measured between two positions occupied by a pic in a time window of minutes were calculated while in our msd analysis -minute long trajectory segments were considered.remarkably,msd revealed the presence of an underlying active transport mechanism.to test the possible role of actin filaments,pic nuclear trafficking was analyzed in cells treated with latrunculin b (actin polymerization inhibitor).preliminary results suggest that the disruption of actin function impairs the active nuclear movement of pics. second harmonic generation microscopy reveals sarcomere contractile dynamics of cardiomyocytes n. prent , c. a. greenhalgh , r. o. cisek , j. aus der au , s. elmore , j. h. van beek , j. squier , v. barzda department of physics and institute for optical sciences, university of toronto, toronto, canada, department of physics, colorado school of mines, golden, usa, department of molecular cell physiology, vrije universiteit, amsterdam, netherlands cardiomyocytes, like other striated muscles, exhibit strong inherent second-order nonlinear optical properties that make them exemplar for live cell dynamic studies with second harmonic generation (shg) microscopy. laser scanning shg microscopy with an incident wavelength around µm, enables fast imaging for extended periods of time with negligible tissue damage. strong shg signal originates from the anisotropic (a-) bands which are comprised of regularly arranged myosin molecules, while actin molecules, the main constituent of the isotropic (i-) bands, produce negligible shg. consequently, the alternating bands along the myofibril are clearly visualized, therefore, enabling the determination of individual sarcomere lengths and the study of sarcomere length dynamics during macro-scale contractions. shg intensity is shown to positively correlate to sarcomere length, which leads to the development of real-time inherent force sensors for in vivo myocytes. rich sarcomere dynamics can be observed during myocyte contraction, which can be used for medical diagnostic purpose of muscular degenerative diseases. imaging cellular communication in insulin secretion ex vivo and in vivo d. w. piston vanderbilt university, nashville, tennessee, u.s.a. the islet of langerhans is the functional unit responsible for glucose-stimulated insulin secretion (gsis), and thus plays a key role in blood glucose homeostasis. the importance of the islet is demonstrated by the proven ability of islet transplants to reverse type i diabetes pathologies in human patients. we are interested in understanding the multicellular mechanisms of islet function, and their role in the regulation of blood glucose under normal and pathological conditions. in many ways, the islet appears to function as a syncytium, which exhibits synchronous behavior of membrane action potentials, ca + oscillations, and pulsatile insulin secretion across all β-cells in the islet. in other ways, the islet works as individual cells, especially in the regulation of gene transcription. using our unique quantitative optical imaging methods and novel microfluidic devices, the dynamics of these molecular mechanisms can be followed quantitatively in living cells within intact islets. these investigations utilize transgenic and tissue-specific knock-out mouse models with demonstrated phenotypes, as well as traditional biochemical and molecular biological approaches. do retinal rod outer segments carry out oxydative phosphorylation? i. panfoli , p. bianchini , d. calzia , s. ravera , a. morelli , a. diaspro biology dept., university of genova, italy, lambs-microscobio res.centre, university of genova, italy, neuroscience and brain technology dept., i.i.t. genova, italy visual transduction in vertebrate retinal rod outer segments (os) is very energy demanding. however, atp supply in os, that are devoid of mitochondria, is still puzzling. by a proteomic analysis we identified in purified bovine os disks, proteins involved in vision, as well as the respiratory chain complexes i to iv and f f o -atp synthase, whose activity was comparable to that of mitochondria and sensitive to specific inhibitors. rhodamine fluorescence quenching experiments showed the presence of a proton potential difference across disks. disks consumed oxygen. confocal laser scanning and transmission electron microscopy showed that cytochrome c oxydase and atp synthase are localized on disks. mitochondrial vital dyes stained os ex vivo, and disks. rhodopsin and mitotracker fluorescence co-localized in os. data, suggestive of an aerobic metabolism in os, point to the existence of "mitochondrial inner membrane-like membranes" ectopically producing atp through oxidative phosphorylation, with respect to mitochondria. new scenarios open on the patho-physiology of many retinal diseases associated to mitochondrial dysfunction. -live cell imaging -abstracts unmixing using lifetime-excitation multidimensional confocal fluorescence microscopy data s. schlachter , s. schwedler , a. esposito , a. d. elder , g. s. kaminski schierle , c. f. kaminski cambridge university, u.k., universität bielefeld, germany fluorescence imaging provides a powerful tool to probe biological systems, enabling the high resolution investigation of the localization, interaction and biochemical modification of biomolecules. multidimensional imaging, in particular, is a growing application of confocal and other forms of fluorescence microscopy. it seeks to measure not only fluorescence intensity in three spatial dimensions, but also other features of fluorescence emission, such as lifetime and fluorescence spectra. although multi-dimensional fluorescence microscopy has been demonstrated before, little attention has so far been paid to the problem of data interpretation and representation when dealing with such large datasets. we present here an instrument capable of recording fluorescence lifetime and excitation data by combining tcspc for lifetime determination and a supercontinuum excitation source for extracting excitation spectra. these technologies permit the acquisition of d and d images with lifetime and excitation spectrum information at every pixel. we demonstrate a method whereby the multidimensional datasets acquired with this instrument are processed to yield biologically relevant parameters, (e.g. unmix fluorophores in a multiply labelled sample). our method relies on a global analysis approach and uses ab plots for displaying multidimensional data. we demonstrate the instrument & processing method on dye and multiply labelled biological samples. advanced neuroimaging with diffractive optical elements p. saggau baylor college of medicine, houston, texas, usa studying neural systems is complicated by the large number and small size of its cellular elements. the traditional way of exploring neuronal function by electrical monitoring with micropipettes is increasingly replaced by molecular imaging. fluorescent molecules allow optical monitoring of neuronal signaling with cellular and often subcellular resolution. in addition to monitoring activity, analyzing the functional properties of individual neurons or neuronal populations requires their controlled activation. optical approaches are well-suited, including molecular photolysis of inert precursors and light activation of engineered proteins that control ionic membrane currents. to fully utilize optical techniques for exploring neural systems requires more than adequate spatial resolution to distinguish neuronal elements and sufficient temporal resolution to induce and/or monitor neuronal signaling. because of the non-linear and non-stationary nature of the studied system it is necessary to access many neuronal sites simultaneously. in modern imaging, wide-field illumination and imageformation are often replaced by patterned excitation and nonimaging photon collection. in many instants, diffractive illumination schemes can substitute for conventional reflective or refractive designs. in particular, the availability of programmable diffractive optical elements has made this an attractive alternative, since they permit highly versatile microscope designs and support a significant increase in the overall spatio-temporal resolution. i will present an advanced imaging approach using diffractive optical elements to analyze structure and function of live neurons in brain slices and intact cortex. efficient evaluation of fret in image cytometry with acceptor photobleaching and ratiometric methods j. roszik, d. lisboa, j. szöllősi, g. vereb department of biophysics and cell biology, university of debrecen, debrecen, hungary fluorescence resonance energy transfer (fret) is a powerful technique that can be applied to study nanoscale intra-and intermolecular events and interactions of molecules in situ in biological systems. a robust, easy to use, self-controlled fret method, independent of donor and acceptor concentration and stochiometry, is acceptor photobleaching fret, which requires only simple image mathematics. another approach with more complicated calculations is the intensitybased ratiometric method, which is not based on destroying the acceptor fluorophores, making it applicable for following molecular interactions in live cells. as the need for using fret in image cytometry for evaluating molecular interactions increases, we have undertaken to develop softwares for these methods involving the calculation and usage of all correction factors needed to obtain reliable energy transfer efficiencies. correction possibilities of the acceptor photobleaching method include unwanted photobleaching of the donor, fluorescent photoproduct of the acceptor after photobleaching, cross-talk of unbleached acceptor into the donor channel and partial photobleaching of the acceptor. in the case of intensity-based ratiometric fret, we can correct for all channel cross-talks and calibrate the fret efficiency calculations. both programs provide registration and semiautomatic processing, and they are freely available. the pendrin (slc a ) gene is responsible, when mutated, for the pendred syndrome, a recessive disorder characterized by sensorineural hearing loss often accompanied by thyroid disfunctions. pendrin is an anion exchanger. the way it works and its role in different tissues, owing to the lack of known isoforms, is matter of wide research and debate. we focused on a still unexplored pendrin function, that is most important in the inner ear: cellular volume control and cl − fluxes regulation. we used hek cells over-expressing wild type pendrin or a mutated isoform together with the eyfp. we challenged cells with hypo-osmolar solutions and followed their volume variations in time. taking advantage of the confocal optical sectioning we independently measured cell volume and fluorescence intensity. in this way, given the dependence of eyfp fluorescence from [cl − ] and ph (measured with snarf ) we could estimate at the same time cl − fluxes, volume and ph variations. the contemporary measurements of the three variables, not yet reported in living cells, allowed to assess the role of pendrin in volume regulation and evidenced its participation to cl − fluxes as compared to the mutated isoform or controls. particle tracking inside the cell largely benefits of the ability to spatially and temporally mark specific structures to follow their "signalling" over a "dark" background as made possible since the advent of the photo-activatable markers. in terms of spatial confinement of the photo-activation process, the use of multiphoton excitation provides several favourable aspects compared to single photon confocal microscopy in photomarking biological structures to be tracked: the confined excitation volumes, of the order of magnitude of subfemtoliter, due to the non-linear requirements provide a unique control of the excitation and consequently photoactivation in the d space. in this context photoactivation experiments can be used to assess quantitative information about the binding kinetics of a macromolecule expressed in different cellular compartments. in this work we extended to photoactivation procedures and models originally developed for the quantitative analysis of frap experiments and we evaluated, for different proteins of medical interest (rac-pagfp), the diffusive behaviour in the cytoplasm and the binding kinetics at the large endosomes. the results are compared with standard photobleaching experiments, in order to evidence the gained sensitivity obtained with photo-activatable proteins. motile plaques involved in stress fiber assembly revealed by high-speed spm for living cells k. tamura, t. mizutani, h. haga, k. kawabata division of biological sciences, graduate school of science, hokkaido university, kita-ku, sapporo, japan stress fibers, which are contractile actin cables aligned in a highly-ordered manner, are important for cell migration, mechanical support of plasma membranes and extracellular matrix organization. although stress fibers are dynamically disassembled and assembled again in cells, it is poorly understood how actin filaments are organized into cables with highly-ordered alignment for stress fiber assembly. for investigation of actin cytoskeletal dynamics during actin cable formation, we performed time-lapse observation of actin cytoskeleton in lamella of living fibroblasts by using scanning probe microscopy (spm). high-speed spm observation revealed that motile plaques defined front-side ends of new actin cables and that preexisting mesh-form actin networks were remodeled into new actin cables. directional order analysis of movement of plaques and pharmacological experiments clarified that plaques were driven by myosin-ii-based retrograde actin flow, indicating that plaques are associated with actin cytoskeleton. immunofluorescence experiments showed that plaques were localized on foci of vinculin, a component of cell-substratum adhesions, suggesting that plaques can bind to extracellular substrata via vinculin. based on these results, we propose a model for actin cable formation that motile plaques initiate remodeling of preexisting actin networks into actin cables aligned in a direction of actin flow by associating with extracellular substrata. abscisic acid (aba) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. recently, aba was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells and by human and murine pancreatic β cells. aba autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin (ptx)-sensitive receptor/g-protein complex, cyclic amp, cd -produced cyclic adp-ribose and intracellular calcium. here, the aba receptor on human granulocytes and on rat insulinoma cells is identified as the lanthionine synthetase c-like protein lancl . co-expression of lancl and cd in the human hela cell line reproduces the aba-signaling pathway. the ptx-sensitive g protein coupled to lancl is identified as g i by transfection of cd + /lancl + hela with a chimeric g protein (gα q/i ). identification of the mammalian aba receptor will enable the screening of synthetic aba antagonists as prospective new anti-inflammatory and anti-diabetic agents. investigation of post-thaw damage of s.cerevisiae yeasts using fluorescent dyes -dab and -dab t. s. dyubko , i. a. buriak , v. d. zinchenko , i. f. kovalenko state scientific institution "institute for single crystals", national acadey of sciences of ukraine, kharkov, ukraine, institute for problems of cryobiology and cryomedicine, national academy of sciences of ukraine, kharkov, ukraine we investigate applicability of the fluorescent probes -dab and -dab available from seta biomedicals (www.setabiomedicals.com) to study the post-thaw damage of saccharomyces cerevisiae yeast cells. the leaving cells stained with these dyes have bright fluorescence in the yellow and red region, respectively. however, the freezing followed by post-thawing causes cell damage, which results in a change of fluorescence intensity. in the native living cells the dyes are preferably localized in the cell membranes and organelles which are highly fluorescent. partially damaged cells have even brighter fluorescence as compared to the living cells. however, their cell ultra-structure is not well-distinguished in the fluorescence mode. ultimately destroyed cells are almost non-fluorescent: the cell membranes are not visible in the fluorescent mode and their organelles are only weakly fluorescent. the number of intensively fluorescing cells with partially destroyed membranes, which were obtained by freezing to - • c, is lower compared to those frozen at - • c. -dab and -dab enable not only to distinguish damaged and undamaged cells, but also allow quantitative estimation of the extent of damage in membranes by cryogenic effects. the thylakoid membrane is a structured network in higher plants which is organised into stacked granal thylakoids that are interconnected by single 'stromal' lamellae. we have studied the mobility of a resident protein of the stromal lamellae, hcf , part of the tat protein translocase. hcf -green fluorescent protein fusion (gfp) was targeted into thylakoids and studied using photobleaching approaches. we show that small regions fail to recover significant levels of hcf -gfp fluorescence within minutes after photobleaching. autofluorescence from the photosystem ii light-harvesting complex (lhcii) in granal stacks likewise fails to recover over this time scale. although the thylakoid membrane is a single continuous entity, these data show that both hcf -gfp and lhcii are constrained within this network. since the hcf homologue, tatb, is highly mobile in the escherichia coli plasma membrane, we believe that the stromal lamellae take the form of distinct domains that are effectively constrained by boundaries within the thylakoid network. cytomechanical modifications induced by drugloaded carriers uptake by breast cancer cells the novel opportunities offered by the nanotechnologies have attracted great interest in the development of novel biomaterials for targeted drug delivery in cancer research. the desired features of pharmaceutical drug delivery for intravenous administration are their small size, biodegradability, high drug content, prolonged circulation in the blood and the ability to target required areas. in this work we have compared efficacy of different type of carriers having complementary properties for pharmaceutical delivery in cancer therapy. drug nano-colloids encapsulated by combination of layer by layer (lbl) techniques and ultrasonication, phytochemical encapsulated artificial oleosomes and drug-loading clay/carbon nanotubes have been used for uptake into breast cancer cells. analysis of viscoelastic response of neoplastic cells induced by cargo-carriers uptake has been carried out by a combination of high resolution optical and scanning force microscopy techniques. furthermore, the effects of drug-reservoir carriers on the cytoskeleton (re)organisation of neoplastic cells were further investigated by confocal microscopy using different fluorescent probes. mapping diffusion by raster image correlation spectroscopy (rics) with analog detection time lapse and flow cytometry data integrated in a common cell cycle proliferation model p. ubezio, v. colombo, m. lupi, f. falcetta istituto di ricerche farmacologiche "mario negri", milano, italy we present a cell cycle simulation tool, connecting the basic proliferation process to the cell population data obtained by time lapse and flow cytometry. the computer program is a general framework within which cell cycle progression can be interactively modeled at the desired level of complexity, including g /s/g m cell cycle phases with variable duration, g phase, cell subpopulations belonging to different generations or differentiation stages, distinct block and cell death parameters for each phase. in this way, we achieved a detailed rendering of cell proliferation of normal or tumor cell populations, additionally including cytostatic and cytotoxic effects of treatments. the program gives as output simulated measures, reproducing those obtained by absolute cell counting, growth inhibition tests, flow cytometric dna histograms and cell cycle percentages, pulse continuous-labeling studies, time-lapse intermitotic times and generation-wise analyses. each technique provides a piece of information related to the underlying proliferation process, catching only some of the phenomena in play, and the measures often cannot be univocally interpreted. we used the cell cycle simulator to fit together time lapse and flow cytometry time courses measures, to achieve a detailed reconstruction of the cell cycle proliferation of an ovarian cancer cell line in vitro after x-ray exposure. -live cell imaging - detection of functional modes in protein dynamics j. s. hub , b. l. de groot deptartment of cell & molecular biology, uppsala university, sweden, computational biomolecular dynamics group, max-planck-institute for biophysical chemistry, göttingen, germany proteins frequently accomplish their biological function by collective atomic motions. yet the identification of a collective motions related to a specific protein function from, e.g. a molecular dynamics trajectory, is often non-trivial. here, we propose a novel technique termed`functional mode analysis' that aims to detect the collective motion that is directly related to a particular protein function. based on an ensemble of structures, together with an arbitrary`functional quantity' that quantifies the functional state of the protein, the method detects the collective motion that is maximally correlated to the functional quantity. the functional quantity could, e.g., correspond to a geometric, electrostatic, or chemical observable, or any other variable that is relevant to the function of the protein. two different correlation measures are applied. first, the pearson correlation coefficient that measures linear correlation only; and second, the mutual information that can assess any kind of interdependence. detecting the maximally correlated motion allows one to derive a model for the functional state in terms of a single collective coordinate. the new method is illustrated using various biomolecules, including a polyalanine-helix, t lysozyme, trp-cage, and leucine-binding protein. logic estimation of the optimum source neutron energy for bnct of brain tumors boron neutron capture therapy (bnct) is a promising method for treating the highly fatal brain tumor; glioblastoma multiform. it is a binary modality; in which use is made of two components simultaneously; viz. thermal neutrons and boron- . a new concept was adopted for estimating the optimum source neutrons energy appropriate for different circumstances of bnct. four postulations on the optimum source neutrons energy were worked out, almost entirely independent of the rbe values of the different dose components. four corresponding conditions on the optimum source neutrons energy were deduced. an energy escalation study was carried out investigating different source neutron energies, between . ev and . mev. mcnp b monte carlo neutron transport code was utilized to study the behavior of these neutrons in the brain. the deduced four conditions were applied to the results. a source neutron energy range of few electron volts (ev) to about kev was estimated to be optimum for bnct of brain tumors located at different depths in brain. the results were discussed. v. l. cruz, j. ramos, j. martinez-salazar instituto de estructura de la materia. csic cannabinoid receptors are an important class of g protein coupled receptors. in particular cb and cb have received considerable interest because they mediate a variety of physiological responses in the central nervous and immune systems. their tertiary structure is still unknown. experimental information suggests the presence of both, an active and an inactive conformation. cb and cb share a common structural framework consisting in a seven transmembrane α-helix bundle connected by three extracellular and three intracellular loops. however, the knowledge of structural differences between both receptors may serve to design new ligands to activate/deactivate selectively only one of those receptors. in the present research, we report a multi-nanosecond molecular dynamics simulation of both receptors in solution, starting from a structure obtained by homology modelling using the x-ray determined bovine rhodopsin protein. we look for differences in the behaviour of cb and cb during the simulation process to shed light about those structural features which can be important for ligand selectivity. in this sense, we observed that cb tend to present a more flexible and opened helix bundle than cb . thus, it is expected than the former receptors would present less steric hindrance for ligand binding. we expect these results will be useful to design more selective ligands. self-assembly and equilibration of bola-lipids membranes studied by molecular dynamics m. bulacu, s. j. marrink groningen university, the netherlands bola-lipids consist of two monopolar, twin-tailed lipids that are held together by chemical linkage between one or both ends of the tails from one lipid and the corresponding ends from the other one. membranes formed by these lipids or by their mixtures with monopolar lipids are known to have additional mechanical stability while retaining membrane fluidity. this is traditionally attributed to the fact that, in the membrane phase, the bola-lipids have predominantly spanning configuration (the two polar heads are positioned at opposite membrane-water interfaces) in detriment to looping configuration (both head groups are located in the same membrane-water interface). we perform molecular dynamics simulations, using the coarse grained martini forcefield. we start with self-assembly simulations of bola-lipids followed by bilayer equilibration. an artificial pore is created in the membrane that significantly increases the flip-flop mobility of the lipids and hasten equilibration. the membrane properties are characterized (area per lipid, thickness, order parameter, pressure profile) with emphasis on the spanning/looping ratio. our study can help designing new artificial membranes, with higher stability under extreme conditions. -multiscale simulation - evidence for proton shuffling in a thioredoxinlike protein during catalysis d. narzi , s. w. siu , c. u. stirnimann , j. p. grimshaw , r. glockshuber , g. capitani , r. a. böckmann theoretical and computational membrane biology, center for bioinformatics, saarland university, germany, institute of molecular biology and biophysics, eth zürich, switzerland, structural and computational unit, embl, heidelberg, germany, paul scherrer institute, villigen psi, switzerland proteins of the thioredoxin (trx) superfamily catalyze disulfide-bond formation, reduction and isomerization in substrate proteins both in prokaryotic and in eukaryotic cells. all members of the trx family with thioldisulfide oxidoreductase activity contain the characteristic cys-x-x-cys motif in their active site. here, using poisson-boltzmann-based protonation-state calculations based on -ns molecular dynamics simulations, we investigated the catalytic mechanism of dsbl, the most oxidizing protein known to date. we observed several correlated transitions in the protonation states of the buried active-site cysteine and a neighboring lysine coupled to the exposure of the active-site thiolate. these results support the view of an internal proton shuffling mechanism during oxidation crucial for the uptake of two electrons from the substrate protein. intramolecular disulfide-bond formation is probably steered by the conformational switch facilitating interaction with the active-site thiolate. a consistent catalytic mechanism for dsbl, probably conferrable to other proteins of the same class, is presented. our results suggest a functional role of hydration entropy of active-site groups . the role of water in zn(ii)-abeta( - ) complexes beta-amyloid (aβ) peptides are the main component of amyloid fibrils detected in the brain of alzheimer patients. fibrils display an abnormal content of cu and zn ions whose binding to aβ-peptides has been recently studied by x-ray absorption spectroscopy [ ] and interpreted in terms of ab initio simulations. in order to perform such simulations it is of the utmost importance to find a compromise between the need of having a realistic description of the actual physical system and the difficulty of dealing with too many atoms and electrons. what is usually done is removing solvent (water molecules), thus studying the system in the so called "gasphase". in this work we investigate the relevance of water in the zn-aβ − coordination mode. relying on a combination of classical [ ] and quantum chemistry [ ] methods we find a significant difference in the zn coordination geometry depending on whether water is present or not. this information is exploited in building a full model system for subsequent car-parrinello simulations where two aβ peptides in water are in interaction in the presence of zn. [ although experiments which can directly probe the mechanical properties of proteins have only been performed recently, it is clear that the complex folds of polypeptide backbones lead to very heterogeneous mechanical behavior. this heterogeneity is likely to play an important role in protein function and interaction and it would be useful to be able to predict what mechanical (and dynamical) properties will result from a given structure. we have been using coarse-grain elastic network models to investigate this question and have found that these models are able to link specific mechanical properties to a number of functional features including enzymatic active sites, folding nuclei and changes in behavior due to point mutations. these models are also adapted to looking at complex formation between proteins and how specific recognition is achieved, while being fast enough to be applied to very large numbers of interactions. refinement of protein model structures using biasing potential replica exchange simulations s. kannan, m. zacharias school of engineering and science, jacobs university bremen, d- bremen, germany comparative protein modeling of a target protein based on sequence similarity to a protein with known structure is widely used to provide structural models of proteins. frequently, the quality of the target-template sequence alignment is non-uniform along the sequence: parts can be modeled with a high confidence, whereas other parts differ strongly from the template. in principle, molecular dynamics (md) simulations can be used to refine protein model structures but it is limited by the currently accessible simulation time scales. we have used a recently developed biasing potential replica exchange (bp-rex) md method (kannan, s. zacharias, m. proteins , , - ) to refine homology modeled protein structure at atomic resolution including explicit solvent. in standard rex-md simulations several replicas of a system are run in parallel at different temperatures allowing exchanges at preset time intervals. in a bp-rexmd simulation replicas are controlled by various levels of a biasing potential to reduce the energy barriers associated with peptide backbone dihedral transitions. the method requires much fewer replicas for efficient sampling compared with standard temperature rexmd. bp-rexmd simulations on several test cases starting from decoy structures deviating significantly from the native structure resulted in final structures in much closer agreement with experiment compared to conventional md simulations. m. s. p. sansom, p. j. stansfeld, c. l. wee, k. balali-mood department of biochemistry, university of oxford, u.k. coarse-grained molecular dynamics simulations may be used to probe the interactions of membrane proteins with bilayers and their component lipids on an extended (∼ µs) timescale ( ) . conversion to atomistic resolution allows more detailed protein/lipid interactions to be examined. this multiscale approach will be examined via three examples: (i) interactions of a small ion channel toxin (vstx ) we present a new competitive approach for the treatment of biomolecular flexibility to provide an alternative to the limitations of current methodologies such as molecular dynamics and normal mode analysis. this method, called static mode method, is based on the "induced-fit" concept and is aimed at mapping the intrinsic deformations of a biomolecule subject to any external excitations: direct mono or multi-site contact, electrical etc... the algorithm allows obtaining a set of deformations, each one corresponding to a specific interaction on a specific molecular site, in terms of force constants contained in the energy model. such a process can be used to explore the properties of single molecular intrinsic flexibility, as well as to predict molecular docking or molecule/surface interactions. from a modelling point of view, the interaction problem can be expressed in terms of reactive sites between the interacting entities, the molecular deformations being extracted from the pre-calculated static modes of each separated ones. the first applications of our method have focused on the intrinsic flexibility of biomolecules like nucleic acids and proteins. more recently, this new methodology allowed us to investigate the folding of the region - of the amyloid β-peptide, via the docking of a zinc ion on the reactive sites of the molecule. conformational study on a myelin basic protein fragment: molecular dynamics simulations in membrane e. polverini , g. harauz dipartimento di fisica, università di parma, parma, italy, department of molecular and cellular biology, university of guelph, guelph, ontario, canada myelin basic protein (mbp) is a multifunctional protein of the central nervous system whose principal role is in maintaining the compactness and integrity of the myelin sheath, the multilamellar membrane wrapped around nerve axons. however, mbp also interacts with other proteins such as cytoskeletal and signalling proteins, adapting its structure to the different roles. mbp is a candidate autoantigen in the human demyelinating disease multiple sclerosis. this study investigated at atomic detail the conformation of a highly conserved central fragment of mbp, constisting of two consecutive regions with different relevant functionalities. the first one is associated with the membrane and comprises the primary immunodominant epitope in multiple sclerosis; the second one was predicted to be a ligand for sh -domains of signalling proteins. molecular dynamics simulations were perfomed in the presence of dodecylphosphocholine micelle, starting from a structure extrapolated from experimental data (harauz and libich, curr. protein pept. sci., ). the results confirm the experimetal hypothesis, showing, in the micelle, a stable alpha-helix anchored to the membrane for the first region and, for the proline-rich second one, a poly-proline type ii helix pointing outwards, ready to interact with the signalling proteins. multiresolution modelling of drug and hormone permeability through a lipid bilayer traditional atomic-level (al) modelling of biomembranes is time-consuming, and hence limited in the range of systems and phenomena that can be simulated. to alleviate this problem, we designed a coarse-grain (cg) representation where each lipid molecule, in reality consisting of more than atoms, is modelled with only cg sites. our cg technique proves two orders of magnitude less demanding of computational resources than traditional al methodology. a unique feature of our approach is that the cg potentials are directly compatible with standard al models, thus facilitating the simulation of multiresolution systems, where the "chemically-sensitive" components (e.g., the solutes in membrane permeation studies) are modelled atomistically, while the surrounding environment is coarse-grained. in this contribution, we present a summary of our multiscale methodology, together with its application to the permeation of large molecules -drugs and steroid hormones. the calculated permeabilities are compared to the available experimental measurements and al simulation data. molecularlevel insights regarding the permeation mechanism are obtained and rationalised. -multiscale simulation - the processes of life involve a variety of events that occur on different scales, ranging from a fewÅ/ps of the triggering steps of the biochemical reactions, up to their macroscopic effects in cells and organs. intermediate steps involve the structural rearrangement of the bio-molecules (∼nm/ - ns), their aggregation, folding (∼ nm-µm/ µs-ms), internal cell diffusion and dynamics (µm-mm/ms-hours), evidently requiring a multi-scale modeling approach. here the multi-scale approaches are first briefly illustrated with a particular attention to the issue of matching the different resolutions, which is essential to achieve a coherent descripition. the focus is then fixed on the coarse grained (cg) models, typically spanning the nm-µm and µs-ms scale, and in particular on their minimalist -simplest and computationally cheapest -versions [ , ] . a parameterization strategy that combines accuracy and predictive power within these models is presented here and applications are shown to relevant cases including the proteins involved in the hiv replication, the green fluorescent proteins and examples of macromolecular complexes. [ ] controlled degradation of collagen is an important process in tissue remodeling and wound healing. collagenase cleaves fibrillar collagens about three quarters of the distance from the amino-terminus. even though the determination of the cleavage site and the collagenase structure took place decades ago, the mode of action of collagenase on collagen is not clear. to understand the mechanism of collagenase activity on collagen, the structure, stability and dynamics of collagen, its conformation around the cleavage site and the possibilities of conformational rearrangements between the two domains in collagenase was explored using md simulation. the results of principal component (pca) and normal mode (nm) analysis of the collagen and collagenase suggests that the c-terminal domain of collagenase recognizes the collagen, and then the n-terminal catalytic domain undergoes rearrangement on the substrate (with the help of linker regions). the sda software for carrying out brownian dynamics simulations of protein-protein diffusional association with the help of biochemical constraints is being used to predict the mode of interaction of collagen and collagenase. different conformations obtained from pca and nm analysis are being used for the docking. the predicted structures of the complex will help us to understand how collagenase recognizes and binds collagen specifically. acknowledgment: daad, germany, csir-srf india, and the klaus tschira foundation. membrane aoration by antimicrobial peptides combining atomistic and coarse-grained descriptions d. sengupta, a. j. rzepiela, n. goga, s. j. marrink university of groningen, the netherlands antimicrobial peptides (amps) comprise a large family of peptides that include small cationic peptides, such as magainins, which permeabilize lipid membranes. previous atomistic level simulations of magainin-h peptides show that they act by forming toroidal transmembrane pores. however, due to the atomistic level of description, these simulations were necessarily limited to small system sizes and sub-microsecond time scales. here, we study the long-time relaxation properties of these pores by evolving the systems using a coarse-grain (cg) description. the disordered nature and the topology of the atomistic pores are maintained at the cg level. the peptides sample different orientations but at any given time, only a few peptides insert into the pore. key states observed at the cg level are subsequently back-transformed to the atomistic level using a resolutionexchange protocol. the configurations sampled at the cg level are stable in the atomistic simulation. the effect of helicity on pore stability is investigated at the cg level and we find that partial helicity is required to form stable pores. we also show that the current cg scheme can be used to study spontaneous poration by magainin-h peptides. over-all, our simulations provide a multi-scale view of a fundamental biophysical membrane process involving a complex interplay between peptides and lipids. the varkud satellite (vs) ribozyme is the largest of the nucleolytic ribozymes, and the only one for which there is no crystal structure. we have determined the overall architecture of the complete vs ribozyme using small-angle x-ray scattering in solution. the substrate stem-loop docks into the tertiary fold of the ribozyme, allowing an intimate loop-loop interaction to occur. this brings two key nucleobases a and g into close proximity of the scissile phosphate, and we believe that these nucleobases are involved in general acid-base catalysis of the phosphoryl transfer reactions. this is supported by functional group substitution, and the ph dependence of the reaction rate for the natural and modified rna. although possessing totally different folds, the functional elements of the vs and hairpin ribozymes are topologically and mechanistically very similar if not identical. this has probably arisen by convergent evolution. other nucleolytic ribozymes have diversified to employ hydrated metal ions and even small molecules to participate in genera acid-base catalysis. by contrast, the larger ribozymes seem to have adopted a different, metalloenzyme, catalytic strategy. why these different groups have evolved different catalytic chemistries is an interesting challenge to our understanding of biocatalysis. comparison of dna and sirna binding and nuclease protection by non-viral vectors for gene delivery j. lam, k. witt, a. j. mason, l. kudsiova, m. j. lawrence pharmaceutical science division, king's college london, uk the biggest obstacle for the success of gene therapy is delivery. with the discovery of rnai, the use of sirna to regulate gene expression as potential therapy has attracted much attention recently. in contrast to dna, sirna delivery does not require nuclear entry. with one less barrier to overcome, the delivery of sirna seems to be easier. it is frequently assumed that comparable delivery strategy could be used for both dna and sirna. in fact the two types of nucleic acids are fundamentally different with distinct properties, which impact their delivery strategies. in the present study it was found that most non-viral delivery vectors including polymers, peptides and lipids were generally more efficient in binding with dna than sirna as shown in gel retardation assay. the inferior sirna binding is possibly due to the rigid structure of sirna, resulting in weaker electrostatic interaction with the cationic vectors. surprisingly, it was observed that all the vectors studied offered better nuclease protection for sirna than dna despite poorer sirna binding. either the nuclease protection for sirna is easier to achieve due to its small size, or the gel retardation assay did not truly reflect the binding efficiency as the weaker sirna complexes may dissociate during electrophoresis. not only the delivery strategy, the results between dna and sirna study must also be carefully adapted and interpreted. single molecule studies of spliceosomal rnas u and u z. guo, d. rueda department of chemistry, wayne state university, detroit, michigan, u.s.a. splicing is an essential step in the maturation reaction of mrna, in which intervening introns from exons. the spliceosome is a dynamic assembly of small nuclear rnas and > proteins that catalyzes splicing. u and u are the only snrnas strictly required for splicing. major conformational changes are expected to take place during spliceosomal assembly and catalysis. we have developed a single-molecule fluorescence assay to study the structural dynamics of a protein-free u -u complex from yeast. our previous data have revealed a -step large amplitude conformational change of the u -u complex. the st step is a mg + -induced conformational change where helix iii and the u -isl are in close proximity in low mg + and separated in high mg + . the nd step corresponds to the formation of the highly conserved helix ib. we now examine the role of the highly conserved bases in the folding dynamics of the u -u complex. the data show that u and the acagag loop play an important role in stabilizing the interaction between helix iii and the u -isl. we hypothesize that u flips out of the u -isl and interacts with the acagag loop to bring them in close proximity. our results raise the interesting possibility that this interaction plays an important role in bringing the ' splice site and the branched a into close proximity of u , which may be critical for catalysis. a structure-based approach for targeting hiv- genomic rna dimerization initiation site s. freisz, s. bernacchi, p. dumas, e. ennifar ibmc -cnrs, strasbourg, france all retroviral genomes consist in two homologous single stranded rnas. hiv- dimerization initiation site (dis) is a conserved hairpin in the ' non-coding region of the genomic rna and essential for viral infectivity. the dis initiates genome dimerization by forming a kissing-loop complex, further stabilized into an extended duplex upon interaction with the ncp nucleocapsid protein. x-ray structures of the dis kissing-loop and extended duplex revealed similarities with the bacterial s ribosomal rna a-site, which is the target of aminoglycoside antibiotics. as a result, aminoglycosides also bind the hiv- dis as shown by our x-ray structures of the dis kissingloop bound to aminoglycosides. using fluorescence, uvspectroscopy and microcalorimetry, we further characterized hiv- dis/aminoglycosides interactions. we found that the affinity of aminoglycosides for the dis was higher than for their natural target, the s a site. they strongly stabilize the dis kissing-loop, blocking its conversion into the duplex form. finally, we also solved x-ray structures of the dis duplex bound to aminoglycosides, revealing an important conformational change following drug binding. these structures show that it is possible to target the hiv- dis dimer before and after the ncp -assisted rna maturation with the same molecule. altogether, our studies create the basis for a rationally driven design of novel potential drugs targeting the hiv- genome. the core protein of hepatitis c virus is a multifunctional protein of aa, consisting of a hydrophilic n-terminal domain with three basic domains (bd -bd ) responsible for the interactions with rna and a hydrophobic c-terminal domain. the n-terminal domain exhibits nucleic acid chaperone properties similar to those of the nucleocapsid protein from hiv. here, we characterized the mechanism of the chaperone properties of a peptide e corresponding to the bd and bd clusters of the n-terminal domain. to this end, we monitored the promotion by this peptide of the annealing of dtar, the dna analogue of the transactivation response element to its complementary sequence, ctar dna, taken as models. the annealing involves two second-order kinetic components that are activated by at least three orders of magnitude by peptide e. this activation was correlated with the ability of peptide e to destabilize the lower half of dtar stem. using, ctar and dtar mutants, the two kinetic components were assigned to two pathways which are connected with the fast annealing of the terminal bases of ctar to dtar and slow extended duplex formation, limited kinetically by the nucleation of central segments of ctar and dtar stems. structure and conformational dynamics of a unique dead box helicase m. rudolph , m. linden , r. hartmann , d. klostermeier hoffmann-la roche, basel, switzerland, biozentrum, univ. of basel, switzerland, univ. of marburg, germany dead box helicases couple atp hydrolysis to rna structural rearrangements. t. thermophilus hera consists of a helicase core and a c-terminal extension (cte) with a putative rnase p motif. crystal structures show that the cte is bipartite, forming a highly flexible dimerization motif with a novel fold and an additional rna-binding module. we provide a first glimpse on the orientation of an rna-binding domain outside the helicase core. in a structure-based model for the complete hera dimer, the rna-binding sites of the helicase cores face each other, allowing for inter-subunit communication. the plasticity of the dimerization motif allows for drastic changes in the juxtaposition of the helicase cores within the dimer. in single molecule fret experiments we identified fragments of the s rrna and rnase p rna as substrates for hera. both substrates switch the hera core to the closed conformation and stimulate the intrinsic atpase activity. rna binding is mediated by the cte, but does not require the putative rnase p motif. atp-dependent unwinding of a short helix in s rrna suggests a specific role for hera in ribosome assembly, in analogy to the e. coli and b. subtilis helicases dbpa and yxin. in addition, the specificity of hera for rnase p rna may be required for rnase p rna folding or rnase p assembly. simultaneous action of two hera subunits on the same rna molecule may be important for efficient rna remodeling in vivo. structural basis for the encapsidation process of turnip yellow mosaic virus m. petersen , j. hansen , s. s. wijmenga nucleic acid center, department of physics and chemistry, university of southern denmark, odense, denmark, physical chemistry/biophysical chemistry, radboud university, nijmegen, the netherlands formation of hairpins with internal loops with c c and c a mismatches in the ' untranslated region is a common characteristic among the plant viruses belonging to the tymovirus genus. turnip yellow mosaic virus possesses two such hairpins, hp and hp . hp , and in particular the presence of the c c and c a mismatches in its internal loop, is important for initiation of the encapsidation of the viral genome. the encapsidation occurs under acidic conditions at the neck of invaginations of the chloroplast membrane. we have now determined the d structures of revertants involved in the evolutionary pathway using nmr spectroscopy. these structures reveal how the grooves in the hairpin become increasingly positively charged in successive generations of evolution. the similarity between the ccca mutant and the wild-type hairpin (hp ) is striking and explains why this mutant gives rise to a viable virus. in addition, a characteristic tilt of the backbone is observed upon occurrence of a protonated c c base pair. both the positively charged major groove and the kink in the rna backbone appear to be crucial for interactions with the viral capsid. at neutral ph, the structure of hp resembles the watson-crick base paired mutant which explains why encapsidation only occurs under acidic conditions. a. percot , j. vergne , m.-c. maurel , s. lecomte ladir, umr , thiais, france, lbeam, upmc, paris, france, cbmn, umr , pessac, france the existence of "rna world" as an early step in the history of life increases the interest for the characterization of these biomolecules. the studied hairpin ribozyme is a self-cleaving/ligating motif found in the minus strand of the satellite rna associated with tobacco ringspot virus. surface-enhanced raman spectroscopy (sers) was successfully used to detect sub-picomole quantities of nucleic acids. sers takes advantage of the strongly increased raman signals generated by local field enhancement near metallic (typically au and ag) nanostructures. sers spectra of dna or rna are strongly dominated by stockes modes of adenine. through an interaction of adenyl residues with silver colloid, adenyl raman signal is times increased compared to raman scattering. in controlled conditions, sers signal is proportional to the amount of free residues adsorbed on the metal surface. upon rna cleavage, residues are unpaired and free to interact with metal. in the present study, we proposed to follow the cleavage reaction of hairpin ribozyme (hpr ) using the sers signal of the liberate adenyl residues. as the sers signal is proportional to the adenyl residues, reactivity of hr was monitored by measuring the raman intensity of the fragment liberated during the cleavage of hairpin. the results obtained were compared with the electrophoresis method performed on the same sample and similar results were obtained. -rna world - eur biophys j ( ) the development of arrays for biomolecular recognition for a broad range of applications in biomedical diagnostics is receiving a constantly increasing attention. the design of efficient protein biochips, however, requires the optimization of protein immobilization protocols for improving the device sensitivity. moreover, innovative platforms for in-vitro detection in highly diluted, small volume samples need to be developed, for which standard micro-fabrication techniques are not suitable. therefore, the development of nano-scale platforms for protein and antibody detection is essential. we report here on a novel approach for the fabrication of multiple protein nanosensors using atomic force microscopy based nanografting and dna-directed immobilization (ddi), which takes advantage of the specific watson-crick hybridization of oligonucleotide-modified proteins to surfacebound complementary oligomers. using nanografting, single-stranded dna nano-structures with well defined local environments were fabricated on a flat surface. dna-protein conjugates were then anchored on the engineered binding sites by ddi in a single chemical operation and detected by the corresponding topographic height increase of the relevant patches. immunological assay were used to demonstrate the biochemical functionality of the immobilized proteins, proving the specificity of biomolecular recognition of our nanodevices, in the micro-molar to pico-molar concentration range. dna accessible surface area and indirect protein-dna recognition: study by bioinformatical approach o. p. boryskina , m. y. tkachenko , a. v. shestopalova , m. y. tolstorukov institute of radiophysics and electronics nas of ukraine, harvard-partners center for genetics and genomics, usa revealing the mechanisms of protein-dna recognition is essential for understanding the regulation of many cellular processes. there is growing evidence that recognition through sequence-specific contacts (direct readout) can be enhanced by recognition via dna sequence-dependent deformability (indirect readout). the role of changes in dna accessible surface area (asa) in distorted dna configurations in complexes with proteins is not fully understood yet, even though such changes and related changes in polarity of dna surface are among key factors of indirect readout. to fill this gap we have developed a publicly-available internet database of protein-dna complexes, which integrates the data on dna conformational parameters with information on asa of dna atoms in the minor and major grooves, protna-asa. the database has been used to analyze the effect of changes in dna backbone configuration on asa of dna atoms in major and minor grooves. we observe that sugar puckering and conformation of torsion angle γ affect the accessibility of dna atoms in both grooves to a noticeable extent. we also report sequence specific preferences of the nucleotides for structural domains of γ. these results can shed new light on the mechanisms of indirect protein-dna recognition. a. arakelyan biology dpt., yerevan state university, yerevan, armenia the influence of ligand, irreversibly binding with dna, on the isotherm of adsorption of ligand binding with dna reversibly has been modeled theoretically. the isotherm of adsorption of etbr on dna in presence of cis-ddpt has been considered at low concentrations of cis-ddpt and etbr.. the isotherm of adsorption of etbr on dna has linear form in scatchard coordinates at low degrees of occupation. the comparison with experimental isotherm permits to estimate the parameters of etbr binding with dna. with taking into account the pseudo-ring structures formation with partially molten regions we consider two types of binding regions, linear and ring at low degrees of occupation. the isotherm of adsorption of etbr on dna and also variation of the number of bounded etbr molecules are calculated with taking into account these two types of binding regions. it was shown that at low concentrations cis-ddp, bounding with dna, changes the isotherm of ligands adsorption. the linear in scathcard coordinates isotherm of adsorption transforms into non-linear isotherm. the degree of transformation depends on the fraction of dna in the ring regions, on the ratio between number of etbr binding cites in the ring and linear regions, and also on the binding constants for these regions. it was shown that low concentrations of cis-ddp affect the dependence of dispersion on the concentration of ligands, changing both the magnitude of maximum and its position. tunable nanoconfinement structures for dna manipulation e. angeli, l. repetto, g. firpo, c. boragno, u. valbusa nanomed labs, advanced biotechnology center and physics department, university of genoa, italy nanostructures, such as nanochannels [ ] or nanoslits [ ] , have been successfully used to confine and stretch dna molecules, offering interesting opportunities of investigation on conformational changes induced by confinement, physical and biological properties, etc. the integration of these nanostructures on lab-on-chip systems has shown their great potential for applications such as dna sieving or single molecule manipulation [ ] , [ ] . arrays of nanochannels fabricated, using a focused ion beam (fib) on a silicon master, are replicated using elastomeric materials, such as poly(dimethylsiloxane) (pdms), and soft-lithography techniques. the cross-section of these flexible polymeric nanoconfinement structures can be reversibly and dynamically tuned, in order to vary biomolecules transport characteristics and confinement conditions of trapped dna molecules. moreover, these nanochannels, with tunable cross-section, are used to study and exploit the sieving mechanism of "entropic recoil" [ ] for the separation of long dna chains. [ ] mannion jt, et al., ( ) biophys. j., , : - . [ ] jo k, et al., ( ) pnas, , : - . [ ] fu j, et al., ( ) nat. nanotechnol., : - [ ] huh d, et al., ( nat. mater., : - . the nucleocapsid protein ncp of hiv- is characterized by two conserved zinc fingers and plays crucial roles in the virus, through its binding to nucleic acids. ncp is required for efficient proviral dna synthesis, by promoting the initiation of reverse transcription and the two obligatory strand transfers. using fluorescence techniques as well as fcs and nmr, we investigated the chaperone properties of ncp on the primer binding sites (pbs) and transactivation response elements (tar) sequences involved in the two obligatory strand transfers. we showed that ncp binds mainly to the (-)pbs loop, which results in an extension of the loop and a destabilization of the upper base pair of the stem. these structural changes chaperone a kissing complex with the complementary (+)pbs loop and its further conversion into an extended duplex. in contrast, the ncp -promoted annealing of ctar-tar results from the destabilization of the bottom of ctar stem, which favors the invasion of the tar stem. by developing new fluorescence methodologies to site-specifically characterize these interactions, we further showed that ncp slows down the ps to ns conformational fluctuations of its nucleic acid targets and freezes the local mobility of the bases contacted by the zinc fingers. exploring dna orientation in flow e. l. gilroy, m. r. hicks, a. rodger department of chemistry, university of warwick, uk dna is one of the most important biomolecules. in order to undertake its biological role the dna needs to fold and unfold for which its structure and flexibility are crucially important. we have studied the characteristics of dna in flow to probe its structure. flow aligned linear dichroism (ld) is a technique that uses light polarised parallel and perpendicular to an orientated sample. it can be used to measure how aligned a sample is, and the orientation of any interacting molecules. to create this alignment, the sample is placed between two concentric cylinders where one is spun to create a shear flow. the longer and more rigid the molecule is the better the orientation and the signal. as the sample is in flow there are other factors than need to be considered when using ld. these include viscosity and temperature. there are many methods to measure the viscosity of a sample of dna at varying temperatures. these include viscometer, rheometer and dynamic light scattering. the effects temperature has on viscosity and the sample itself need also to be considered. the findings of all these studies have been reported and show the significance that viscosity and temperature have on dna ld measurements. possible applications of using ld to study dna have also been discussed, showing the importance of the use of ld and in the results shown. a.-m. florescu, m. joyeux laboratoire de spectrométrie physique, université joseph fourier grenoble , france we present a dynamical model for simulating non-specific dna protein interactions, which is based on the "beadspring" model for dna with elastic, bending and debye-hückel electrostatic interactions, and where the protein interacts with the dna chain through electrostatic and excluded-volume forces. we study the properties of this model using a brownian dynamics algorithm that takes hydrodynamic interactions into account and obtain results that partially agree with experiments and predictions of kinetic models. for example, we show that the protein samples dna by a combination of three-dimensional diffusion in the solvent and one-dimensional sliding along the dna chain. this model evidences the presence, in a certain range of values of the effective protein charge, of facilitated diffusion, i.e. a combination of the two types of diffusion that leads to faster than -dimensional diffusion sampling of dna. moreover, the analysis of single sliding events shows that the number of base pairs visited during sliding is comparable to those deduced from single molecule experiments. in contrast to kinetic models, which predict an increase of the number of different base pairs visited by the protein proportional to the square root of time, our model however suggests that this number increases linearly with time until it reaches a value that is close to the total number of dna base pairs in the cell (published in j. chem. phys. , ( )). use of md simulation to identify the critical radiation-induced lesions of a dna binding protein n. chalabi, s. goffinont, n. garnier, d. genest, orléans cedex , france a key step in the regulation of gene expression, dna structuring and dna repair is the binding of some proteins to specific dna sequences. we have shown previously that ionizing radiation destabilizes such dna-protein complexes mainly through damage to the protein. for the typical lactose operator -repressor system we have shown by fluorescence measurement and mass spectrometry that upon irradiation, all the four tyrosine residues of the dna binding domain (called headpiece) are oxidized into , dihydroxyphenylalanine (dopa). a circular dichroism study revealed a global conformational change and the destabilization of the headpiece. to decide which lesion is critical for the induction of these effects, a molecular dynamics simulation study was undertaken in parallel with a site-directed mutagenesis one. each tyrosine residue of the headpiece was replaced by another amino-acid that mimics the damaged tyrosine. the most common amino-acid used in site-directed mutagenesis being alanine, we have replaced one tyrosine ( , , or ) in the nmr-based structure of the headpiece from pdb databank ( lqc) by an alanine. the conformational stability of each tyr→ala mutant has been studied by molecular dynamics simulation (md) and compared to that of the native sequence and of the different tyr→dopa mutants. the mammalian high mobility group protein at-hook (hmga ) is a nuclear protein associated with mensenchymal cell development and differentiation. disruption of its normal expression pattern is directly linked to oncogenesis and obesity. our laboratory has utilized a variety of biochemical and biophysical methods to investigate the molecular mechanisms of hmga recognizing at-rich dna. using a pcr-based selex procedure, we discovered that hmga is a sequence-specific dna-binding protein and recognizes the following two sequences: '-atattcgcgawwatt- ' and 'atattgcgcawwatt- ', where w is a or t. using a double-label emsa assay, we found that hmga binds to at-rich dna as a monomer. using isothermal-titrationcalorimetry, we demonstrated that hmga binds to at-rich dna with very high binding affinity whereby the binding of hmga to a-tracts is entropy-driven and to alternate at sequences is enthalpy-driven. this is a typical example of enthalpy-entropy compensation in which the hydration difference between hmga -dna complexes is a main reason for the compensation. interestingly, the binding of hmga to different at-rich sequences is accompanied with a large negative heat capacity change indicating an important role of solvent displacement and charge-charge interaction in the linked folding/binding processes. partition of gibb´s free energy of drug-dna complexation we report a computation methodology, which leads to the ability to partition the gibb's free energy for the complexation reaction of aromatic drug molecules with dna. using this approach it is now possible to calculate the absolute values of the energy contributions of various physical factors to the dna binding process, whose summation gives a value that is reasonably close to the experimentallymeasured gibb's free energy of binding. application of the methodology to binding of various aromatic drugs with dna provides an answer to the question 'what forces are the main contributors to the stabilization of aromatic ligand-dna complexes' ? we report the effects of khz ultrasound irradiation of double-stranded dna solutions under conditions of transient cavitation. a new method was developed for studying these effects which is based on combination of two procedures: ultrasound irradiation of the solutions of double-stranded dna fragments and subsequent analysis of the high resolution denaturing gel electrophoresis data. statistical treatment of the data on the mobility of '-end-labelled restriction fragments with known sequence allowed us to discover the phenomenon of sequence specific ultrasonic cleavage of dna. our analysis results in the following conclusions: ) all steps with '-ward cytosine [ '-d(cpn)- '] have significantly higher cleavage rates than others; the intensity of cleavage diminishes in the order cpg > cpa ≈ cpt > cpc; ) the cleavage rates of all steps depend on the type of flanking base pairs; ) the cleavage rates of the complementary base pair steps are not identical. thus, subtle sequence specific conformational and physical-chemical variations modulate the reaction of sugar-phosphate single bonds on the ultrasound exposure. a theory of the mechanisms on the simultaneous binding of two aromatic drugs to dna it has long been recognized that certain combinations of dna-binding aromatic drugs, x +y, lead to synergistic biological effects. considering x as a basic ligand and y as an added ligand, the change of the integral biological response of x in the presence of y has been interpreted in terms of two mechanisms: the interceptor and protector action of y on x. this mechanisms have been characterized by two criteria, r d and a d , reflecting the removal of x from dna by y (biophys. chem., , vol. , pages - ) . in this work we develop the theory of the interceptor-protector action of a mixture of two biologically-active dna-binding aromatic drugs. the theory is based on solution of a general system of mass balance equations in the three-component system x -y -dna with respect to the two factors, r d and a d . the outcome is a set of expressions enabling estimation of the change in biological response of x on addition of y as a function of equilibrium parameters under different restrictions. the results are in good agreement with known in vitro data on caffeine+antibiotic action in leukemia cell lines. the influence of mn + ions on the structure of natural calf thymus dna was studied by raman spectroscopy. measurements were done at room temperature and ph . ± . , in the presence of the physiological concentration of mm na + ions, and in the presence of mn + concentrations that varied between and mm. no condensation of dna was observed. dna backbone conformational changes were not detected in the whole concentration range of mn + ions as judging from the raman spectra. no evidence for dna melting was identified. binding of manganese(ii) ions to the charged phosphate groups of dna, stabilizing the double helical structure, is indicated in the spectra. as judged from the marker band of dc near cm − , altered nucleoside conformations in dc residues are supposed to occur, in the mn + concentration range of - mm. binding of divalent ions to n of guanine and, possibly, in a lesser extent to adenine was observed as judging from the raman marker bands at , , and cm − . toward rapid dna sequencing -ab initio study of nucleotide sandwiched between au( ) plates c. morari, d. bogdan, i. turcu national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania recently a new technique for dna sequencing based on measurement of transversal conductive properties of a single strained dna molecule has been proposed. such a method would allow single-base resolution by measuring the electrical current perpendicular to the dna backbone. until now, it is still not clear if the electrical signals obtained for the four nucleotide can be clearly distinguished by a hypothetical experimental setup. several factors -like the influence of the pentose group or the presence of water -may influence quite strongly the electrical signatures of the four bases. therefore, in order to obtain a working device, the understanding and detailed description of the conduction mechanisms through dna bases connected to a metallic electrode is essential. the goal of theoretical studies in this field is to describe the electric signatures of dna bases from a theoretical point of view. our study is focused on the detailed description of the electronic structure of dna's base pairs "squeezed" between two au plates. while such a geometrical model closely mimic the sequencing devices proposed in the literature, it allows us to compute meaningful physical properties such as density of states, charge transfer and orbital localizationby using "ab initio" methods. the results allow us to give qualitative prediction over the potential use of such a device in the dna's sequencing technology. multinuclear platinum complexes represent a new class of anticancer agents, distinct in dna binding and antitumor activity from their mononuclear counterparts. bbr as a first representative of this class has undergone phase ii clinical trials for treatment of ovarian and lung cancers. the structure of this trinuclear pt drug consists of two trans-ptcl(nh ) units bridged by a trans- the main lesions formed by multinuclear pt complexes in dna are long-range intra-and interstrand cross-links (cls) bridging two guanines separated by up to four base pairs. since interstrand cls can prevent dna strand separation interfering with critical cellular events they represent a serious obstacle in cell survival. in order to contribute to understanding the biological effects of dna interstrand cls of bbr , we analyzed the effect of the single, site-specific , -interstrand cl formed by this metallodrug between two guanine bases on opposite strands in the '- ' and '- ' direction on the thermal stability and energetics of short dna duplexes. the results demonstrate that , -interstrand cls of bbr in both '- ' and '- ' directions exist as two distinct conformers that are not interconvertible and affect thermodynamic stability of the dna differently. side-by-side and end-to-end attraction of doublestranded dna c. maffeo , b. luan , a. aksimentiev university of illinois at urbana-champaign, urbana, usa, ibm watson research center, yorktown heights, usa genomic dna is densely packed inside cell nuclei and viral capsids. such close packing suggests that electrostatic repulsion between negatively charged dna in the condensed states is balanced by counterion-induced attraction. several theoretical models have been proposed to explain dna attraction, however, specific microscopic mechanisms are not known. here, we report all-atom molecular dynamics simulations of the effective force between double-stranded dna in side-by-side and end-to-end orientations. in the side-by-side orientation, the dna molecules were found to form a bond state in the presence of magnesium ions. in the bond state, the dna molecules contact each other via their negatively charged phosphate groups, bridged by magnesium ions. the maximum attractive force in the side-by-side orientation is about pn per dna turn. in the end-to-end orientation, a strong ( > pn) attractive force was observed at short (< . nm) end-to-end distances regardless of the electrolyte concentration. the presence of a phosphate group at the 'ends of the fragments was found to direct dna end-to-end self-assembly and produce bound states resembling a continuous dna molecule. the computed potentials of the mean force suggest that the end-to-end attraction, rather than being mediated by counterions, is likely caused by hydrophobic and van der waals interactions between terminal nucleobases of the fragments. doxorubicin (dox) is an anticancer antibiotics with a four-membered ring system containing an anthraquinone chromophore, and an aminoglycoside. it has good anticancer activity against a wide spectrum of tumors and is one of the most extensively used antitumor chemotherapeutic compounds currently in clinical use. interestingly, conversion of dox to pyrrolinodoxorubicin analog (p-dox) exhibits - times higher toxic effects in human breast cancer cell line (nagy, a. et al., pnas, ( ) . molecular mechanisms responsible for this enormously enhanced cytotoxicity have not been entirely clarified. there is good evidence that a key component of the mechanism of action of dox is its intercalation into dna and the formation of dox-dna adducts. therefore, we have examined in detail, using the methods of molecular biophysics, dna interactions of p-dox in cell-free media and compared these results with the same studies focused on the parental dox. we find distinct differences between dna interactions of dox and p-dox and suggest that these differences are responsible at least in part for different biological effects of these two anticancer drugs. design of a microfluidic devices for the detection of oligonucléotides by sers designing fast and efficient analytical tools allowing the detection of free dna or rna at very low concentration within small volumes, without specific molecular labeling, remains a major issue of significance to perform diagnostic or to detect pathogen agents. our strategy is to use surface-enhanced raman scattering (sers) to probe selectively the spectral signature of each base in polynucleotides. sers takes advantage of the strongly increased raman signals of species when adsorbed on adapted silver colloids. we already demonstrated that adenyl raman signal of pa in presence of silver colloids is times enhanced compared to bulk signal. we plan to use nanoliter droplets of uniformed size that form spontaneously in microchannels when two immiscible fluid streams merge. these tiny droplets are almost ideal reactors as they create homogeneous control condition. we will design an optimized channels network platform that result in droplets production hosting both nucleotides and silver colloids: internal fluids recirculation provide fast and efficient mixing, favoring base adsorption on silver nanoparticles. sers will be used to determine the chemical composition of the droplets. nicks represent the most common damage in dna which occurs naturally in living cells. structural properties of nicked dna fragments have been an object of numerous studies due to its special role in reparation processes. here we report experimental results covering ultrasound irradiation of nicked dna solutions. several single-stranded nicks were produced into one strand of dsdna fragments by the nicking enzyme bst i. we have quantitatively estimated the ultrasonic cleavage rates in nicked dna fragments with known sequences using the polyacrylamide gel electrophoresis. computer analysis of the cleavage pattern in the '-end labeled and primarily intact strand reveal cleavage enhancement in the regions of about b. p. up and down the nicks which were initially produced into complementary strand. the intensity of cleavage near the nicks is (in average) about times higher than cleavage in the same sites of the intact dsdna fragments. at the same time, the cleavage rates in positions beyond the regions of the nick markedly grow weak even comparing to the sequence-specific cleavage of intact double-stranded dna fragments. thus, the presence of the nick serves as an expressive structural indignation, which exceeds modulation of the structure caused by the base-pair sequence and is capable of absorbing mechanical stresses applied to the nearby sites of the molecule. comparing the native and an irradiated lactose repressor-operateur complex by md simulation g. naudin, n. garnier, d. genest, rue charles sadron, orléans cedex , france the function of the e. coli lactose operon requires the binding of a protein, the tetrameric repressor, to a specific dna sequence, the operator. the formation of this complex involves the interaction of at least one protein dimer with the operator sequence. this occurs via the dna-binding domains (called headpieces) of the two constitutive monomers. we have previously shown that upon irradiation with gamma rays the complex is destabilised mainly because the repressor losses its dna binding ability. radiation-induced lesions were identified that may be responsible for this deleterious effect: all tyrosine residues of the headpieces are oxidized into , -dihydroxyphenylalanine (dopa). in order to unravel the mechanisms leading to the observed destabilization of the operator-repressor complex, we compare by md simulations two complexes: -the native complex formed by a dimer of two headpieces and a fragment of dna with the operator sequence and -the damaged complex in which all tyrosines are replaced by dopa. analysis of these trajectories shows a loss of stability and binding energy as well as changes in the structure of the damaged complex with respect to the native one. by comparing precisely the evolution of the two complexes we can explain how the oxidation of the tyrosine residues of the headpieces into dopa may trigger the destabilization of the complex. local conformation of confined dna studied using emission polarization anisotropy in nanochannels with dimensions smaller than the dna radius of gyration, dna will extend along the channel. we investigate long dna confined in nanochannels with dimensions down to * nm, using fluorescence microscopy. studies of the statics and dynamics of the dna extension or position in such confinements as a function of e.g. dna contour length, degree and shape of confinement as well as ionic strength has yielded new insight into the physical properties of dna with relevance for applications in genomics and fundamental understanding of dna packaging in e.g. viruses. our work extends the field by not only studying the location of the emitting dyes along a confined dna molecule but also monitoring the polarization of the emission. we use intercalating dyes whose emission is polarized perpendicular to the dna extension axis, and by measuring the emission polarized parallel and perpendicular to the extension axis of the stretched dna, information on the local spatial distribution of the dna backbone can be obtained. we will discuss results in shallow ( nm) and deep ( nm) channels and describe how the technique can be used to investigate non uniform stretching of a single dna molecule. raman spectroscopy of dna modified by new antitumor nonclassical platinum complexes o. vrana, v. kohoutkova, v. brabec institute of biophysics as cr, královopolská , cz- brno, czech republic platinum anticancer agents (cisplatin, carboplatin, oxaliplatin) are in widespread clinical use especially against testicular, ovarian and head and neck carcinomas. there is a large body of experimental evidence that dna is the critical target for the cytostatic activity of cisplatin. platinum complexes form several types of adducts, which occur in dna with a different frequency and differently distort the conformation of dna. clinically ineffective trans isomer of cisplatin (transplatin) also covalently binds to dna bases. the trans geometry in dichloridoplatinum(ii) complexes was activated by various ways. the replacement of at least one amine ligand by planar amine ligand represents example of such activation. raman spectroscopy is powerful technique for examining both structural and thermodynamic properties of nucleic acids in solution. interactions of cis-and trans-pt(ii) complexes having nonplanar heterocyclic amine ligand such as piperidine, piperazine and -picoline with dna have been investigated by laser raman spectroscopy. raman difference spectra reveal that the pt(ii) complexes induce great structural changes in b-dna and indicate disordering of b-dna backbone, reduction in base stacking and base pairing and specific metal interaction with acceptor sites on purine residues. acknowledgement: this work was supported by grant as cr, iqs . a. v. vargiu , a. magistrato , p. carloni , p. ruggerone cnr-infm-slacs and physics dept., university of cagliari, cagliari, italy, cnr-infm-democritos and sissa/isas, trieste, italy, iit and sissa/isas, trieste, italy the minor groove of dna is the target of several anticancer agents, which interfere with replication and translocation processes, leading to cell death. the molecular recognition event is a key step to achieve detailed knowledge of the interactions behind selectivity and affinity of a ligand towards a particular nucleic acids sequence. recognition is a multiroute process which can involve many steps before the formation of the most stable adduct. in particular, many studies have pointed out the importance of events like sliding along the groove and dissociation (which is a relevant step in the translocation among different sequences) for the affinity and the specificity of minor groove binders. despite this, no studies on the dynamics of this event were reported in the literature. in this contribution i present our recent work on the subject. umbrella sampling and metadynamics were used in the framework of classical md to characterize mechanisms andfree energy profiles of molecular recognition routes by the antitumoral agents anthramycin, duocarmycin and distamycin. our results are in very good agreement with the available experimental data, and provide insights on the influence of factors like size, charge and flexibility on the molecular recognition process. amplification of oligonucleotide sequence recognition using bioresponsive hydrogels s. tierney, b. t. stokke biophysics and medical technology, dept. of physics, the norwegian university of science and technology, ntnu, no- trondheim, norway we describe development and characterization of oligonucleotide functionalized hydrogels for amplifying the molecular recognition signal occurring on hybridization between dioligonucleotides. the µm radius hemispherical hydrogels were integrated on a high resolution interferometric fiberoptic readout platform supporting determination changes in the optical length of the hydrogel with nanometer resolution. the hydrogels were designed with hybridized dioligonucleotides grafted to the polymer network as network junctions in addition to the covalent crosslinks or oligonucleotides grafted to the network chains. the probe oligonucleotide destabilizing the junction point by displacement hybridization yielded an optical signal about five times as large as for binding within the hydrogel design with a comblike grafted oligonucleotide. the optical signal was found to be dependent on the concentration of the probe, the sequence and matching length between the probe and sensing oligonucleotide. concentration sensitivity applied as specific label-free detection of oligonucleotide is estimated to be in the nanomolar region. the current design support detection in excess of x sequences. amplification of the molecular recognition employing the developed oligonucleotide imprinted hydrogel for label-free sensing of probe oligonucleotide sequences or taking advantage of the oligonucleotide sequence designed as aptamers for determination of other types of molecules are discussed. tracing t-cells by paramagentic nanoparticles in the brain of the rat model of als d. bataveljic , g. vanhoute , g. bacic , p. andjus inst. for physiology and biochemistry, univ. of belgrade, serbia, bio imaging lab, univ. of antwerpen, belgium, school of physical chemistry, univ. of belgrade, serbia amyotrophic lateral sclerosis (als) is a devastating neurological disorder affecting upper and lower motoneurons. since immune disbalance is known to be an important manifestation of the disease we were particularly interested in following the labeled immune cells in the familial als rat model, hsod- g a . a t -or t -weighted mri protocol was used with a mini surface coil placed over the skull of the anesthetized animal in a . t wide bore magnet. in order to compare this approach to standard high field small animal imaging a . t mri system was also used. there was a congruence of images of lesions in the brainstem at the two field strengths. it was confirmed with gd-dtpa contrast that the blood brain barrier (bbb) is compromised at the interbrain level. in order to study immune cell infiltration rats were i.v. injected with magnetically labeled antibodies against helper cd + or cytolytic cd + killer t cells. by combined t , t and t * weighted imaging cd + lymphocyte infiltration was observed in the brainstem-midbrain region while the cd + cells were more confined to the brainstem region. comparison of mri of labelled cd + vs. cd + lymphocytes reveals the relevant cellular inflammatory mechanism in als. the appearance of the mri signal from the latter t cell type points to the mechanism of bbb disruption as suggested from a recent study on the role of cd + t cells in a model of multiple sclerosis. emodin uptake study in u- mg cells using optically trapped surface-enhanced raman scattering probes s. balint , s. rao , p. miskovsky , d. petrov icfo -the institute of photonic sciences, barcelona, spain, department of biophysics, university of p.j.Šafárik, košice, slovakia emodin ( , , -trihydroxy- -methylantraquinone) is a photosensitizing pigment present in plants of herbal laxatives. emodin inhibits nuclear transcription factor-κb activation and induces free radical production in human mononuclear cells resulting in its antiviral and anti-cancer activity. the uptake and distribution of emodin inside the u- mg cell line is studied by combining optical tweezers and surfaceenhanced raman spectroscopy (sers). sers greatly enhances the spectrum of an otherwise weakly scattering material which is achieved by attaching nano-sized silver colloids to micron-sized dielectric beads. the distribution of emodin in the cell is studied by simultaneously trapping and exciting the sers bead and scanning it across the membrane while recording the emitted light. secondly, the beads are statically placed inside the cell and excited at certain intervals in order to track the migration of emodin through the membrane. the results give new insight in to the metabolic pathways of emodin and demonstrate a new imaging and detection technique that is fast and less invasive than current standards. acknowledgement: miin fis - (spain), fundació cellex barcelona, nadacia spp (slovakia), apvv- - (slovakia) amphotericin b (amb) is a polyene antibiotic that has been widely used for treatment of systemic fungal infections. the main mechanism of biological mode of action of amb is considered to be associated with formation of ionic membrane pores or channels in the lipid membranes. the aim of this work was to study the influence of the k + and na + ions on the aggregation process of amb in aqueous medium. the analysis of electronic absorption and fluorescence spectra of amb shows that the increasing k + concentration have influence on the level of aggregation of the drug much more than the same amount of na + ions. this effect is especially noticed at neutral ph values. the rls technique was used to study aggregation of amb in solution, in the environment of the k + and na + ions. the application of this technique makes it possible to study the electronically coupled chromophores, especially molecular aggregates. the results of the atr-ftir and raman spectroscopic studies also support this conclusion. these results provide a better understanding of the interaction between k + and na + ions and antibiotic which has not been previously considered to be significant for biological action of amb. g-quadruplexes: combining theory with experimental spectroscopic methods guanine-rich oligonucleotides can form unique tetrameric structures with four coplanar guanine bases, known as gquadruplex motifs. the g-quadruplexes have been found in vivo in the terminal parts of telomeres and other genomic regions. ligands, specifically binding to the g-quadruplex regions inhibit telomerase activity and thus can play an important role in the cancer therapy. most recently, the potential use of these structures has been tested in biosensors and nanotechnology industry. in the present work we use the infrared spectroscopy, including the relatively novel technique of the vibrational circular dichroism (vcd), in a combination with molecular dynamics and quantum chemistry computational methods to investigate the structure and spectroscopic response of the quadruplexes formed by the d(g) and selfassociated dgmp. we obtained a good agreement between the computed and experimental spectra, confirming that the proposed geometrical models are realistic. the vcd technique appears especially convenient for the studies as it can detect the liquid-crystalline phases of the g-quadruplexes by an anomalous enhancement of the signal. j. bogdanovic pristov, a. mitrovic, k. radotic, i. spasojevic institute for multidisciplinary research, belgrade, serbia fructose, due to its high antioxidative capacity, represents a significant component of non-enzymatic defense of some plants against cold-provoked oxidative stress. in the present study, we have investigated role of fructose in seasonal adaptation of picea omorika (pančić) purkinye to cold. this endemic coniferous species is exposed to subfreezing temperatures that range from - to - • c during the autumn/winter and high temperatures exceeding • c during the summer. characteristic epr signal of free or weakly bound mn + was used as an indicator of oxidative status of needles, since coldrelated oxidative damage leads to mn + release from photosystem ii. it was observed that prooxidative conditions developed in the autumn, at the beginning of cold season, which corresponded to significant increase of fructose level. total sod, as well as mnsod activity also rose significantly higher in the autumn. observed activation of antioxidative system (non-enzymatic and enzymatic) led to adaptation of needles to cold, as oxidative status during winter was decreased and similar to the status of needles in cold-free seasons. calyx of held: sted nanoscopy of a glutamatergic terminal p. bingen , t. m. staudt , c. kempf , h. horstmann , j. engelhardt , t. kuner , s. w. hell german cancer research center / bioquant, heidelberg, germany, max planck institute for biophysical chemistry, göttingen, germany, institute for anatomy and cell biology, university of heidelberg, heidelberg, germany the calyx of held, a large glutamatergic synaptic terminal in the auditory brainstem circuit has been increasingly employed to study presynaptic mechanisms of neurotransmission in the central nervous system. a highly detailed model of the morphology and distribution of cytoskeleton, synapsin, synaptic vesicles, calcium sensors, mitochondria, the presynaptic membrane and its active zones is derived by colocalization analysis of these different key elements of synaptic transmission in the rat brain. the various cellular components are visualized with subdiffraction resolution by stimulated emission depletion (sted) microscopy. imaging individual structural elements exhibit a focal plane resolution of < nm inside µm thick tissue sections. three-dimensional shim and pem to study collagen arrangement and crimping pattern p. bianchini , m. franchi , l. leonardi , a. diaspro lambs-ifom microscobio research centre and department of physics, university of genova, genova, italy., italian institute of technology (iit), genova, italy, department of human anatomical sciences and physiopathology of the locomotorapparatus, university of bologna, italy ligaments have been generally described as multifascicular structures with collagen fibre bundles cross-connecting to each other or running straight and parallel with crimps. a different collagen array and crimping pattern in different ligaments may reflect a different mechanical role. aim of this study was to relate the d collagen arrangement and crimping pattern by backward and forward second harmonic imaging microscopy (shim) and -photon excitation microscopy ( pem). shim on a laser-scanning system is a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of tissue architecture. although it is a coherent process the multiple scattering through the tissue give us the capability to acquire signal in both backward and forward direction [ ] . shim and pem were combined in a dual-mode nonlinear microscopy to find out collagen fibre arrangement and crimping pattern. both polarization dependence and differences between forward and backward signals allowed to yield information on local structure [ ] . currently used tcspc flim systems are characterised by high counting efficiency, high time resolution, and multiwavelength capability. the systems are, however, restricted to count rates on the order of a few mhz. in the majority of applications, such as fret or autofluorescence, the photostability of the samples limits the count rate to much lower values. the limitation of the count rate is therefore no problem. however, if flim is used for ion concentration measurements or imaging of chlorophyll in plants the available count rates can exceed the counting capability of a single tcspc channel. we therefore developed a flim system that uses eight fully parallel tcspc channels. by using a polychromator for spectral dispersion and a multichannel pmt for detection we obtain multi-spectral flim data at a rate of several frames per second. we will demonstrate the application of the system to dyamic changes of the fluorescence lifetime of chlorophyll in living plants. -imaging and spectroscopy - scattering effects on non linear imaging of thick biological samples f. cella , z. lavagnino , a. diaspro lambs-ifom, microscobio research center, university of genoa, italy, iit, italian institute of technology, genoa, italy non linear optical scanning microscopy became a useful tool for living tissue imaging. biological tissues are highly scattering media and this leads to an exponential attenuation of the excitation intensity as the light travels into the sample. while performing imaging of biological scattering tissues in two photon excitation ( pe) regime, the localization of the maximum pe intensity was found to shift closer to the surface and the imaging depth limit appears strongly limited by near surface fluorescence . in this work we computed illumination and photobleaching intensity distribution in order to characterize the effects induced by scattering. simulations of pe illumination and photobleaching intensity profiles have been performed for different scattering coefficients and at different focus depth. furthermore imaging of fluorescent immobile sample (polyelectrolyte gel) allowed to perform an experimental test on thick turbid media. results confirm that under these conditions no photobleaching effects due to scattering occur close to the surface. [ ]ying et al., appl. opt. , ( ) . [ ] theer p. and denk w, j. opt. soc. am. a. ( ) [ ]mazza d. et al., appl. opt. ( ) . biospectroscopic probes for real time measurement of hydrogen-deuterium exchange p. carmona , m. molina instituto de estructura de la materia (csic), madrid, spain, escuela universitaria deÓptica, madrid, spain isotopic exchange has long been used for the analysis of biomolecular structure and dynamics. hydrogen-deuterium exchange rates depend on ph, temperature and biomolecular environment. this is due to hydrogen bonding, low solvent accessibility, and steric blocking. time resolved measurement of hydrogen-deuterium exchange for subsequent d correlation spectroscopic analysis can, then, be very useful to obtain structural information from the said viewpoint. we have developed a microdialysis quartz cell for use in conjunction with raman spectroscopy to investigate hydrogen-isotope exchange reactions of biomolecules. the system requires only µl volumes of the initial substrate and perturbing effluent solutions. we have obtained a d o efflux rate of k d = . ± . min − with the greatest mwco ( kda) used here, which involves that an exchange rate of . min − is the limiting rate that could be resolved with the said cell system. analogous results have been obtained using an infrared biospectroscopic microdialysis probe. the use of the method described here has the advantage of avoiding sample dilution (and subsequent signal loss) involved in the known stop flow methods. acknowledgements: the authors gratefully acknowledge financial support from the spanish ministerio de ciencia e innovación (project ctq - /bqu). polarized transient absorption to resolve electron transfer between tryptophans in dna photolyase photoactivation of dna photolyase comprises electron transfer through the chain fadh • -w -w -w . photo-excited fadh • abstracts an electron from the tryptophan residue w in ∼ ps (monitored by transient absorption spectroscopy). the subsequent electron transfer steps (from w to w •+ and from w to w •+ ) are difficult to resolve experimentally, because electron transfer between chemically identical species does not give rise to net absorption changes. to overcome this difficulty, we make use of the fact that polarized excitation (pulse laser) induces a preferential axis (that of the excited flavin transition) in the system (photoselection), and that w and w form different angles with that axis (known from the crystal structure). thus, polarized detection should allow distinguishing between them. using polarized "classical" transient absorption on a nanosecond time scale and the pump-probe technique on a picosecond scale, we demonstrate the feasibility of the method and provide evidence that electron transfer from w to w •+ is faster than the ps time constant of the initial electron transfer from w to excited fadh • . synchrotron based fourier transform infrared (sr-ftir) microspectroscopy was applied to investigate apoptotic death of u- mg cells induced by the photosensitizer hypericin (hyp), in using different transport systems (hyp alone vs. hyp/ldl complexes) and incubation protocols. the differences between ir spectra of non-treated and hyp treated cells are mainly manifested in the positions of amide i and amide ii vibrational bands of proteins. these vibrational shifts are attributed to the protein structure changes from dominantly alfa-helix, in the non-treated cells, to beta-sheets and random coil structures, which prevail h and h after photodynamic treatment, respectively. the observed conformational changes of proteins can be explained as the consequences of the processes leading to apoptosis as was verified by flow cytometry experiments. the results confirm suggestion that ir spectroscopy can be successfully applied for the detection of early apoptotic processes. a. k. de, d. goswami indian institute of technology kanpur, india molecular fluorescence has been an indispensable tool in modern day optical imaging. one of the state-of the-art challenges in fluorescence microscopy is having better depth resolution as embodied by the confocal and multi-photon laser-scanning microscopic techniques. however, each technique bears its own limitation in having sufficient out-of-focus signal for the former while the low non-linear photon absorption cross-section for the latter. for confocal microscopy using one-photon excitation, we have shown how the clever choice of pulsed illumination instead of continuous-wave excitation leads to a gigantic enhancement in fluorescence that also has immediate applications in microscopy. moreover, single-photon illumination with ultrafast pulses leads to a novel way of achieving axial resolution along with numerous other advantageous applications e.g. reduced photo-bleaching of the chromophore. on the other hand we have thrown new insight demonstrating that the use of mode-locked laser pulses in multi-photon microscopy induces severe solvent-induced photo-thermal damage and prescribed methods to get rid of it. besides, the use of pulse pair excitation in multi-photon microscopy leads to probe and control the dynamics of fluorophores which has crucial role in selective excitation of fluorophores from quantum control perspectives. all these cutting edge research works will be presented in addition to our recent work on application of laser pulse shaping in multi-photon microscopy. interkingdom signalling in pseudomonas aeruginosa b. davis , r. jenson , p. williams , p. o'shea institute of biophysics, imaging and optical science, university of nottingham, u.k., institute of infection, immunity and inflamation, university of nottingham, u.k. quorum sensing is the process through which some bacterial species coordinate cell-cell communication. pseudomonas aeruginosa; the pathogen responsible for over % of chronic lung infections and the leading cause of mortality in cystic fibrosis patients, expresses two major classes of quorum sensing molecules characterized as n-acyl homoserine lactones and -alkyl- -quinolones. these compounds, in addition to their signalling roles have also been found to possess virulent properties, not only against competing species of bacteria such as staphylococcus aureus but also eukaryotic cells such as t-lymphocytes. the process of bacterial quorum signalling molecules influencing eukaryotic cell activity is termed 'interkingdom signalling'. to date it has been suggested that the quorum sensing molecules outlined above act on eukaryotic cells through interactions with an as yet unidentified plasma membrane or cytosolic receptors. this project is directed towards developing an understanding of how these compounds elicit eukaryotic response through a combination of membrane based interactions at physiologically relevant concentrations. particular emphasis is placed on studies of ligand binding with membrane microdomains in and the consequent downstream signalling are also considered. this work is significant as it will not only lead to a better understanding of pseudomonas infection, but may also lead to the discovery of new classes of agents for the treatment of infective diseases. a xas study of the sulphur environment in human neuromelanin and its synthetic analogues neuromelanin is a complex molecule accumulating in the catecholaminergic neurons that undergo a degenerative process in parkinson's disease. it was shown to play an either protective or toxic role depending on whether it is present in the intraneuronal or extraneuronal milieu. in the present study x-ray absorption spectroscopy is employed to investigate the sulphur binding mode in natural human neuromelanin, synthetic neuromelanins and in certain structurally known model compounds, namely cysteine and trichochrome c. based on comparative fits of human and synthetic neuromelanin spectra in terms of those of model compounds, the occurrence of both cysteine-and trichochrome-like sulphur coordination modes is recognized and the relative abundance of these two types of structural arrangement is determined. data on the amount of cysteine-and trichochrome-like sulphur measured in this way indicate that among the synthetic neuromelanins those produced by enzymatic oxidation are the most similar ones to natural neuromelanin. c. cremer kirchhoff-institute for physics, university of heidelberg, germany here we report on "spectral precision distance/position determination microscopy (spdm) with physically modifiable fluorochromes (spdm phymod ) to analyse the spatial distribution of single nuclear proteins and dna sequences at the macromolecular optical resolution level. like other methods of "spectrally assigned localization microscopy" (salm), spdm phymod is based on labelling 'point like' objects (single molecules) with different spectral signatures, spectrally selective registration and high precision localization monitoring by far field fluorescence microscopy. the intranuclear spatial location of single molecules was determined up to a density up to ca. molecules/µm of the same type, and distances down to - nm were nanoscopically resolved. quantum dots (qds) are semiconductor nanoparticles with increasing application as fluorescent markers in biology.we investigated structure of the cell walls of different species complexed with cdse qds using fluorescence microscopy, fluorescence spectroscopy and ftir techniques. in the experiments we used the cell walls isolated from three distinct plant species: arabidopsis thaliana, acer sp. and picea omorika. we studied both unlabeled and cdse-labeled cell walls. fluorescence spectroscopy and microscopy were used for detection of qds alone or complexed to the cell walls. emission spectra were deconvolved using the nelder-mead algorithm in matlab . . we calculated approximate probability distribution (apd) for positions of spectral component maxima. there was certain difference between unlabeled cell walls and those complexed with qds. the ftir spectra also show some difference between the complexed and pure cell walls. the results show that structure was changed, but not significantly in reaction with cdse qds. these results are promising in context of use of qds as labels in cell wall studies. the characterization of the complex of cell wall structure with qds is a part of the study of nanoparticles application in investigations of plant materials. modulating the response of single neurons and neuronal networks with biophysical stimuli f. difato, a. maccione, l. berdondini, f. benfenati, a. blau italian institute of technology, department of neuroscience and brain technologies, genoa, italy during differentiation, cell processes create connections with other cells to form tissue capable of performing complex tasks. biophysical constraints provide necessary inputs for cellular organization in living organism . to better understand how biophysical conditions influence tissue development, it is necessary to bridge the gap between experiments on single cells and complex tissues , . to achieve this goal we pair optical tweezers with electrophysiology measurements . by adopting neuronal networks as a biological model, neuronal signal transmission can be recorded either by patchclamp electrophysiology or microelectrode arrays (meas). dissociated neurons will be cultured on meas to record neuronal network activity at different sites of the network while applying spatio-temporally defined biophysical stimuli to individual neurons. a. diaspro , k. cortese , p. bianchini , c. gagliani , c. tacchetti iit -italian institute of technology, morego, genova, italy, microscobio, university of genoa, italy correlative light/electron microscopy (clem) is becoming increasingly frequent in molecular and cellular biophysics. we successfully applied the method to analyze the d structure of rough and smooth russell bodies used as model systems. the major advantages of this approach are the following: (i) the ability to correlate several hundreds of events at the same time, (ii) the possibilitˆto perform d correlation, (iii) the potential to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the effective combination of the high data analysis capability of flm with the high precisionaccuracy of transmission electron microscopy in a clem hybrid morphometry analysis. we have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic d reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach. the relevance of the presented approach lies in two important key elements, namely: the development of optical nanoscopy methods and the potentiality for exploring different correlative frameworks like optical nanoscopy vs. optical microscopy adding scanning force microscopy techniques. multidrug resistance is a well known phenomenon which limits effectiveness in treating malignancy with chemotherapy by modifying the internalization and/or externalization flow of the drugs through the cancerous cells. combined chemotherapies, such as mvac, are therefore currently used in bladder cancer treatment. however, about % of patients do not respond this chemotherapy because of inherent or acquired drug resistance. we developed a non invasive predicative test on urinary cells to estimate the chemotherapy effectiveness before treatment, based on the fluorescence emission of mvac. we first studied the mvac photophysical properties in solution and using five cell lines: a drug sensitive cancer cell line mgh-u s, its multidrug resistant subline mgh-u r, a not tumorigenic cell line sv-huc- , its tumorigenic counterpart mc-sv-huc t- and a cell line from transitional cell carcinoma t . the results revealed a penetration and localization of the drug depending of the cell line type, allowing us to find a specific fluorescence signature for the identification of mvac resistant cells. similar data have been obtained for cytospined fixed culture cells and patients urinary cells. -imaging and spectroscopy -abstracts multilayered photoresist system as a ghost model for biological samples in confocal microscopy. l. ferrari, f. cilloco, f. r. bertani, s. selci istituto dei sistemi complessi cnr rome we have realized a bilayer photoresist system as a model to perform optical characterization in the vis-ir range of multilayered complex biological specimen. our goal is to obtain structural and spectroscopic reference parameters from the identification of reflectance pattern features within a novel project granted by miur (skintarget, ideas firb). our model is composed by a nm thick layer (shipley photoresist ) with a refractive index n= . - . , wavelength dependent, and a nm tick hsq (hydrogen silsesquioxane) layer with a refractive index n around . , both spin-coated over a glass substrate. we present the analysis of reflectance pattern that can be obtained by a confocal laser reflective system as compared to the expected values as coming from analytical calculations, using a matrix approach, or a microscopic electromagnetic finite element analysis. interfacial roughness and consequent optical scattering are analyzed using a neural network approach. translational biophysics: multimode imaging for preclinical and clinical applications d. l. farkas cedars-sinai medical center, los angeles, usa our focus is where light and patient meet, and improvements yielding better outcomes. surgery is moving towards minimally invasive intervention, where biophotonics represents a major area of hope and growth. the translation of useful laboratory-derived knowledge into clinical practice has been hampered by the difficulty of detecting, characterizing and monitoring molecules and cells in the human body, especially dynamically. advanced bi ophotonic imaging is best suited for studying such entities, but has been lagging in clinical acceptance, in spite of major advances, and a clear need for the kind of resolution (spatial, temporal, spectral) and specificity that it alone can offer. biophysics-based new strategies are needed to address this challenge. the development of biophysical methods for translational medicine will be reviewed, with emphasis on our recent advances. our approach is a multimode one -combining methods to achieve early, quantitative detection of abnormalities. with imaging fulfilling its dual role of better describing anatomy and physiology, intrasurgical histopathologyequivalent molecular and cellular imaging is achievable in vivo, as is a closer spatio-temporal connection between imaging and intervention. some application areas to be covered: cancer (early detection by spectral reflectance/autofluorescence; progression quantitation by oct; nano-and targeted chemotherapy assessment in vivo); neurobiology (imaging fast calcium transients and alzheimer's plaques); hyperspectral mie scattering imaging for in situ displasia; design and use of an advanced multimode imaging endoscope with in vivo delineation of hirschsprung's disease for better intervention; monitoring of stem cell fate in vivo. immobilization of liposomes in a sol-gel matrix: a fluorescence confocal microscopy study r. esquembre , s. n. pinto , j. a. poveda , m. prieto , c. r. mateo ibmc, universidad miguel hernández, elche, spain, cqfm, instituto superior técnico, lisbon, portugal immobilization of liposomes shows interesting applications in protein biology, membrane biophysics and biosensor technology. previous fluorescence spectroscopy works revealed that the entrappement of zwitterionic phospholipid liposomes in a silica sol-gel matrix alters the thermodynamic properties as well as the fluidity of the lipid bilayer. interactions between the polar head of phospholipids and the porous surface of the host matrix could be responsible of such behaviour. in order to get more insight into this possibility we have immobilized, for the first time, giant unilamelar vesicles (guvs) and the shape and size of these structures as well as the possible existence of lipid domains have been visualized through fluorescence confocal microscopy. this technique allows for direct observation of the effect of encapsulation on an individual liposome, in contrast to the averaged information given by macroscopic spectroscopic techniques. liposomes composed of pure popc or dopc as well as mixtures dopc/dppc were labelled with the fluorescent probes bodipy, rhd-pe and rhd-dope. preliminary results shows that only the smallest guvs ( - µm) survive to the encapsulation process but often with a slight loss of its sphericity, probably due to pressures suffered during the matrix gelation. however no change in the gel/fluid phase proportion has been observed for immobilized dopc/dppc guvs, regarding to solution. solvent fluctuations play a key role in controlling protein motions and functions. here, we have studied how the reaction catalyzed by the light-activated enzyme protochlorophyllide oxidoreductase (por) couple with solvent dynamics (g. durin et al, biophysical j. ( ) , . to simultaneously monitor the catalytic cycle of the enzyme and the solvent dynamics, we designed temperature-dependent uv-visible microspectrophotometry experiments, using flash-cooled nano-droplets of por. the temperature-dependant formation of the first two intermediates in the por reaction were measured, together with the solvent glass transition temperature (t g ) and the build-up of crystalline ice. we find that formation of the first intermediate occurs below t g and is not affected by solvent dynamics, whereas formation of the second intermediate occurs above t g and is influenced by solvent dynamics. these results suggest that internal protein motions drive the first step of the por reaction whereas solvent slaved motions control the second step. we propose that the concept of solvent slaving applies to complex enzymes such as por. amphotericin b (amb) is one of the main polyene antibiotics widely used to treat deep-seated fungal infections. the mechanism of biological action of amb is most probably directly related to the ability of the drug to form hydrophilic pores in the membrane core, thus affecting physiological transport of ions. the effects of amb-cu + complexation are demonstrated by the electronic absorption and fluorescence spectra. the absorption spectra of amb in water (ph= ) after the injection of water solution of copper(ii)sulfate display a complex structure with hypsochromic-and bathochromic-shifted bands indicative of formation of molecular aggregates of the drug. formation of the electronically coupled chromophores of amb, especially aggregates, was analyzed at different cu + concentrations by the rls (resonance light scattering) technique. intensity of the fluorescence emission spectrum (characteristic of the dimeric form of amb) decreases after the amb-cu + complex formation. this effect of the formation of the amb aggregated structures by amb-cu + are different from the spontaneous molecular aggregation process, as deduced from the spectroscopic analysis. morfo-functional asymmetry of the olfactory receptors of the honeybee apis mellifera l e. frasnelli , g. anfora , f. trona , f. tessarolo , r. antolini , g. vallortigara cimec, centre for mind/brain sciences, university of trento, italy, iasma research and innovation center, fondazione e. mach, s. michele all´adige (tn), italy, biophysics and biosignals lab., dept. of physics, university of trento, italy lateralization, i.e. the different functional specialisation of the left and right side of the brain, has been documented in many vertebrate and, recently, invertebrates species. in the honeybee apis mellifera l. olfactory memory seems to involve at first the use of the right antenna. the present study investigated physiological and anatomical differences between left and right antennae of honeybees. electroantennographic responses (eag) were recorded from the left and right antennae of honeybees to linalool, a floral volatile compound, and isoamyl acetate, an alarm pheromone, at doses (from , to µg). the number of sensilla on the left and right antennae was recorded by scanning electron microscopy (sem). each antenna segment, from insects, was observed from different viewpoints in order to image the whole antenna surface and compute the number of sensilla. the tested compounds induced higher eag responses on the right than on the left antenna at every dose. sem showed that the placoidea olfactory sensilla were slightly more abundant on the right antenna surface than on the left one. results suggest an asymmetry in the peripheral odour perception mechanism in the honeybee a. mellifera. super-resolution imaging of dna through single molecule switching of intercalating cyanine dyes c. flors, c. n. j. ravarani, d. t. f. dryden school of chemistry, university of edinburgh, u.k. a growing trend in far-field super resolution fluorescence microscopy involves the replacement of photoactivatable fluorophores by common dyes such as cy, atto or alexa [ ] . it has been shown that these dyes can blink in useful timescales for single-molecule based imaging by adding suitable buffers. this strategy greatly simplifies the sample preparation and imaging scheme, enabling its application to a wider range of biological systems. we have explored if a similar approach might be useful to study dna topology using intercalating cyanine dyes such as yoyo- . there are two main advantages of this approach: i) dna labelling with intercalating dyes is straightforward, and ii) the free dye in solution is essentially non-fluorescent, greatly reducing the fluorescence background. we show that yoyo- can blink in the absence of oxygen and in the presence of cysteamine, which allows its application to nanoscale imaging. we exemplify its use by imaging λ-dna and a puc plasmid. we also explore the compatibility of several intercalating dyes with biological systems such as enzymes or cells. our results suggest that dna intercalating dyes are a promising option for fluorescence super-resolution studies of dna topology. seeing more in total internal reflection fluorescence (tirf) microscopy r. fiolka, a. stemmer nanotechnology group, eth zürich, zürich, switzerland total internal reflection fluorescence (tirf) microscopy is an effective widefield imaging tool that selectively excites a very thin sample layer within the evanescent excitation field at the glass-water interface. lateral resolution of standard tirf microscopy is limited to approx. nm using green emission, which can be insufficient for a large class of biological investigations. additionally, the evanescent excitation field is prone to light scattering, creating out of focus blur in the final image. we present several techniques that address these mentioned shortcomings of standard tirf microscopy. using evanescent standing waves, the lateral resolution in tirf microscopy can be extended by a factor of . , reaching nm. we further show techniques to reduce the blur induced by light scattering of the evanescent field. finally, we demonstrate optical sub-sectioning capabilities in tirf microscopy by acquiring several images with different penetration depth of the evanescent field and applying suitable post processing algorithms. thereby the obtainable z-resolution exceeds the classical limit of widefield microscopy, and object structures lying within the evanescent field can be reconstructed. the natural photosentizer hypericine (hyp) exhibits potent properties for tumor diagnosis and photodynamic therapy. evidences of hyp release from ldls prior to passive diffusion within cells are addressed in this study. fluorescent properties of hyp have been used for dynamic studies of its interaction with low-density lipoproteins (ldls) and u glioma cells. subsequent non-specific staining of intracellular membranes compartment were observed by mean of colocalization fluorescent imaging studies. it was shown, that monomers of hyp are only redistributive forms. increasing of hyp concentration leads to the formation of non-fluorescent aggregates within ldls as well as within the u cells, and can preclude its photosensitizing activities. in all experiments, hydrophobic character of the molecules appears as the driving force of its redistribution process. acknowledgment: this work was supported by the slovak res. and dev. agency contracts no. lpp- - . we also kindly thank the synchrotron soleil for using the detection system of the disco beamline. ledgf/p switches from a dynamic to a tight chromatin interaction upon binding to hiv- integrase j. hendrix , z. debyser , y. engelborghs laboratory of biomolecular dynamics, katholieke universiteit leuven, belgium, laboratory of molecular virology and gene therapy, katholieke universiteit leuven, belgium human transcriptional co-activator ledgf/p is hijacked by hiv- integrase (in) during the replication of hiv. little is still known about the molecular complex of these two proteins in the living cell. in this work we first studied the cellular chromatin interaction of egfp-tagged ledgf/p with tunable-focus fluorescence correlation spectroscopy (tf-fcs) and show that ledgf/p is in equilibrium between a free brownian motion and a very slow movement on the chromatin. being dependent on the size of the laser focus, this slow movement represents a continuous associationdissociation-reassociation process that is governed by diffusion. concentration-dependent continuous photobleaching measurements (cp) furthermore revealed the existence of high-affinity chromatin binding sites. next, we co-expressed mrfp-tagged in and confirmed its intracellular interaction with ledgf/p by fluorescence cross-correlation spectroscopy (fccs). interestingly, cp and fluorescence recovery after photobleaching (frap) indicated that the affinity of this complex for chromatin is exceptionally high. by twophoton fluorescence lifetime imaging ( p-flim) we verified if the cellular stoichiometry was altered when the proteins were expressed together. we believe that this work is useful for the understanding and targeting of hiv-replication. biophysical identification of orf from clavulanic acid biosynthesis cluster as a cyp l. s. goto , c. o. hokka , j. f. lima , o. r. nascimento , a. p. u. araújo grupo de engenharia bioquímica, ufscar, br, grupo de biofísica molecular "sérgio mascarenhas", usp, br streptomyces clavuligerus produces the clinically important β-lactamase inhibitor clavulanic acid. biosynthesis related genes reside in three gene clusters, one of these, named clavulanic acid gene cluster, includes most of the known clavulanic acid biosynthetic enzymes. the penultimate step along clavulanic acid biosynthesis remains unclear. required transformation involves at least two events: oxidative deamination and double epimerization of ( s , s )-clavaminic acid into ( r, r)-clavaldehyde. downstream the known part of clavulanic acid cluster lays orf , a putative gene encoding a tentative cytochrome p -like protein which knockout has been proven deleterious to clavulanic acid biosynthesis. should such protein exist, it would be candidate to fulfill the clavulanic acid pathway missing step. in this work, orf encoded protein is characterized aiming to place it as a real p . for this task, molecular cloning and recombinant expression of orf were accomplished. purified protein was submitted to spectroscopic measurements such as circular dichroism and electron paramagnetic resonance which indicate p features, including catalytic relevant heme iron redox states and homolytic peroxide scission mechanisms. further, peroxide reaction adducts were characterized by spin trapping. this work is supported by fapesp. muscle structure and gabaergic innervations in the limbs of barnacle cyprid l. gallus , s. ferrando , c. gambardella , a. diaspro , p. bianchini , p. ramoino , v. piazza , g. tagliafierro libiom, dibio, università di genova, italy, ifom-lambs/microscobio, difi, università di genova, italy, dipteris, università di genova, italy, ismar cnr, venezia, veneto, italy balanus amphitrite is a sessile crustacean that settles at the larval stage of cyprid. in this stage we studied the gabaergic innervation of limb striated muscular fibers, by immunohistochemistry. the second harmonic generation (shg) and -photon excitation ( pe) microscopy were used to set out the muscle structure and its relationship with nerve terminal. sections were observed at a multimodal nonlinear microscope composed by the leica clsm. the laser system used is a ti:sapphire chameleon-ultra (coherent inc, santa clara, ca, usa), tunable between nm and nm and characterized by a pulse width of fs delivered at a repetition rate of mhz by means of a homebuilt set-up (bianchini p. and diaspro a. j biophotonics : - ). the z-stacks were performed in order to obtain the -dimensional distribution of the muscular fibers. in the posterior ganglion gad immunoreactive (ir) motor neurons were arranged in clusters near the emergence of the limb nerves. gaba and gababr ir neuromuscular junctions (nj) were localized in the limb muscle fibers; vgat ir cells surrounded each limb muscles. these results suggest that gaba plays a key role in the regulation of limbs movement. the shg was very useful to outline the relationship between nerve terminals and limbs muscle fibers. giant unilamellar vesicles (guvs) are very useful model membrane systems to study many aspects of lipid-lipid and lipid protein interactions, particularly employing fluorescence microscopy related techniques (bagatolli ) . the use of this model system can be particularly useful to study aspects related with the lateral structure of bacterial membranes using the aforementioned approach (i.e. fluorescence microscopy). bacterial cells have a size close to the resolution limit of optical microscopy and details about the organization of their membranes are not easy to achieve using such technique. recently a new electroformation method to prepare guvs composed of compositionally complex mixtures under physiological conditions was developed in our laboratory (montes ). in the present work we further extended this electroformation method to prepare guvs composed of bacterial lipid extracts and lipopolysaccharide (lps). in our experiments we used e.coli lipid extract to prepare small vesicles containing various lps species, from smooth strains and rough mutants. suvs were used as starting point to electroform guvs using various types of buffers with high ionic strength. the successfully obtained bacterial-guvs were used to study the interaction of these membranes with known lps-binging proteins (lung surfactant protein d, sp-d) and peptides (polymyxin b). our results remark the usefulness of this particular bilayer models to perform studies mimicking bacterial membranes. mechanical properties of polymeric membranes probed by afm m. kocun , i. mey , w. müller , m. maskos , a. janshoff georg-august universität, institute for physical chemistry, göttingen, germany, johannes gutenberg universität, institute for physical chemistry, mainz, germany many biological cell functions are dependent on the mechanical properties of the membrane. polymeric membranes that mimic native cell membranes are valuable research tools which can be used to better understand the physics of biological membranes. we have investigated free standing artificial membranes prepared from polybutadiene-b-polyethylene oxide (pb-b-peo). the membranes were prepared from vesicles ruptured on porous silicon substrates. these polymeric membranes were studied by confocal laser scanning microscopy (clsm) and atomic force microscopy (afm). force indentation curves were performed on the membranes and theoretical models were used to extract elasticity constants from the results. the study of polymeric membranes can give insight to the function of biological membranes, furthermore polymeric membranes can be used to create new hybrid systems by incorporating biological (lipids, proteins), artificial (polymers, dyes) and inorganic (nanoparticles) components. site-directed spin labeling study of the lightharvesting complex cp the topology of the long n-terminal domain of the photosynthetic light-harvesting complex cp was studied using electron spin resonance (esr). cp is a minor antenna complex of the photosystem ii (psii), a multisubunit protein complex. wild-type cp protein containing a single cysteine at position and nine single cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. in all cases the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. the spin label esr spectra were analyzed in terms of a multi-component spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. these results permit to trace the structural organization of the long n-terminal domain of cp . we took the crystal structure of light-harvesting complex ii (lhcii), major antenna complex of psii available in pdb as a starting point and constructed a model for cp based on esr data. present opportunities and future developments in soft x-ray transmission and emission microscopy b. kaulich, a. gianoncelli, v. babin, m. kiskinova elettra -sincrotrone trieste, s.s. km . in area science park, i- trieste-basovizza, italy soft x-ray transmission and emission imaging and spectromicroscopy are bridging the gap to other microscopy techniques in terms of lateral resolution, penetration depth and chemical sensitivity. the novel soft x-ray spectromicroscopy approach of the twinmic instrument at the elettra synchrotron radiation facility is combining several imaging modes for morphology characterization, such as scanning, projection and full-field imaging with several contrast techniques including brightfield, darkfield, differential phase and interference contrast at sub- nm lateral resolution. complementary chemical information is provided by chemical imaging and micro-spectroscopy using the photon-in/photon-out xray absorption and x-ray fluorescence. unique for twinmic is the low-energy x-ray fluorescence setup operated at - ev photon energies, which allows simultaneous analysis of the morphology, and the distribution of light elements on cellular and sub-cellular level. in the presentation the principles of the methods used in the twinmic instruments the performance and potentials of the instrument will be demonstrated by several examples of applications in the field of human, animal and plant biology, biophysics and chemistry, physiology and genetics. the potential impact of microscopy techniques using free-electron x-ray lasers on biophysics will be illustrated by the diproi project at fermi@elettra. -imaging and spectroscopy - fluorescence anisotropy and afm used as tools to characterized porin´s reconstituion in luv´s s. c. lopes, i. sousa, p. eaton, p. gameiro requimte, faculdade de ciências, universidade do porto, rua do campo alegre, - porto, portugal a major requirement to perform structural studies with membrane proteins is to define efficient reconstitution protocols that assure, not only, a high incorporation degree in preformed liposomes, but also a protein directionality and topology that mimics its in vivo conditions. for this kind of studies, protein reconstitution in membranes systems via a detergent-mediated pathway is usually successfully adopted, since detergents are generally used in the initial isolation and purification of membrane proteins. in this study we report the reconstitution of ompf in preformed dmpc and e. coli liposomes using two different techniques for detergent removal: ( ) exclusion chromatography and ( ) incubation with detergent adsorbing beads. the incorporation degree was determined by bicinchoninic acid assay and fluorescence anisotropy was used to determine ompf effect on the structural order of membrane lipids. these results show that protein insertion in membranes depends both on the technique used to remove detergent and on the lipids used to prepare the liposomes. moreover more anisotropy and atomic force microscopy studies will allow a better characterization of bacterial model system membranes. the wavelength dependence of the luminescence for different sized aunp by -photon clsm k. li, m. schneider pharmaceutical nanotechnology, saarland university, campus a , d- saarbrücken, germany gold nanoparticles (aunp) with different sizes can exhibit original luminescent properties if excited with pulsed nearinfrared laser light which makes them a suitable object to be detected in biological tissues. the main obstacle is to distinguish between the object of interest (emitted light) and the autofluorescence from the sample which limits the scope of application of aunp. therefore, our aim is to characterize the luminescent properties of such nanoparticles regarding their excitation and emission. in this study, the excitation and emission spectra of aunp with different sizes below nm in an excitation range of nm to nm were investigated. our study shows the emission spectra curves of aunp are broadband spectrum and vary with the changing of excitation wavelength from nm to nm. the results also suggest the minimum laser power necessary to trap the aunp depends on particle size and excitation light wavelength and a maximum power above which the particles are destroyed. in all the experiment above, to avoid the simultaneous effects from slide and cover slip is a must. rocking, tumbling, and sliding: real-time nanomotion of a membrane-bound virus p. kukura , h. ewers , c. mueller , a. renn , a. helenius , v. sandoghdar laboratory of physical chemistry, eth zurich, ch- zurich, switzerland, institute of biochemistry, eth zurich, ch- zurich, switzerland the interaction of a virus with its receptors in the plasma membrane is decisive for its infection of cells. optical studies have revealed that after binding, virus particles move laterally on the membrane, but the complexity of the cellular environment and the drawbacks of fluorescence microscopy have prevented access to the molecular dynamics of early virus-host couplings. here, we examine a model system, in which single simian viruses (sv ) interact with their gm ganglioside receptors in supported lipid bilayers. we employed scattering interferometry and single molecule fluorescence localization to visualize the vectorial rotational motion of virions. at low receptor concentration, we observed sliding and tumbling of single virions during rapid lateral diffusion. in contrast, at increased receptor concentration the virions repeatedly underwent periods of standstill, reminiscent of their behavior prior to endocytosis. by an unprecedented combination of millisecond time and nanometer spatial resolutions, we revealed that during these immobile periods, the virions rock back and forth among nanoscopic spots separated by nm. our insights, together with the structure of the viral capsid, suggest aggregation of receptors in nanodomains and recurrent swap of binding between receptor molecules and neighboring viral protein pentamers. herein, we have developed different membrane probes binding spontaneously to the outer plasma membrane leaflet and showing sensitivity to membrane properties such as surface charge and phase state. the first two types of probes were based on -hydroxyflavone fluorophore. one of them showed a high sensitivity to the surface charge and to the phase state of lipid bilayers, while the other was only sensitive to the surface charge. the third type, which was based on nile red fluorophore, was sensitive only to the phase state. surprisingly, the probes that were sensitive only to the surface charge did not respond to apoptosis, while the other two types of probes showed significant spectroscopic response to it. moreover, the latter exhibited response to cholesterol depletion, which was similar to that observed on apoptosis. thus, according to our data, the intact living cells present a remarkable fraction of the cholesterol-rich domains, while apoptotic loss of the transmembrane asymmetry decreases it dramatically. these probes represent a new tool for quantification of surface charge and cholesterol-rich domains in cell membranes. -imaging and spectroscopy - due to its porosity and unique optical properties porous silicon (psi) is an attractive template to develop biomaterials and biosensors. porous silicon microcavity (psimc) structures were prepared then functionalized for covalent protein attachment of glucose oxidase (gox) or solubilized bacteriorhodopsin (br). functionalization and protein infiltration was monitored by specular reflectometry sensitive to change in refractive index, when a molecule is attached to the large internal surface of psi. protein infiltration into the porous scaffold was confirmed by edx spectroscopy and the structures were imaged by biphoton microscopy. second harmonic generation and enhanced two-photon excited fluorescence emission from porous silicon was observed when resonantly exciting the structures. in addition, when the microcavities were infiltrated with gox or br, the proteins acted as a very efficient internal two-photon-excited fluorescence emitter, hence protein infiltration enabled the in-depth visualization of the porous structure by taking advantage of the optical sectioning capacity inherent to the non linear optical microscopy technique. patterning of bio-molecules: methods for characterization of neuron-substrate interfaces we investigated how to use and improve micro printing techniques to obtain molecules patterns on cell culture substrates. with micro contact printing (µcp), we generated geometrically defined depositions of poly-d-lysine (pdl) using poly-dimethylsiloxane stamps and optimized neuronal culturing conditions. rat and mice hippocampal neurons grown on those patterns showed to be alive and functional. we then applied µcp to study axonal development by combining cell culture assays with atomic force microscopy (afm) to investigate in more detail the molecule deposition on the surface and to measure morphological changes in the growth cone (gc) during the early phases of neurite development. we found distinct shapes of the gcs depending on whether they were growing on l adhesion molecule patterned by indirect-µcp or on pdl coated surfaces. we also attempted to transfer such patterns on multi electrode arrays in order to constrain neuronal cell bodies on the electrode area and to improve electrophysiological recordings from neuronal networks. other patterning techniques were therefore explored using a nano-drop printing system. patterned surfaces were analyzed with afm and scanning electron microscopy to combine different approaches aimed to the improvement and characterization of the printing techniques. insights on the mechanism of electron transfer in complex i a. l. maniero , c. bergamini , m. bortolus , r. fato , s. leoni , g. lenaz università degli studi di padova, padova, italy, università degli studi di bologna, bologna, italy complex i (nadh dehydrogenase) plays a central role in cellular energy production, transferring two electrons from nadh through a series of iron-sulfur clusters (fes) to ubiquinone (coenzyme q); the electron transfer is coupled to the translocation of protons across the membrane. the fes center n is the last acceptor in the electron-transfer chain, but the mechanism through which the enzyme couples the e − reduction of the fes centers to the e − reduction of ubiquinone (q→sq→qh ) is unclear [ ] . in our experiments, submitochondrial particles were treated with different inhibitors, in the absence or in the presence of different quinone analogues, and nadh addition initiated the electron transfer. we assess by epr (electron paramagnetic resonance) spectroscopy the relative abundance of the reduced n center and of the semiquinone radical, and coupled epr data to enzymatic activity assays and to fluorescence measurements on the effect of inhibitors on reactive oxygen species (ros) production. we identify two different classes of inhibitors showing different effects on ros production. moreover the redox state of the complex has shown to depend both on the inhibitors and on the quinone analogues. a possible mechanism of the electron transfer, that can explain the experimental findings, will be presented. conventional fluorescence microscopy suffers from a resolution limit imposed by the diffraction of light. stimulated emission depletion (sted) microscopy overcomes this resolution barrier without being limited by the wavelength by taking advantage of the photophysics of the observed sample into the image formation process, and has proven to be a powerful approach for exploring relevant biological issues. the outstanding resolution of sted microscopy is achieved by drastically minimizing the spatial extent of the focal region from which fluorescent molecules can emit signal in the sample. so far, mainly complex and relatively expensive lasers systems providing pulsed beams have been used to inhibit the fluorescence in the outer area of the focal region. here we report on the development of a new setup based on a fast beam scanning confocal microscope using compact turn-key and inexpensive continuous wave lasers. the great potential of this simple configuration is demonstrated for a selection of commonly used fluorescent markers. characterization of hepatitis b antigen particles by atomic force microscopy each year, over one million people die from hepatitis b virusrelated chronic liver disease, including cirrhosis and hepatocellular carcinoma. the major surface antigen of hepatitis b virus (hbsag) is a cysteine-rich, lipid-bound protein with amino acids. recombinant hbsag produced in yeast can self-assemble into -nm immunogenic spherical particles that are used in licensed hepatitis b vaccines (protein/lipid ratio is / in mass). hbsag size and shape have been mainly investigated by transmission electron microscopy after negative staining of the particles. however, under these conditions, no details of the particle surface can be obtained because of the shadowing effect due to the uranium salts. here we describe new structural insights of hbsag particles using atomic force microscopy (afm) performed under physiological conditions. we applied atomic force microscopy to define structural details of the surface organization with a resolution in the nanometer range. as expected, the diameter of hbsag particles is , ± , nm in average. the surface of these particles clearly shows the presence of protuberances that most probably correspond to proteins. indeed, reduction and alkylation induces the disappearance of the protuberances. the number of the protuberances estimated from afm micrographs is about per spherical particles. a biophysical study of equine herpesvirus- entry into cells g. mckenzie , p. o'shea , j. kydd , c. rauch school of veterinary medicine and science, university of nottingham, uk, institute of biophysics, imaging and optical science, university of nottingham, uk equine herpesvirus- (ehv- ) can cause respiratory disease in young horses, varying degrees of paralysis and abortion during the later stages of pregnancy. furthermore, there has been a recent increase in the number of outbreaks involving paralysis of thoroughbred horses in training. virus disseminates rapidly after initial infection via cell associated viraemia. controlling this may prove crucial in combating the pathogenicity of the disease. we have investigated the cellular interactions of ehv- . initially, binding of ehv- to fluorescein phosphatidylethanolamine (fpe)-labelled phospholipid vesicles of various compositions was examined. a variety of microscopy techniques were then employed to study the events surrounding the binding of virus to equine peripheral blood mononuclear cell (pbmc) membranes. confocal microscopy images have highlighted possible colocalisation of ehv- with membrane 'rafts'. total internal reflection fluorescent microscopy was then used to identify viral binding at the membrane with high contrast images, able to observe single virus particles. establishing the viral entry pathway into pbmcs would allow drugs that target this process to be employed, reducing clinical viraemia. the interpretation of in-vivo binding rate measurements allows inferring the molecular interactions that regulate cellular functions. fluorescence recovery after photobleaching (frap) is a widely used tool to quantify binding reactions in vivo. however the lack of "golden standards" for these measurements requires the measured binding rates to be validated with other techniques. we present a new fluorescence correlation spectroscopy (fcs) method to measure the in vivo bound fractions and residence times for molecules that interact with an immobile substrate. we apply this method to measure binding of mutants of the transcription factor vbp (vitellogenin binding protein) to the dna. comparison of fcs with frap results in comparable estimates of the measured diffusion constants and bound fractions. however, fcs provides an estimate of the vbp residence time at the dna, while frap does not. this limitation in the analysis of vbp is due to the larger photobleaching volume used in frap, if compared to the observation volume of an fcs experiment. in sum, we present a method to measure binding rates with fcs. substantial agreement of this method with frap is shown. however, further validation on tightly bound molecules will be necessary to assess if frap and fcs agree in the measurement of residence times. -imaging and spectroscopy - we have investigated the changes in the mechanical properties of the zona pellucida (zp), a multilayer glycoprotein coat that surrounds mammalian eggs, that occur after the maturation and fertilization process of the bovine oocyte by using atomic force spectroscopy. the response of the zp to mechanical stress has been recovered according to a modified hertz model. zp of immature oocyte's shows a pure elastic behaviour. mature and fertilized oocyte's zps evidence, instead, a transition from a purely elastic behaviour, which occurs when low stress forces are applied, towards a plastic behaviour has been observed. the high critical force necessary to induce deformations, that well supports the non-covalent long interactions lifetimes of polymers, increase after the cortical reaction. afm images show that oocytes' zp surface appear to be composed mainly of a dense, random meshwork of nonuniformly arranged fibril bundles more wrinkled surface characterize marure oocytes with respecto to immature and fertilized oocytes from a mechanical point of view, the transition of the mature zp membrane toward fertilized zp, through the hardening process, consists in the recovery of the elasticity of the immature zp, while maintaining a plastic transition that, however, occurs with a much higher force with respect to that required in mature zp. dynamical behavior of ribose and deoxyribose supercooled water solutions s. e. pagnotta , s. cerveny , a. alegria , j. colmenero centro de fisica de materiales, centro mixto csic-upv/ehu, san sebastián, spain, departamento de fisica de materiales, universidad del pais vasco (upv/ehu), facultad de quimica, san sebastián, spain, donostia international physics center, san sebastián, spain ribose and '-deoxyribose are probably the most widespread monosaccharides in nature. they can be extensively found in ribonucleic acid (rna) and '-deoxyribonucleic acid (dna), respectively, where they form, together with a nitrogenous base and a phosphate group, a peculiar building-block structure called nucleotide. in the present work, the relaxation dynamic of ribose and deoxy-ribose water solutions at different concentrations has been studied by broadband dielectric spectroscopy and differential scanning calorimetry in the temperature range of - k. two relaxation processes are observed for all the hydration levels; the slower (process i) is related to the relaxation of the whole solution whereas the faster one (process ii) is associated with the reorientation of water molecules in the mixture. as for other polymeric water solutions, dielectric data for process ii indicate the existence of a critical water concentration above which water mobility is less restricted. moreover, according with these results, atr-ftir measurements of the same sugar solutions showed an increment in the intensity of the oh stratching sub-band close to cm − as water content increases. the regulation of the formation of cytoskeletal protein complexes by actin-binding proteins m. nyitrai, a. vig, t. kupi, z. ujfalusi, s. barkó, g. hild university of pécs, faculty of medicine, department of biophysics, pécs, hungary in living cells various groups of proteins are associated to supramolecular actin filament structures, often in a nucleation factor dependent manner. for example, actin structures associated with formins can bind tropomyosin and profilin, while those polymerised by the nucleation of the arp / complex bind cofilin and myosin i. the molecular mechanisms underlying the regulation of the formation of these protein complexes is still ambiguous. we have shown recently that formins can bind the actin filaments and change their conformational state. subsequent binding of other actin-binding proteins, such as tropomyosin and myosin, can reverse these changes. it appears that the reversal effect assumes that the actin-binding protein binds the filaments in a well-defined and specific binding site. the altered conformational state of the actin filaments observed after the binding of these proteins provides a possible explanation for the modified affinity of the filaments for other-actin binding proteins. based on the results available so far we assume that the affinities are modified differently by different nucleation factors, and the conformational changes introduced to actin by actin nucleation factors can serve as the molecular bases for the regulation of the formation of actin based intracellular protein complexes. experiments are currently in progress to test and further corroborate the existence of such regulatory mechanisms in living cells. correlation between sub-cellular distribution of photoactive drug hypericin (hyp), determined by applied delivery systems (hyp vs hyp/ldl), and mode of the cell death is addressed in this study. co-localization of hyp with mitochondria, lysosomes and golgi apparatus in u- mg glioma cells was determined by confocal laser scanning microscopy in using organelle specific fluorescent dyes as well as by time resolved fret experiments. flow cytometry experiments were realized to study a photodynamic effect of hyp ( nm/ jcm − ) on cells. significant differences in the proportional representation of live, apoptotic and/or necrotic cells were observed for different types of delivery systems of hyp hours after hyp ( x − m) photoactivation. conclusions: i) sub-cellular distribution of hyp depends on using delivery systems, ii) the mode of cell death depends more on concentration of hyp inside cells, than on different type of delivery systems (for non selective wide-field cell illumination), iii) fluorescence lifetime is sensitive parameter to study sub-cellular distribution of hyp. novel time-resolved spectroscopic methods have been used to investigate the interactions between a fluorescently-labelled mutant of the peptide melittin and supported lipid bilayers, formed by self-assembly at a silica-water interface via vesicle deposition. time-resolved evanescent wave-induced fluorescence spectroscopy (trewifs) is a surface-selective technique in which the evanescent field from a pulsed laser source is used to photoexcite fluorescent species at an interface. the resulting fluorescence decay kinetics, measured using time-correlated single-photon counting, report on the micro-domains experienced by those fluorescent species at an interface. extending trewifs to time-resolved evanescent wave-induced fluorescence anisotropy measurements (ew-trams) provides dynamic rotational information of a fluorescent species, reporting on its mobility at an interface. presented here are trewifs and ew-trams data obtained from the fluorescence of an alexa -labelled melittin mutant interacting with a dipalmitoylphosphocholine bilayer at room temperature, physiological ph and ionic strength. the results provide new insights into the conformation, location and motion of cytolytic peptides interacting with cell membranes. optical tweezers are used to controllably apply forces to red blood cells and the resulting chemical and structural changes are monitored using raman spectroscopy. the forces are applied in vivo and mimic that which the cell undergoes mechanically as it passes through vessels and smaller capillaries. the first result presented will be spectroscopic evidence of a transition between the oxygenation and deoxygenation states of hemoglobin that is caused by the stretching of the red blood cell. the transition is due to mechanically induced enhancements of hemoglobin-membrane and hemoglobin neighbor-neighbor interactions. the latter, lesser known effect is further studied by modeling the electrostatic binding of two of the protein structures using molecular dynamics methods. secondly, polarized raman spectroscopy is utilized to study the packing and ordering of the hemoglobin proteins in the red blood cell as it is stretched. depolarization ratios for a number of heme group modes change, indicating that the applied force additionally packs and orders the proteins inside the cell which further demonstrates the role of cell deformation in the oxygen transport kinetics. acknowledgements: this work was supported by miin fis - (spain) and fundació cellex barcelona fret microscopy is widely used to study protein-protein interactions in living or fixed specimens. currently, most commonly used visible fluorescent proteins for live-cell fret studies are the cerulean and venus variants of the cyan and yellow fluorescent proteins. even though this fret pair appears to be ideal for monitoring protein-protein interactions, the most commonly used fixed laser wavelengths do not excite cerulean at peak absorption. recently, we characterized an ideal donor, the monomeric teal fluorescent protein (mtfp), which is excitable using the commonly available ( ) opt. june/july, ). we used teal as a donor for various red fluorescent proteins as acceptors including tdtomato, mko , morange , mtagrfp, mkate. we have employed a "fret standard" genetic construct to minimize variability in separation distances and positioning of the fused donor and acceptor fps. using spectral fret imaging and fluorescence lifetime measurements in living cells expressing the fused proteins, we have characterized both sensitized acceptor emission and the change in the donor lifetime distribution as a result of quenching for each of the fused fp pairings. our results indicate that some red fps are better acceptors than others in terms of quenching the teal donor and sensitizing the emission of the acceptor indicating a fret event. s. m. perepelytsya, s. n. volkov bogolyubov institute for theoretical physics, nasu, -b metrologichna str., kiev, , ukraine stability of the dna double helix is determined by na + counterions, neutralizing the negatively charged phosphate groups of the macromolecule backbone. but under spatial conditions they may be replaced by much heavier ions, for example cs + . to determine the influence of heavy counterions on internal dynamics of the double helix the conformational vibrations of na-and cs-dna are studied. for this purpose the model of conformational vibrations of dna with counterions is used [perepelytsya s.m., volkov s.n., eur.phys.j. e. , , ] . as the result the frequencies and amplitudes of vibrations for b -dna with na + and cs + counerions are calculated. the frequencies of internal modes of the double helix are about , , and cm − . the frequencies of ion-phosphate modes are about and cm − for na-and cs-dna respectively. the calculated amplitudes of vibrations show that light counterions not disturb the dna internal dynamics, but heavy counterions make move all structure elements of the double helix. using the valence-optic approach the intensities of the dna vibrations in raman spectra are calculated. the calculations show that the ion-phosphate mode in cs-dna spectra is prominent, in contrast to na-dna spectra, where it has very low intensity. obtained results describe the intensity increase of the band cm − in cs-dna spectra that was observed in [bulavin l.a., et al., arxiv: . v ]. s. soria , f. quercioli , r. mercatelli , f. bianco , i. cacciari , g. righini centro studi e ricerche "e. fermi", p. del viminale , rome, italy, ifac-cnr, istituto di fisica applicata "n. carrara", via madonna del piano , sesto fiorentino (fi), italy, isc-cnr, istituto dei sistemi complessi, via madonna del piano , sesto fiorentino (fi), italy we report on the application of a simple white light source based on the supercontinuum generation from commercial photonic crystal fibres to confocal fluorescence microscopy and fluorescence lifetime imaging (flim) microscopy. the coherent white light can be tuned by varying the wavelength and intensity of the pump, a ti:sapphire laser. there are several advantages jn the use of sc sources: spatially coherent white radiation, tuning ranges of approximately nm, high brightness, a robust compact system (potentially all-fibre) and relatively low cost. being pulsed, sc sources are suitable for flim. we have used this system for measuring foerster resonance energy transfer (fret) in order to study interactions between ions channels and proteins of membrane within lives cells resolving the quaternary structure of plague f capsular antigen a. soliakov , j. r. harris , m. r. hicks , a. rodger , r. woody , a. watkinson , j. h. lakey cell and molecular biosciences inst., newcastle univ., uk, chemistry dept., univ. of warwick, coventry, uk, biochemistry and molecular biology dept., colorado state univ., usa, pharmathene uk, billingham, cleveland, uk most gram-negative pathogens express multi-subunit fibres on their surfaces that mediate host cell attachment, biofilm formation, invasion of host defenses and protection against phagocytosis. here we have studied capsular antigen fraction (or caf ), secreted through the conserved chaperone/usher pathway by the plague agent yersinia pestis [ ] . caf is highly immunogenic and is used in a recombinant subunit vaccine against plague. recent immunological studies indicated vaccines containing polymeric caf have higher protective efficacy than vaccines containing its monomeric variant. this difference in protective efficacy was attributed to the quaternary structure. however the quaternary structure of caf has not been characterized [ ] . in our study we have used transmission electron microscopy of negatively stained specimen and linear dichroism spectroscopy to determine the quaternary structure of recombinant caf . whereas electron microscopy revealed morphology of caf fibres bound to the surface of a carbon coated grid, linear dichroism gave the orientation of subunits in flow aligned caf fibres in solution. our results show recombinant caf comprises extended linear fibres and circularized fibres, both of which have high degree of conformational freedom. despite the vast application of lambert-beer law in biology, this empirical law cannot accurately describe the light absorption process of molecules with a long-lived excited state. this family of molecules includes most fluorescent molecules, which are very important in biology. lambert-beer law is in fact only valid at very low intensity of incident light and very low concentration of chromophore. if the system doesn't meet these conditions, it falls into a nonlinear regime when it is affected by a phenomenon which we call "dynamic photobleaching": the depletion of chromophores from the first layers, due to their transition to the excited state, leads to a sub-exponential propagation of light in the medium [ ] . this phenomenon leads to the necessity of a new formula for the light absorption dynamics which depends on the lifetime of the excited state of the chromophore. the predictions of the theory were successful in describing the absorption dynamics of azobenzene [ ] , but now they have been tested also on a biologically relevant molecule like chlorophyll. the results indicate that the absorbance is affected by the intensity of the incident light, and it is therefore a non reliable way of determining, for example, the concentration of the molecule. annular pupil filter to improve spatial highfrequency signal to noise ratio in linear and non linear microscopy e. ronzitti , v. caorsi , a. diaspro lambs, microscobio, department of physics, university of genoa, genoa, italy, semm, ifom-ieo, university of milan, milan, italy, neuroscience and brain technologies department, iit, genoa, italy shot-noise significantly affects and deteriorates the imaging capabilities of typical two-photon excitation and confocal laser scanning microscope, especially in biological applications where the detected signal can be remarkable slight [ ] . in particular, shot-noise substantially influences the spatial high-frequency range inducing a remarkable reduction of the optical transfer bandwidth. the insertion of an annular filter on the microscope objective lens in the illumination light pathway is here proposed to retrieve the high frequencies information loss [ ] .the electromagnetic interference effect induced by the filter insertion, gives a redistribution of the optical transfer function. in particular, the microscope frequency response in filter scheme exhibits an enhancement of signal to noise ratio at the high frequencies able to recover the high frequencies hampered by shot-noise [ ] . the goal of biomarker studies is to develop simple noninvasive tests that identify disease states. focus is beginning to shift from identification of individual biomarkers to identification of biomarker panels comprising multiple targets of different molecular species. there are, however, no current technologies available that allow for a comprehensive and simultaneous analysis of the expression levels of multiple cellular components, i.e. proteins and rna. surface plasmon resonance (spr) polaritons are surface electromagnetic waves that propagate in a direction parallel to the interface between a metal surface and an external medium e.g., liquid. these oscillations are very sensitive to any change of this boundary, a phenomenon that has been exploited to facilitate label-free, real-time detection of biological interactions, e.g. protein binding interactions. we are utilising the power of spr to develop technologies that facilitate diagnostic procedures for complex diseases such as alzheimer's disease and chronic obstructive pulmonary disease, through identification and detection of patterns of biomolecules indicative of disease. our approach will facilitate better disease characterisation, improve early detection strategies and aid drug discovery. surface generated fluorescence detection by supercritical angle confocal microscopy d. verdes, m. rabe, s. seeger institute of physical chemistry, university of zürich, winterthurerstrasse , zürich, switzerland a two channel confocal microscope for surface and simultaneously in solution fluorescence detection is presented. the microscope's core element is a parabolic mirror objective that collects the fluorescence above the critical angles for total internal reflection (tirf) of the water/glass interface. an aspheric lens incorporated into the parabolic mirror is used for diffraction limited focusing and collecting the fluorescence at low angles with respect to the optical axis. this low angles excitation approach is technically straightforward and gives an advantage over high numerical objectives that require very high angles for tirf illumination. by separating the collection of the fluorescence into supercritical and subcritical angles, two detection volumes highly differing in their axial resolution are generated at the interface. the surface selectivity of the detection volume is obtained on the basis of the dipole emission profile near a dielectric interface. its angular distribution is highly anisotropic and consists of a superposition of traveling and evanescent waves, which both are detected using the parabolic mirror objective. unlike with objective tirf microscopy, the parabolic mirror objective achieves easily diffraction limited excitation/detection volume at the water/glass interface. the objective optical performance is shown by measuring the actin cytoskeleton of cultured cells, fret energy transfer within adsorbed clustered proteins and single molecules detection. differential polarization laser scanning microscopy. anisotropy in biological samples g. steinbach , i. pomozi , g. garab biological research center, hungarian academy of sciences, szeged, hungary, pi vision bt., budapest, hungary differential polarization (dp) spectroscopy provides unique information on the anisotropic organization of biological samples [tinoco et al. ann rev biophys biophys chem : ]. however, anisotropy is often a microscopic property. the dp-lsm, constructed in our laboratory enables us to image the main dp quantities: linear and circular dichroisms (ld&cd), also in fluorescence detection ( . further applications include: periodic structure of isolated amyloids, anisotropy variations in cell walls related to drought resistance, and strong anisotropy of 'artificial chlorosomes', nanorods of synthetic porphyrins. dp-lsm might thus represent a novel tool in the better understanding of highly organized molecular macroassemblies. t. m. staudt , p. bingen , c. kempf , h. horstmann , j. engelhardt , t. kuner , s. w. hell german cancer research center/ bioquant, heidelberg, germany, max planck institute for biophysical chemistry, göttingen, germany, institute for anatomy and cell biology, university of heidelberg, heidelberg, germany the calyx of held, a large glutamatergic synaptic terminal in the auditory brainstem circuit has been increasingly employed to study presynaptic mechanisms of neurotransmission in the central nervous system. a highly detailed model of the morphology and distribution of cytoskeleton, synapsin, synaptic vesicles, calcium sensors, mitochondria, the presynaptic membrane and its active zones is derived by colocalization analysis of these different key elements of synaptic transmission in the rat brain. the various cellular components are visualized with subdiffraction resolution by stimulated emission depletion (sted) microscopy. imaging individual structural elements exhibit a focal plane resolution of < nm inside µm thick tissue sections. -imaging and spectroscopy - r. worch, t. weidemann, p. schwille biotec, biophysics group, technische universität dresden, germany interleukin- (il- ), a small soluble protein, is a principal regulatory cytokine, playing an important role during the maturation and clonal expansion of antigen specific b-cells in mammals. at the plasma membrane, il- is recognized by a receptor that consists of two single spanning transmembrane proteins: a high affinity il- r alpha chain, and a low affinity il- r gamma chain. it is still controversial by which molecular mechanism the signaling complex is fully activated: dimerization of chains, large conformational change upon il- binding or a combination of both. moreover, the influence of the lipid environment in which the activation takes place is poorly characterized. to address these issues we aim to reconstitute the receptor component in artificial membrane systems to study the various mutual interactions by means of fluorescence-based techniques, mainly fluorescence correlation spectroscopy. due to reduced background in a chemically defined system this may provide details not yet accessible in the living cell. k. wicker, s. sindbert, r. heintzmann randall division of cell and molecular biophysics, king's college london, se ul london, u.k. fluorescence confocal microscopy, an indispensable tool of modern biology, allows the imaging of live fluorescent specimen with high lateral as well as axial resolution. through the introduction of a sufficiently small confocal pinhole, the lateral resolution can be enhanced compared to that of a wide field microscope. however, this gain in resolution comes at the cost of a decrease in detection efficiency, as light blocked by the pinhole is lost. we present a method for improving the lateral resolution (extending work of sandeau et al. [ ] ) and detection efficiency of scanning microscopes by adding an interferometer with image inversion in one of its arms to the detection pathway [ ] . this surpasses the lateral resolution achievable in a conventional confocal microscope (closed pinhole) while increasing the detection efficiency substantially. point spread function measurements for a uz-interferometer (uzi) are shown. the light in this setup follows threedimensional u-and z-shaped paths and relies on reflections off planar surfaces only in order to achieve image inversion. we achieved an interference contrast of % for white light, and excellent agreement with theoretical predictions. g. vicidomini, a. schönle, j. keller, r. schmidt, c. von middendorff, s. w. hell max planck institute for biophysical chemistry, department of nanobiophotonics, göttingen, germany fluorescence far-field microscopy is a indispensable tool in modern life science. however, the resolution of its standard variants is limited by diffraction to ∼ nm in the focal plane and ∼ nm along the optical axis. to overcome this limit, a new class of super-resolution microscopy techniques has been designed. one example is pi microscopy. to obtain isotropic resolution, pi uses interference of the wave fronts from two opposing lenses. the point spread function (psf) of pi system is characterized by multiple maxima/sidelobes, which replicate the object in the image. therefore image restoration is mandatory to render unambiguous imaging. the situation is further complicated because the positions and the relative heights of the multiple maxima/sidelobes of the psf depend on the phase difference (pd) between the two wave fronts at the focus. if the refractive index of the sample varies in space, this pd becomes a function of position and pi image formation process looses its shift invariance. the pd function (pdf) may not even be known a-priori and must then be estimated from the image, leading to a blind image restoration problem. here, we propose a maximum a-posteriori based method to solve the problem. we either assumed a mathematical model for the pdf that depends on a small number of parameters or allowed for an arbitrary pdf but introduced a smoothing constraint. designer multicomponent lipoplexes have recently emerged as especially promising transfection candidates, since they are from to times more efficient than binary complexes usually employed for gene delivery purposes [ ] [ ] [ ] . here, we show, for the first time, that after internalization binary complexes of lower transfection potency remain in compact perinuclear endosomes, while multicomponent systems have intrinsic endosomal rupture properties that allow plasmid dna to escape from endosomes with extremely high efficiency [ ] . endosomal rupture results in an extraordinarily homogeneous distribution of unbound plasmid dna throughout the cytoplasm and in the nucleus [ ] . fluorescence spectroscopy and stopped-flow technique were utilized for the study of the kinetics of incorporation of hypericin (hyp), a natural photosensitizing pigment, into lowdensity lipoproteins (ldl). triphasic kinetics of hyp association with ldl was observed when solutions of hyp and ldl were mixed together. the most rapid phase of hyp incorporation is completed within tens of msec, while the slowest one lasts - min. the most of hyp molecules are incorporated into ldl in the slowest phase. the kinetics of the incorporation of hyp into ldl particles pre-loaded with hyp were also investigated. the observed decrease of the lifetime and total intensity of hyp fluorescence with the increase of the incubation time of hyp with hyp/ldl complex is a sign of the formation of aggregates and the dynamic quenching of singlet excitation state of hyp inside ldl. to study the kinetics of a transfer of hyp molecules between ldl particles, the time evolution of the stopped-flow and time-resolved fluorescence experiments were investigated after the mixing of the complex hyp/ldl= : with appropriate amounts of free ldl. for each final hyp/ldl ratio the increase of the lifetime and total intensity of hyp fluorescence was observed. the half-time of this process is similar to that one of the slowest phase of hyp incorporation into free ldl. a. bosca , r. magrassi , g. firpo , l. repetto , c. boragno , u. valbusa italian institute of technology, genova, italy, nanomed labs (difi-cba), genova, italy the technique of choice to measure the electrophysiological activity of neuronal cells is the so called patch-clamp method because of its precision and sensitivity; this procedure could play a major role also in the investigation of the behavior of a biological neuronal network and so represents an important tool for understanding its functionality and for screening the effects of drugs and compounds on it. the final aim of this project is the development of a planar patch clamp device suited to measure simultaneously the electrical activity of cultured neurons associated in a network. this device will be made of polymeric disposable material and will include a microfluidic perfusion system. in order to create a smooth micro sized pore in a thin polymeric membrane we exploited the prototyping capabilities of focused ion beam etching and we extended the air moulding technique by combining it with soft moulding, obtaining micro structured substrates with the requested features. we also subjected our substrates to a chemical treatment capable of rendering its surface stable and hydrophilic and we verified that it makes them suitable for neuron culturing. use of lipoamino acids for nasal delivery of therapeutic proteins c. bijani , s. sice , j. elezgaray , c. degert , o. broussaud , e. j. dufourc physica pharma, pessac, france, umr cbmn, cnrs-université bordeaux , iecb, pessac, france most of the therapeutic proteins are clinically administered through an intravenous injection several times a day / week. because the repeated injections are not convenient and cause pain in patients, an alternative route of administration is desirable such as oral or nasal. unfortunately, proteins are easily degraded by proteolytic enzymes in the gastrointestinal tract and, therefore, have a low bioavailability when administered via oral route. physica pharma has gained experience in forming sprayable solutions combining lipoamino acids (laa) and small therapeutic organic compounds with the aim to improve their intranasal absorption. in the present work we investigated the ability of laa to complex large molecule such as human erythropoietin, human growth hormone and salmon calcitonin in order to form easily sprayable colloids. these three proteins are used respectively to treat anaemia, growth problem and hypercalcemy. circular dichroism and dynamic light scattering were used to further characterize the laa-protein colloids. a specific molar ratio of laa versus protein was found for wich the proteins keep their secondary structure and have an overall isotropic size slightly increased. molecular dynamics show that proteins are indeed coated with laa. such a complex is shown to pass very easily through a culture of nasal cells growth at confluence. in this study we describe two systems based on soft matter designed for the drug delivery and for the replacement of synovial fluid in osteoarticular pathologies. (i) a new class of temperature sensitive hydrogels pva/poly(ma m nipaam n ) shaped as microspheres obtained with a water-in-water emulsion photocrosslinking reaction. microparticles of pva/poly(ma m nipaam n ) with m:n theoretical molar ratios equal to : ; : , : have been studied in terms of average size and responsiveness to temperature characterized by confocal laser scanning microscopy (clsm), dynamic light scattering (dls) and differential scanning microcalorimetry (dsc). (ii) a physical network based on hyaluronic acid with a small extent (degree of substitution: %) of hydrophobic moiety grafted on the backbone, hyadd , has been characterized in order to account for the influence of thermal treatment on the stability of the hydrogel. dynamic light scattering (dls) and small angle neutron scattering (sans) have been used for dynamic-structural characterization of hyadd hydrogels. diffusion of macromolecular probes has been studied by fluorescence recovery after photobleaching (frap) to study the mesoscopic texture of the hydrogel and molecular dynamic (md) simulations were used to approach the time evolution of the physical junction points and of chains clusters. multi-scale estimation of water soluble diffusivity in polysaccharide gels g. eisele , s. bertini , d. paganini , l. piazza ronzoni institute, milan, italy, distam, university of milan, italy diffusion properties in gels play important role in food and biotechnological applications. an attractive goal is to design gels in such a way that the active molecules are delivered by the material according to specific release sequences. the transport of macromolecules within polymeric gels depends: on the obstruction effects of the surrounding gel strands, on the molecular interactions between the gel and the solute, on the interactions between the solute molecules themselves and the interactions between the solute and the solvent. physical polysaccharide gels were evaluated in this work to probe diffusion over both microscopic and macroscopic distance scales. physical gels from agar and starch were investigated by high and low resolution nmr techniques in order to characterize their structures. obstruction effects of the surrounding gel strands were considered by studying diffusion of glucose. local diffusion, due to brownian motion, was quantified by low resolution nmr spectroscopy. the fickian diffusion coefficient was measured by modelling experimental concentration-distance curves obtained by means of a two-compartment diffusion-cell. diffusion coefficients depend on the viscoelastic properties of the gel matrix and on water-polysaccharide interactions. j. l. cuellar, g. koehler, m. fischlechner, e. donath medizinische institut für physik und biophysik, leipzig, germany rather than being just pathogens, from the view point of biotechnology and materials science viruses can be regarded as nanocontainers in which nucleic acids have been enclosed in a protective self assembled protein cage. in some viruses an additional lipid-protein envelope wraps the capsid shell. the capsid is constituted of several copies of one single protein subunit or just a few, arranged in a regular fashion showing icosahedral symmetry. the mechanical properties of the cage are important for understanding the infection mechanism involving the release of the encaged genome. by means of atomic force microscopy, it becomes possible to probe such material properties by nanoindentations of single viral particles. it is very interesting to learn how strong or brittle a virus can be. here we have studied the mechanical properties of empty rubella virus particles (rlps) due response of external applied forces. we found that rlps are extremely soft comparable to that of some rubber materials. a peculiarity of the rubella virus is that the capsid is considerably smaller than the surrounding shell filling only a fraction of the lumen provided by the envelope. the envelope in rubella has a distinguishable response on the material properties of the virus. deformation and fracture of the capsid requires comparatively larger forces. our results indicate that the ph is a major factor influencing on rubella particle material properties. this can be related to the infection mechanism. pentavalent antimony (pa) (glucantime, sanofi-aventis or glaxo) are the mainstream agents of choice for leishmaniasis treatment. in therapeutic doses the pa treatment has cardiac side effects like electrocardiographic (ecg) alterations, that include qt segment prolongation, t wave flattening or inversion, inversion of st segment, p, r and t waves amplitude reductions, torsade de pointes arrhythmias and sudden death by cardiac arrest. the objective of this study was to characterize the arrhythmogenic potential of pa. we used guinea-pig to assess the chronic effects of pa therapeutic dose on corrected qt interval, qt dispersion, ventricular action potential (ap) amplitude and duration at % and % of maximal repolarization and survival rate. guinea-pig received daily mg/kg pa or saline for days. eight lead ecg were recorded before and in the last treatment day. at the end the animals were killed and the left ventricle papillary muscles excised for ap recording with the intracellular microelectrode technique. our results of chronic pa treatment showed significant increase of qrs complex duration, qt interval duration, qt dispersion and incidence of t wave flattening or inversion and arrhythmias. the ap analysis demonstrated prolongation at % duration. the treatment was lethal in % of the animals. we concluded that pa is a proarrhythmic drug that upon chronic use may causes arrhythmias and mortality by disturbances in the ventricular repolarization process. m. m. khvedelidze , t. j. mdzinarashvili , t. partskhaladze , n. nafee , m. schneider ivane javakhishvili tbilisi state university, tbilisi, georgia, institute of molecular biology and biological physics, tbilisi, georgia, biopharmaceutics and pharmaceutical technology, saarbrücken, germany, pharmaceutical nanotechnology, saarbrücken, germany we have studied the thermodynamical properties of chitosan-coated nanoparticles (cnp) and non-coated nanoparticles (np) and have gained some insights about plga nanoparticles' properties using supersensitive differential microcalorimetry. the experiments show that in a wide ph interval the changes in transition temperature did not take place. it was shown that such nanoparticles could be used in acidic surrounding for drug transfer. stability and their other properties are less depended on either the particles were in bidistilled or deionized water, or the suspension of particles were located in buffer. to determine the interaction of plga nanoparticles with dna. in the case of dna presence in cnp solution the calorimetric experiments show that the heat absorption peak is constricted, what biophysically means that interaction between them takes place. for more exact determination the contribution of cnp in spectrum, we have compared the spectra of pure dna with the spectrum of the same concentration dna plus cnp. the optimal ratio for dna loading onto the cnp was found to be : . protein-based "epoxy-like" physical hydrogels for stem cell transplantation s. c. heilshorn, c. wong po foo, j. s. lee materials science and engineering, stanford univ., u.s.a. stem cell transplantation has emerged as a promising therapy for multiple injuries and diseases; however, cell survival after transplantation is often poor and unpredictable. we hypothesize that co-injection of stem cells encapsulated within an optimized physical hydrogel will enhance viability. whereas, current physical hydrogels require a shift in environmental conditions (e.g., ph, temperature) to initiate the sol-gel phase transition during encapsulation, our newly designed molecular-recognition gels do not require environmental triggers. instead, these "physical epoxy-like" gels consist of two components that undergo a sol-gel transition upon mixing due to specific hydrogen bonding. the gel viscoelasticity is predictably tuned through precise variation in the molecular-level design of the two components, created using recombinant protein techniques. the design of the two components is based on simple polymer physics considerations and utilizes bio-mimimcry. adult neural stem cells or mesenchymal stem cells are encapsulated within these gels with high viability at constant physiological conditions. the gels promote the growth and differentiation of neural progenitors into neuronal phenotypes, which adopt a d-branched morphology. the gels are further optimized for use in the central nervous system by tethering neuroprotective peptides to the gel through molecular-recognition sites. these peptides are released-on-demand by cells through the action of proteolytic enzymes. we consider the integration of the portal protein of the bacteriophage virus φ into lipid bilayers of giant unilamellar vesicles (guvs) membranes with the aim of constructing a functional cargo-device able to transport dna and later translocate it outwards. our nano-engineering plan consists of growing guvs from bilayer membranes built up from proteoliposomes previously prepared by extrusion. we have designed two alternative chemical routes for integrating the portal protein in the lipid bilayer, the first considering the native protein and the second a mutant modified with a hydrophobic belt made of histidine-tags. in our contribution we will present details on the different nano-engineering strategies and experimental evidence about the integration of the portal protein in the membrane with an orientation adequate to allow for functional dna translocation. p.-h. guelluy , m.-p. fontaine-aupart , m. hoebeke biomedical spectroscopy, ulg, belgium, laboratory of molecular photophysics, univ. of paris-sud, france ppme is a second generation photosensitizer (ps), and a promising candidate for photodynamic therapy (pdt) treatment. we have previously demonstrated that ppme can be easily and efficiently encapsulated in dmpc liposomes, used as ps-vector. we therefore compared the photophysical and photochemical properties of free and encapsulated ppme incubated with human carcinoma cells. absorption and fluorescence microspectroscopy as well as flim analysis allow evaluating the aggregation state of ppme inside the different cellular organelles and the extracellular medium. confocal microscopy established undoubtedly the colocalization of ppme, by robust probabilistic exclusion method, within mitochondrion (central siege of apoptosis). after ps activation ( h and h), the balance apoptosis-necrosis was double-estimated by facs device and fluorescence confocal microscopy. quantification of hydroxyl radical was purchased by spin trapping-esr spectroscopy and quenching technique. all these techniques required peculiar settings because of the fluorescence activity of ppme. these results allow to ascertain that the vectorization of ppme affords a better cellular penetration and a monomeric state of ppme. the presence of ppme inside mitochondrion orientated the cellular death in apoptosis h following ps-activation. but necrosis is the major actor h after treatment. active substrates to study mechanotransduction j. le digabel, p. hersen, b. ladoux laboratoire matière et systèmes complexes, université paris diderot -cnrs, paris, france cellular processes imply an important coordination of interactions with the extracellular medium. accumulating evidences demonstrate that cell functions can be modulated by physical factors such as the mechanical forces acting on the cells and the extracellular matrix, as well as the topography or rigidity of the matrix. these extracellular signals can be sensed by mechanosensors on the cell surface or in the cell interior to induce various cell responses. we have developed an original approach based on micro-fabricated substrates of polydimethylsiloxane (pdms) to study cell migration. we used a closely spaced array of flexible micropillars (diameter∼ ?m) to map the forces exerted by cells on their substrates. in this case, the micropillars act as passive force sensors. here, we propose to analyze the cell response to an external applied stress by a well-controlled actuation of the substrate. to do so, we used magnetic pillars. such substrates allow us to modify dynamic adhesion conditions of cells and to better understand the coupling phenomena between mechanical sensing and biochemical activity of a living cell. using polyacrylamide hydrogels doped with ferromagnetic iron oxide particles or ferrofluids, we can make magnetic pillars with diameters of to microns while a magnetic field can be locally applied with a magnetic needle. such a technique will be helpful to study the mechanical response of cells to an external force or to local changes in their microenvironment. glucose scavenging activities of pamam dendrimers m. labieniec , t. gabryelak , c. watala university of lodz, deptartment of general biophisics, lodz, poland, glycation is a spontaneous, non-enzymatic modification of biomacromolecules with hexoses, mainly glucose. in terms of its pathophysiological relevance, it targets predominantly proteins, but also nucleotides and phospholipids, and is of major importance to both physiology (ageing) and pathology (diseases with a metabolic background, like diabetes). earlier we demonstrated in vivo that the administration of pamam g to diabetic rats resulted in a significantly reduced blood glucose, as well as the early (hba c ) and late (ages) protein glycation products. in this study we investigated the ability of pamam g ( surface nh groups) and g ( nh ) to inhibit glycation of proteins in plasma, and a model protein -bsa. pamam g and g competed chemically with protein nh for the binding of glucose, and hampered protein glycation. in a high-glucose medium they underwent an excessive glycation themselves. this modification was more effective in pamam g , in which surface nh were more mobile and accessible. pamam modification with glucose rendered these dendrimers less polycationic in aqueous solutions. pamams neither affected bsa conformation nor formed stable complexes with a protein. we conclude that pamams are very effective glucose scavengers. thus, even less toxic pamams of lower generations, like g , may appear useful in further medical applications as the agents attenuating the detrimental effects of sever hyperglycaemia on biomacromolecules. selective drug delivery and novel drug approaches by polyelectrolytes s. krol cbm-cluster in biomedicine, area science park, s.s. , . km; trieste, italy the use of polyelectrolytes in the past was mainly related to targeted drug delivery and nanoparticle preparations for medical applications. but rarely the polyelectrolytes were investigated for the own features as a drug. in our present work we use a special physical feature of cancer cell membranes as a target for a specific polycation. we found that the polycation is selectively up-taken by cancer cells (leukemia, hepatocarcinoma, cancer stem cells) while normal cells remain unaffected. another interesting application of polyelectrolytes are in form of a multilayer coating on the surface of nanogold particles. for this topic, two new approaches were developed for a drug delivery through the blood brain barrier. in the first case, high amounts of creatine were bound to the gold particles and delivered as protective agents for ischemic stroke (viota et al., j colloid interface sci. , ( ): - ). in the second case coated multifunctional gold particles were prepared as a drug for neurodegenerative disease on the basis of protein aggregates. the use of gold as core for coated nanoparticles offers the possibility to study our systems as "theranostics", system which are modified to recognize selectively diseased cells and carry the moieties to treat or destroy the malfunctioning cells. possible treatments can be local hyperthermal therapy by using the particles as amplifier and enhancer or photodynamic therapy with gold as a drug. m. koneracka , v. zavisova , m. muckova , p. kopcansky , a. jurikova , n. tomasovicova , g. lancz , m. timko , m. fabian institute of experimental physics, slovak academy of sciences, košice, slovakia, hameln rds a.s., modra, slovakia, institute of geotechnics, slovak academy of sciences, košice, slovakia the aim of this study was to develop biodegradable and biocompatible paclitaxel loaded magnetic plga nanospheres (nps) suitable for biomedical applications. biodegradable poly(d,l-lactic-co-glycolic acid) (plga) was used as a capsulation material and the magnetic fluid was used as a magnetic carrier. incorporation of magnetic particles and drug in the plga polymer matrix was confirmed by infrared spectroscopy (ftir) and differential scanning calorimetry (dsc). the release of the drug from the prepared nps to the surroundings under the different conditions was studied also. the prepared magnetic plga nps with encapsulated paclitaxel (spherical shape, size - nm) have good stability in the presence of high nacl concentration at • c, the toxicity of prepared samples declared times higher value of lethal dose ld in comparison with pure paclitaxel (ld = mg/kg) and showed the significant response to external magnetic field which is useful from the point of view to achieve pharmaceutically acceptable drug delivery systems for tumour treatment. success of human gene therapy depends upon the development of delivery vehicles or vectors, which can selectively deliver therapeutic genes to target cells with efficiency and safety. many cationic polymers have been used to condense dna by electrostatic interaction into small particles (polyplex), for protecting the dna from degradation and enhancing uptake via endocytosis. polyethylenimine (pei) appears to be one of the most advanced delivery system that can condense dna efficiently forming pei-dna polyplex complex. the physicochemical properties of different molecular weights of pei, such as condensation ability, buffer capacity, time kinetics, ftir and surface charges of the pei-dna complexes may be important factors to obtain a higher transfection efficiency of the polycation vectors. our intent in this study was to characterize pei-dna complexes to see whether these physicochemical properties have any influence on their disposition characteristics and cellular uptake process. we found that pei-dna complexes, obtained by the k pei at n/p ratio > , were more stable in the presence of tissue culture medium & serum, and did not dissociate in nacl easily. key words: polyethylenimine, polyplex, polycations, dna, transfection, gene delivery. effect of nanopatterned substrate on neuronal growth cones activity in the last years an increasing interest has been address to explore, at the level of the single cell, the physical interaction between neurons and micro-and nano-patterned surfaces which mimic the biological environment and can induce specific biological behavior. a consistent number of substrates have been tested (nano-grooves, pillars, gap nanowires) but the effect of nano-topographical features at subcellular level e.g. branching and pathfinding of growth cones(gc)is still unexplored. using nanoimprinting lithography we fabricated gratings on glass with grooves of variable pitch, depth and width all in the nm range. embryonic stem cell derived neurons were seeded on nanostructured and on flat control glasses. we investigate gc morphology with nm resolution by afm. a significant effect on sub-cellular architecture was observed.on nanopatterned substrates % of gc were branched with a large number of long and thin filopodia(average length and height . µm and nm)while on control only %of gc were branched with an higher percentage of long and thick filopodia (average length and height , µm and nm). on the contrary we did not observe a significant influence of the nanopatterning on the alignment and elongation of neurites. in both cases the distribution of angles between axon and filopodia showed a preferential direction at • . in conclusion, the tested nanopatterns do not influence the neurite directions but do enhance the gc morphology and explorative activities. growth enhancement and adhesion control of pc on micropatterned ns-tio thin film c. lenardi , a. v. singh , p. milani c.i.ma.i.na., dip. di scienze molecolari applicate ai biosistemi, università di milano, italy, c.i.ma.i.na., semm, european school of molecular medicine, fondazione ifom, milano, italy, c.i.ma.i.na., dip. di fisica, università di milano, italy cluster assembled nanostructured titanium dioxide (ns-tio ) has been explored as novel substrate for in vitro cell culture. in this work, we report micropatterned ns-tio thin film as putative microdevice for neuron culture and growth. in additions, we show a simple scheme of molecular patterning of bovine serum albumin (bsa) as cell anti-adherent substrate complementary to ns-tio micropattern which favors a selective spatially confined adhesion of neurons. bsa was drop coated and physisorbed over glass coverslip covered with a thin conducting layer of indium tin oxide (ito) and then pmma was spin coated over it using standard protocols. later, using combinations of e-beam lithography and pulsed microplasma cluster source (pmcs), a thin layer of ns-tio was deposited over micropatterned pmma. further, lift off process enabled us to generate complementary micropatterns of hydrophobic bsa (cell repellent) and hydrophilic ns-tio (cell adhesive). cell culture studies have confirmed that pc cells like to grow on ns-tio substrate and not on bsa layer. the technique offers a novel approach for neuronal cell assay applications. gene delivery with chitosan: influence of chain length on intracellular trafficking and dissociation s. lélu , c. d. l. davies , n. reitan , s. p. strand department of physics, ntnu, trondheim, norway, department of biotechnology, ntnu, trondheim, norway chitosan, a cationic polysaccharide presenting low cytotoxicity, is a promising nonviral gene delivery vector. chitosan complexes dna into nanoparticles, and the complexation and cell transfection efficacy are function of the chitosan/dna ratio and of the intrinsic properties of chitosan, i.e. degree of polymerization dpn, charge density, and molecular structure. nanoparticles formed by shorter chitosan (dpn - ) mediated higher transgene expression than nanoparticles based on longer chitosans. the purpose of this work is to relate the dpn of linear chitosan with the cellular uptake, intracellular trafficking and dissociation of chitosan-pdna complexes, measured by confocal laser scanning microscopy and fluorescence correlation spectroscopy (fcs). cells were incubated with oligomers (dpn and ) either free or complexed with plasmid-dna. both chitosan oligomers seem to penetrate cell nucleus in association with or free from pdna. hours after incubation, accumulation of oligomers in cell nucleus is similar for free and complexed dpn , whereas it increases for complexed dpn compared to free, indicating a possible delayed dissociation of the complexes based on dpn in the nucleus and suggesting a dissociation of pdna-dpn in cytoplasm. these data are consistent with previous studies which suggested that longer chitosan chains led to tighter complexes, and hence to a delayed dissociation process and lower transfection efficiencies compared to shorter chitosans. material surface properties greatly influence dna purification and pcr yield in microsystems c. potrich , l. lunelli , l. marocchi , l. pasquardini , g. guella , d. vozzi , l. vanzetti , p. gasparini , c. pederzolli fbk, trento, italy, univ.trento, italy, univ.trieste, italy modern microchip platforms integrate dna purification, target amplification by pcr and dna detection in a single device. combination of these processes minimizes sample loss and contamination problems as well as reduces analysis time and costs. different strategies are available to perform dna extraction on a chip. here we exploited amino-coated materials as a tool for specific binding of dna through the electrostatic interaction between amine groups and nucleic acids. we analyzed the ability of different treated substrates to selectively absorb/desorb the genomic dna with the aim to purify dna from unwanted components. amino-coated substrates were characterized by afm, xps, fluorescence microscopy and absorption spectroscopy to define the surface chemical and morphological properties. the distribution of the dna adsorbed on materials was homogeneous and the eluted dna was tested for pcr. same materials were analyzed for their compatibility with pcr and the use of different enzymes and reagents or proper surface treatments was employed. we established the best conditions for dna amplification in silicon/pyrex microdevices depending on the type and the fabrication method used and on the quality of reagents more than on the passivation treatment or increment in standard taq polymerase concentration. nanoporous alumina fabricated using unconventional acids for enhanced biomolecular physisorption n. patra, m. salerno, r. losso, r. cingolani italian institute of technology, genova, italy in the last decades the available porecell sizes of porous alumina have been extended, but operating conditions to obtain some pore diameters still have to be optimized, particularly below the nm limit. in this range the use of biocompatible porous alumina thin films could find applications in biosensors, after functionalization by appropriate physisorbed biomolecules. while pore ordering and film growth rate are mainly influenced by the electrical parameters of voltage and current, respectively, the typical pore size primarily depends on the electrolyte. in the search for alternative anodization conditions, we have investigated several acids that have never been used before, namely gallic, lactic, propionic, and glycolic acid. the anodizations were carried out in galvanostatic conditions at fixed concentration and durations, by varying the current. atomic force microscopy was used to test the oxidized surface morphology. in particular, lactic and propionic acids demonstrated feasible. lactic acid gave best results for ∼ ma/cm current density, corresponding to roughly constant ∼ v potential, with resulting pore diameter in the nm range, whereas propionic acid performed best for ∼ ma/cm , corresponding to ∼ v, resulting in nm pore diameter. both kind of films looked lightgray, different from the yellowish oxalic acid porous alumina, which can be a hint of lower ion contamination. chitosan-arabic gum nanoparticles as potential vehicles for peptide delivery l. moutinho, s. rocha, m. coelho, m. c. pereira lepae, chemical engineering department, faculty of engineering, university of porto, portugal chitosan (cs) -arabic gum (ga) nanoparticles were produced by an ion-ion interaction process, using different weight ratios of polysaccharides. according to zeta potential (zp) measurements, particles are ionically stable. zp values ranged from to mv for cs:ga ratios of : . and : . , respectively. cs:ga : yielded uncharged nanoparticles and aggregates, showing that, at this ratio, the negative charges of ga neutralize the positive charges of cs. the particle diameters ranged from to nm, as measured by dynamic light scattering (dls), and displayed a slight tendency for decreasing as the ga concentration increased. transmission electron microscopy (tem) images showed numerous spherical nanoparticles. peptides were encapsulated within the nanoparticles, by mixing them with chitosan solution prior to adding ga. this system can protect short peptides from rapid metabolization, prolonging their blood half-life. physical characterization of nanocarriers for drug delivery s. motta , y. gerelli , g. sandri , e. ricci , p. brocca dept. of chem, biochem and biotech for medicine -university of milan, italy, dept. of physics -university of parma, italy, dept. of pharmaceutical chem -university of pavia, italy nanoparticles used as nanocarriers for pharmaceuticals can improve solubility and bioavailability of problematic drugs and protect labile or toxic molecules. fundamental parameters like drug encapsulation efficiency and release kinetics are tuned by the physico-chemical features of the drug/carrier complex. a challenging aspect of the pharmaceuticallyoriented issues resides in the relatively low number of molecules allowed to build up nanosystems with different properties and morphology, according to the specific drug and to the intended therapy. we have studied: ) soybeanlecithine/chitosan nanoparticles for progesterone and tamoxifen delivery; ) solid-lipid nanoparticles (compritol ato, poloxamer , tween and chitosan) for cyclosporine-a delivery via ophthalmic formulation. both void carriers and drug-loaded nanoparticles have been studied, to understand how the structure of the carriers can be modified by the active molecules. several non-invasive physical techniques have been used to achieve a detailed knowledge of the systems: a) dynamic light scattering for the dimensional distribution of the nanocarriers, b) zeta potential to determine surface charge, c) cryo-tem for morphological analysis d) x-ray scattering (in small angle configuration) and dsc to access information on the inner structure. . a. schollier , a. halperin , m. sferrazza , g. fragneto institut laue-langevin, grenoble, france, universite libre de bruxelles, belgium, cnrs-ujf grenoble, france protein adsorption on surfaces is responsible of several unwanted effects in technological and pharmaceutical applications: fouling of contact lenses, clotting in blood containing devices, inflammation around artificial organs for instance. those phenomena can be repressed with certain types of polymer brushes, in particular peg (polyethylene glycol) brushes, and a better understanding of the mechanism of adsorption could lead to improvements in the design of biomaterials. we have developed a theory describing this mechanism and carried out measurements to confirm the theoretical predictions [langmuir , , ] . three cases are predicted: primary adsorption at the grafting surface, secondary adsorption at the outer edge of the brush, and ternary adsorption within the brush itself. we prepared samples with different grafting densities and different degrees of polymerization and used different protein size and concentrations. neutron reflectivity experiments, able to determine the structure and composition of material interfaces with a fraction of nanometer resolution, were carried out and, by using deuterated proteins (anti-freeze protein, dihydrofolate reductase and myoglobin) on different peg compositions and grafting densities, it was possible to locate the proteins in the brush and to distinguish between the different kinds of adsorption : primary adsorption is dominant for short brushes and ternary adsorption for long brushes. effect of powder air polishing on nanocomposite dental materials measured by atomic force microscopy m. salerno , g. derchi , a. m. genovesi , l. giacomelli italian institute of technology, genova, italy, istituto stomatologico tirreno, lido di camaiore, italy in dentistry, airpolishing with glycine and bicarbonate powders is widely used to remove accumulated plaque. however, the resulting teeth and gingival surface roughness, which is a risk factor for further plaque accumulation, has to be considered. in this in vitro study the effect of the above mentioned technique on the surface of a novel nanocomposite dental material is evaluated by means of atomic force microscopy (afm). square specimens ( . × . cm size) were airpolished either with glycine or bicarbonate, using different application times ( , , sec) and distances ( , , mm) . four specimens were evaluated for each timedistance combination, and checked versus untreated specimens (controls). the specimens were imaged with tappingmode afm at two different scan sizes ( × and × µm ) in two different regions for each sample. the surface roughness was measured as the rms of the feature heights. for the sec mm group airpolishing resulted in an increased surface roughness as compared to the controls; however, glycine was associated with a lower roughness than bicarbonate (glycine: ± nm; bicarbonate: ± nm; controls: ± nm; p< . for treated specimens vs controls, p< . for glycine vs bicarbonate, anova test). similar results were obtained for all the other timedistance combinations. the work comprises the design of polysaccharide based nanoparticles for drug delivery. chitosan/arabic gum and arabic gum/maltodextrin nanoparticles were prepared by polyelectrolyte complexation and spray drying [ ] , respectively. dynamic light scattering characterization established that arabic gum/maltodextrin nanoparticles are more polydisperse (diameters ranging from to nm) than chitosan/arabic gum particles (diameters of approximately nm). scanning electron microscopy measurements demonstrated that the particles are spherical and have a smooth surface. the systems are highly stable to forces up to nn as observed by atomic force microscopy. due to their nature they are hydrophilic and biodegradable. the nanoparticles were used to entrap short peptide sequences and antioxidants and were proved to be efficient in maintaining the biological activity of the molecules. kyotorphin (ktp) was found in in bovine brain. despite revealing remarkable analgesic properties, analgesia was only induced after central delivery. this limited ability to cross the blood-brain barrier (bbb) combined with the unknown mechanism of action largely confines its pharmacological use. to surpass these problems, we designed new ktp derivatives: ktp-rc and ktp-rc-lipogen. biophysical studies were carried out using fluorescence methodologies to characterize the peptides' interaction with biomembrane model systems. partition coefficient quantification showed a clear preference of the derivatives towards fluid zwitterionic and anionic membranes. moreover, a relationship between anionic lipid percentage and in-depth insertion in membrane was established. additionally, studies with fluorescent probe di- -anepps revealed different membrane-interaction profiles of morphine and ktp-derivatives, suggesting distinct actions between them. the analgesic efficacy of the compounds was studied in vivo after systemic administration in models of acute and tonic pain. unlike ktp, both ktp-rc and ktp-rc-lipogen displayed high efficacy from doses as low as . and . mg/ g, respectively, indicating bbb crossing. the observed correlation between higher partition/insertion in the membrane and enhanced analgesic action proved the biophysical rationale to be a powerful strategy for early screening in cns drug development. metabolic syndrome (ms) is now regarded as a major risk factor for cardiovascular disease. as prooxidant/antioxidant balance is disturbed in the course of this disorder, it is possible that melatonin may exhibit protective effect against oxidative damage of blood cells. ms was diagnosed according to international diabetes federation definition ( ) . the investigated group consisted of patients before and after the melatonin supplementation ( mg per day for two months) and was compared to control group of healthy individuals on normal diet. our experiments show that erythrocytes from patient group exhibit significantly higher tbars and total cholesterol levels whereas the protein thiol concentration, na + k + atpase and glutathione peroxidase activities were decreased in comparison to those of healthy volunteers. after two month melatonin supplementation, tbars and cholesterol concentrations significantly decreased, whereas the na + k + atpase, catalase and glutathione peroxidase activities increased. the glutathione concentration was also higher. these results show that melatonin supplementation has a protective effect on erythrocytes of ms patients. action of ferritin, a nanoparticle model, on ros formation and glutamate uptake in synaptosomes t. waseem, a. alekseenko, s. fedorovich institute of biophysics and cell engineering, minsk, republic of belarus nanoparticles are currently used in medicine as agents for targeted drug delivery and imaging. however it has been demonstrated that nanoparticles induce neurodegeneration in vivo and kill neurons in vitro. the cellular and molecular bases of this phenomenon are still unclear. we have used the protein ferritin as a nanoparticle model. ferritin contains iron particles (fe + ) with size nm and a protein shell. we investigated how ferritin influences uptake and release of [ c]glutamate and free radical formation as monitored by fluorescent dye dcfda in rat brain synaptosomes. we found that even a high concentration of ferritin ( µg/ml) did not induce spontaneous [ c]glutamate release. in contrast the same concentration of this protein inhibited [ c]glutamate uptake two fold. furthermore ferritin induced intrasynaptosomal ros (reactive oxygen species) formation in a dose-dependent manner. this process was insensitive to µm dpi, an inhibitor of nadph oxidase and to µm cccp, a mitochondrial uncoupler. these results indicate that iron-based nanoparticles can cause ros synthesis and decrease glutamate uptake, potentially leading to neurodegeneration. functionalized carbon nanotubes (cf-cnts) have a promising future as vectors for drug delivery and for neuronal cell growth and communications . it has been demonstrated that cnts can be employed in drug delivery systems. the toxicological properties of cnts result strictly correlated with nanotube solubility . in this study we focused our attention on multi-walled cnts (mwcnts) because they are easy to manipulate and we covalently functionalize them in different ways to increase their solubility.. we grew different derivatives of a dendrimer on mwcnts surface with positive charges at the periphery, in order to study the different solubilisation properties and correlate them to the biological activity in gene therapy . slowdown of - β-peptide aggregation by addition of two synthetic biocompatible polymers p. sciortino , r. carrotta , g. cavallaro , d. bulone , p. l. san biagio ibf-cnr, palermo, italy, ctf dept., university of palermo, palermo, italy fibril deposit formation of amyloid β-protein (aβ) in the brain is a hallmark of alzheimer disease (ad). fibrils formation is triggered by molecular conformational changes and protein-protein interactions involving partially unfolded regions of different aβ peptide molecules. increasing evidence suggests that toxicity is linked to diffusible aβ oligomers, which have been found in soluble brain extracts of ad patients, rather than to the insoluble fibres. new therapeutical approach, based on searching molecules capable of regulating the peptide aggregation, is currently developing. here, we study the effects on the aggregation of aβ - peptide of two different synthetic polymers with structure similar to that of a protein (α,β-polyasparthylhydrazide -pahy and α,β-polyasparthylhydrazide-polyethyleneglycol -pahy-peg). static and dynamic light scattering measurements showed that the aggregation kinetic is slowed down by the presence of both polymers. optical microscopy revealed the presence of aggregates of different dimension in all samples. transmission electron microscopy allowed to establish that all aggregates are made of fibers, as confirmed by fluorescence spectroscopy measurements on thioflavine binding. a. smorodchenko , d. sittner , a. rupprecht , j. goyn , a. seiler , a. u. bräuer , e. e. pohl institute of cell biology and neurobiology, charité-universitaetsmedizin, berlin, germany, german federal institute for risk assessment, zebet, berlin, germany ucp is a member of the mitochondrial anion transporter family and one of the three ucps (ucp , ucp and ucp ) associated with the nervous system. in our previous work we have shown that ucp appears in brain at embryonic day (e ). we hypothesized that the ucp expression can be related to neuronal differentiation. to prove this idea we now investigated protein and mrna levels in two different systems: (i) mouse embryonic stem cells of line d (mesc) and (ii) brains from pre-and postnatal mice. ucp was not present in undifferentiated mesc. during differentiation of mesc in neurons the expression of neuronal marker map and ucp started at the same time -as early as on day in culture -and were increasing simultaneously. (ii) the comparative analysis of gene transcripts prepared from whole embryos, brains and different brain regions (neocortex, hippocampus, cerebellum) demonstrated that levels of ucp mrna were increasing from e till e , reached an expression peak between e and postnatal day (p ) and remained constant in adult animals. in contrast, expression of ucp was increasing permanently until birth, whereas ucp expression was invariant in time. our results suggest that ucp contributes to specific neuronal functions. the plant hormone abscisic acid stimulates the proliferation of human hemopoietic progenitors through the second messenger cyclic adp-ribose s. scarfì , c. fresia , c. ferraris , s. bruzzone , f. fruscione , c. usai , m. magnone , m. podestà , l. sturla , l. guida , g. damonte , a. salis , a. de flora , e. zocchi advanced biotechnology center, genova, italy, dept of experimental medicine, biochemistry section, and cebr, university of genova, italy;, institute of biophysics, cnr, genova, italy, stem cell center, s. martino hospital, genova, italy abscisic acid (aba) is a hormone involved in pivotal physiological functions in higher plants. recently, aba was demonstrated to be produced by human granulocytes, β pancreatic cells and mesenchymal stem cells (msc) and to stimulate cell-specific functions. here we show that aba expands human hemopoietic progenitors in vitro, through a cadpr-mediated increase of the intracellular calcium concentration. incubation of cd + cells with micromolar aba also induces transcriptional effects, which include nf-κb nuclear translocation and transcription of cytokines encoding genes. stimulated human msc produce and release aba at concentrations sufficient to exert growth-stimulatory effects on co-cultured cd + cells, as demonstrated by the inhibition of colony growth in the presence of an anti-aba monoclonal antibody. these results provide a remarkable example of conservation of a stress-hormone from plants to humans and identify aba as a new hemopoietic growth factor involved in the cross-talk between hp and msc. t. golovanova, g. b. belostotskaya sechenov institute of evolutionary physiology and biochemistry, russian academy of sciences, saint petersburg, russia a subpopulation of small cells (volume ± µm ) have been found in the neonatal cardiac myocytes culture whereas the same parameter of the remaining cells was ± µm on the st day. in contrast to main part of hypetrofied cardiomyocytes, the small cells were able to proliferate, form colonies and differentiate spontaneously into cardiac myocytes in the culture. on the th day there were slight, slow ( - beats/min), arrhythmic contractions in the centre of colonies. pulsing cells were not united by common contraction and had individual beating profile. on the - th days, the colonies displayed comprehensive contractile activity with the pulsation rate - beats/min and reached beats/min on the d - th day and beats/min on the - th days in the culture. it has been shown that receptors of the surface membrane and sarcoplasmic reticulum of colony cells gradually mature. by estimating the ca + transition under the specific agonists action: acetylcholine, kcl and caffeine, we have detected the activity of basic structural and functional elements of excitation-contraction coupling in contractile cells inside colonies. the small cells ability to proliferate and differentiate under the influence of near mature cardiomyocytes allows us to put forward a hypothesis, that they belong to a category of resident stem cells. changes in the gating properties of na + channels were studied in es-derived neural stem (ns) cells during in vitro neuronal differentiation with the use of the whole-cell and cell-attached variants of patch-clamp technique. ns cells represent a novel stem cell population remaining stable and highly neurogenic over multiple passages.voltage-clamp recordings during neuronal differentiation of ns cells indicated significant changes in the key properties of na + currents. a voltage-gated and tetrodotoxine-sensitive na + current,absent under self-renewal conditions, was first recorded following application of differentiative agents. current density increased with time of exposure to differentiating conditions. whole-cell and single channel analysis revealed that the observed increase in current density was due at least in part to changes in steady-state activation and inactivation properties. namely, half activation potential shifted from - mv to - mv, while half-inactivation potential shifted from - mv to - mv. furthermore, a contribution to the increase in na + current density could also be given by an enhancement in channel expression, as suggested by an augmentation in the number of single channels per patch area, with increasing neuronal differentiation. interestingly, those changes in na + channel activity well correlate with the capability of ns cells to generate action potentials during in vitro neuronal differentiation. a. viale, g. bonizzi, a. cicalese, f. de franco, c. pasi, s. pece, a. orleth, p. di fiore, p. pelicci european institute of oncology, milan, italy we are characterizing the biological differences between normal and transformed scs. scs are defined by their ability to generate more scs, termed self-renewal, and to produce cells that differentiate (asymmetric cell division). scs, however, possess the ability to expand in number (i.e. during development, in adulthood after injury/disease); this increase is not accounted by asymmetric divisions, in which only one daughter cell maintains sc identity. recent findings in invertebrates indicate that scs can also generate daughter cells destined to acquire the same fate (symmetric cell division). on the other hand, sc quiescence is critical to maintain tissue homeostasis after injury. here we show increased symmetric divisions of cscs in breast tumors (due to inactivation of the p tumor suppressor) and dependency of leukemia development on quiescent leukemia scs (due to transcriptional up-regulation of the cell cycle inhibitor p by leukemia-associated fusion proteins). we suggest that sc asymmetric divisions function as a mechanism of tumor suppression, that sc quiescence is critical to the maintenance of the transformed clone and that symmetric divisions of scs permit their geometric expansion. a. uccelli department of neurosciences ophthalmology and genetics, genoa, italy stem cells are considered as a possible source of cells for tissue repair. in this scenario damaged tissues will be reconstructed by newly formed cells with the result of a recovery of lost functions. thus, the rationale for utilizing stem cells for the treatment of neurological diseases such as multiple sclerosis (ms) stemmed from the idea that they differentiate in neural cells regenerating the damaged tissues at the basis of irreversibile disability. however, while multipotential embryonic stem cells may provide in the future an optimal source of cells competent for myelin and axons repair in ms, their use is still far from being exploitable in a clinical setting. moreover, it is widely accepted that adult stem cells may in vitro transdifferentiate in neural cells but recent experimental data have challenged the biological importance of this event in vivo. nevertheless, several experimental studies have provided new evidences supporting the use of adult stem cells derived form different human tissues for the treatment of ms. in the cases of mesenchymal stem cells and neural stem cells current experimental data support an unexpected therapeutic plasticity mediated by diverse paracrine mechanisms including neuroprotection, induction of local neurogenesis and modulation of the immune response. therefore, current experimental and clinical studies support the use of stem cells for the treatment of neurological diseases such as ms. unravelling the structure of the water splitting site of photosynthesis and implications for mechanism of catalysis j. barber imperial college london, u.k. photosystem ii (psii) is a multi-subunit membrane protein complex which catalyses the oxidation of water to molecular oxygen and reducing equivalents. the reaction occurs at a catalytic centre composed of mn ions and a ca ion, is thermodynamically demanding and generates highly oxidised species. unavoidable side reactions cause detrimental effects on the protein environment leading to the rapid turnover of the reaction centre d protein. to understand the mechanisms of water oxidation and d turnover structural information is required. initially the positioning of various protein subunits and their transmembrane helices were determined by electron microscopy. more recently a refined structure of the cyanobacterial psii unit has been elucidated by x-ray crystallography giving details of specific environments of the redox active cofactors. the implications of these structural studies will be discussed in relation to the unique facets of psii function, particularly the water splitting reaction. importantly this new knowledge is providing a blue print for the design of photochemical catalysts which can mimic the photosynthetic water splitting reaction and thus give hope that new technologies will emerge to provide humankind with a sustainable energy supply. photochemically controlled molecular devices and machines v. balzani department of chemistry "g. ciamician", university of bologna, italy the macroscopic concepts of a device and a machine can be extended to the molecular level. molecular-level devices and machines operate via electronic and/or nuclear rearrangements and, like macroscopic devices and machines, they need energy to operate and signals to communicate with the operator. the extension of the concepts of a device and a machine to the molecular level is of interest not only for basic research, but also for the growth of nanoscience and the development of nanotechnology. if a molecular device or machine has to work by inputs of chemical energy, it will need addition of fresh reactants ("fuel") at any step of its working cycle, with the concomitant formation of waste products that compromise the operation. currently there is an increasing interest in the use of light to power molecular devices and machines. the lecture will illustrate examples of recent achievements [ , , ] , which include molecular wires, switches, plug-socket systems, extension cables, antennas, and light powered nanomotors. biophysics laboratory, institute of botany, azerbaijan national academy of sciences, baku, azerbaijan it is well known that fast component of induction curves of millisecond delayed chlorophyll fluorescence (ms-df) originates via radiative recombination of reaction center with product on the donor side of psii. in view of our previous data (j. photochemistry and photobiology b: biology , ) it was shown that partners for radiative recombination of p q a − localized as a hole on the donor side of psii. depending on ph this hole might be on the camn -cluster or on y z and their recombination with p q a − can be monitored by the ms-df fast component. for analysis of site of damages, photoinhibition of psii particles from spinach at different ph was monitored by ms-df. during photoinhibition of psii ( µmol photons m − s − ) the fast component of ms-df was shown to be more stable at ph . and ratio of the fast component to the steady-state level of ms-df was approximately constant, while at ph . the fast component essentially decreased, the steady-state level increased and ratio of these components rapidly went down. it is possible concluded that strong light damaged of recombination reaction with in the presence ppbq -artificial electron acceptor -the protection of psii from photoinhibition has been observed only at acidic condition. stress factors such as heavy metals, strong light and low temperature were investigated by measurement of transient pictures of ms-df in intact plants, isolated chloroplasts and pure psii particles. the heavy metals action on psii was investigated on wheat seedlings. the targets for toxic action of al, mn and co ions were found to be a q a -q b acceptor side of psii. the damage site for cd + may be partners for recombination with p + -depend on of medium ph -either y z or camn -cluster. investigated ions (mn + , al + , cd + and co + ) have lead to reduction of chlorophyllprotein complexes pigment fund and cd + and co + destroyed also an apoproteins, especially of psii. the photoinhibition of psii was investigated in intact leaves of barley and maize seedlings at low and normal temperature. a very sharp reduction of intensity of ms-df second fast component, possibly y z * p ÿq a − radiative recombination of maize seedlings was observed even after short illumination by strong light at • c. ph dependence photoinhibition of chloroplasts and pure psii particles have shown that strong light damaged of camn -cluster in great degree than y z . hydrogenases are considered as potential energy sources. in particular, the ability of the green alga c . reinhardtii to reduce protons to hydrogen gas upon illumination by means of a [fefe]-hydrogenase is a phenomenon of great scientific interest, as it would need only light and water to generate energy. however, the catalytic activity is strongly inhibited by the o produced during photosynthesis; furthermore, the protein is expressed at very low levels, only in conditions of strict anaerobiosis. this mutually exclusive nature of o and h photoproduction represents a crucial problem in the development of h bio-production. the study of the structurefunction relationship of [fefe] hydrogenases, which would help to clarify the molecular mechanisms underlying both h production and o sensitivity, requires the characterization of purified native and modified proteins, which can be obtained by site-directed mutagenesis. we expressed the algal hydrogenase in the cyanobacterium synechocystis sp. pcc , which holds a bidirectional [nife]-hydrogenase with a well known maturation system ( ) . we obtained two constructs to stably transform synechocystis, enabling it to express the c. reinhardtii hydrogenase in an active form. this suggests that the [nife]-hydrogenase maturation pathway is able to drive the biosynthesis of functional [fefe] enzymes. these data open new perspectives about the indispensable presence of hyde, hydf and hydg auxiliary proteins ( , ) to obtain a correctly folded [fefe]-hydrogenase. the a low energy monomeric chlorophyll of cp to investigate the role of low energy spectral forms in energy transfer among chlorophyll protein complexes, we characterized one of the redmost chlorophylls of psii antenna complexes, the a of cp . pigment stoichiometry of the wild type (cp wt) and mutant lacking the a binding residue (cp a ) reconstituted complexes confirmed the loss of a chlorophyll a. to exclude the possibility of a marked excitonic interaction of the a with other chlorophylls, we quantified the decrease in dipole strength after mutagenesis as a function of the frequency and performed a second derivative analysis of the difference absorption spectrum cp wt minus cp a . these data, along with circular dichroism spectra of both complexes, support the idea that the a is a monomer in cp , in disagreement with recent suggestion of involvement in an excitonic dimer (mozzo et al., bba ) . chla a reorganization energy and inhomogeneous broadening were then determined through a thermal broadening analysis in the - k temperature range. these parameters allowed us to calculate the förster overlap integral of the homogeneously broadened a bandshape, a fundamental factor for energy transfer rate estimations between chromophores. a comparison with foi of chlorophyll in solution suggests that chla a may connect cp complex to psii core favoring energy transfer from cp towards other inner antenna low energy chlorophylls. in oxygenic photosynthetic organisms, reaction centre complexes catalyze electron transport from water to co using light energy absorbed by tetrapyrrole pigments bound to so called "antenna proteins". a major challenge in performing this type of photosynthesis consists on the difficulty of operating "one electron" transport through a multi-step pathway in the chloroplast environment which is the source of oxygen of biosphere. in fact, synthesis of ros, mainly singlet oxygen and superoxide anion and consequent photoinhibition is an intrinsically unavoidable consequence of photosynthesis that must be prevented and/or controlled in order to avoid photodestruction and death. mechanisms involved include chlorophyll (chl) triplet quenching, ros scavenging and controlled heat dissipation of excess chl singlet excited states. the latter process has been studied for over years with little success in elucidating its mechanism. reverse genomics and ultrafast-spectroscopy led to the proposal that the transient formation of carotenoid radical cations, followed by charge recombination might be the underlying mechanism of energy dissipation while three proteins belonging to the lhc superfamily could be the site hosting the reaction. probing the dark cycle to reveal regulatory networks controlling photosynthetic efficiency the food, fibre and fuel needs of an ever-increasing population are one of the challenges facing current society. higher plant photosynthetic efficiency is ∼ % whereas the theoretical maximum is thought to be ∼ - % giving potential for great improvements. it is not however clear where in the photosynthetic process the additional losses are occurring. we are adopting a systems biology approach to reveal the regulatory networks that control photosynthetic efficiency, the challenge being to make quantitative measurements of the input parameters to models of the network of photosynthetic reactions, and also to identify missing physical parameters and processes. the aim being to understand how they interact with other key physiological pathways and what impact they hold over photosynthetic efficiency. we report initial results in this poster from experiments using conventional and novel proteomic methods. e.g. an optical technique based on a non-linear dir method is being developed for proteomic and metabolomic analysis. unlike many conventional proteomic methods, which provide information on the relative levels of various proteins and metabolites, the viability of the dir method as a quantitative, sensitive and high throughput proteomics platform has recently been demonstrated. in collaboration with klug this project will employ dir as a both a proteomic and metabolomic tool. t. morosinotto , p. arnoux , g. saga , g. m. giacometti , r. bassi , d. pignol dip. di biologia, padova, italy, cea, cadarache, france, dip. di biotecnologie, verona, italy violaxanthin de-epoxidase (vde) is the enzyme responsible for zeaxanthin (z) production. the synthesis of this carotenoid in plants exposed to high light conditions is an important photoprotection mechanism, enhancing excess energy dissipation and reactive oxygen species scavenging. the inactive enzyme is normally soluble but, upon activation by low ph, it binds to the thylakoids membrane, where its substrate is found. we present here the first structural data on this enzyme obtained at both acidic and neutral ph. at neutral ph, vde is monomeric with its active site occluded in the lipocalin barrel. upon acidification, the barrel opens up resulting in a functional dimerization of the enzyme. the channel linking the two active sites of the dimer can harbour the entire carotenoid substrate and thus allow the parallel de-epoxidation of the two violaxanthin β-ionone rings, making vde an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate. structural data opened the possibility to investigate deeper this enzyme and further work allowed the identification of its active site, the protein domains responsible of its membrane association as well as key residues involved in the ph dependent conformational change. hydrogen is considered the fuel of the future if produced from sun light-driven water splitting. a hydrogen economy based on genetically modified organisms, immobilized enzymes or biomimetic synthetic catalysts requires a profound knowledge of the structure and function of the respective enzymes in nature. light-induced water splitting is performed by a tetranuclear manganese cluster located in photosystem (ps) ii of oxygenic photosynthesis. the protons generated by ps ii can be converted to molecular hydrogen by the enzyme hydrogenase, which is for example found in green algae and cyanobacteria. [nife]-and [fefe]-hydrogenases are the two main classes of this enzyme. they contain bridged binuclear transition metal cores, which are tuned by a special ligand environment to efficiently convert protons to hydrogen -or vice versa -via a heterolytic mechanism. rhodobacter sphaeroides strain a- isolated from mineral springs in armenia produces h with high rate in anaerobic conditions under light. in our previous work the inhibition of h production by n,n' -dicyclohexylcarbodiimide (dccd), the f f -atpase inhibitor, was shown, and dccd-inhibited atpase activity was determined. therefore it is possible to admit a role of this atpase in h production by r. sphaeroides; otherwise dccd inhibition of hydrogenase is not ruled out. in order to examine the mediatory role of proton-motive force (pmf) or proton atpase in this process transmembrane electrical potential (∆Ψ) and ∆ph are determined and the atpase activity is studied in r. sphaeroides grown under light. pmf was determined under anaerobic conditions in the dark. at ph . the ∆Ψ was of - mv and the reversed ∆ph was + mv, resulting in the pmf of - mv. but ∆Ψ was not affected by dccd. moreover, adenine nucleotide phosphates (anp) content is essential for cell functioning and may result with atpase activity. the percentage of atp was calculated from the total quantity of anp in whole cells, it was . %. a relatively high concentration of adp (∼ %) and amp (∼ %) and accordingly low energetic charge were noted; these might be indicative for the atpase activity, a further study is required. relationship between h production and atpase activity by rh. sphaeroides is suggested; possible mechanisms are discussed. identification of the sites of chlorophyll triplet quenching in relation to the structure of lhc-ii from higher plants. evidence the chl a molecules involved in the triplet-triplet energy transfer to the central luteins in trimeric lhc-ii are identified by time-resolved and pulse epr techniques. the concept of spin conservation during triplet-triplet energy transfer is exploited. the sites with the highest probability to form triplet states, which are quenched by the central luteins, are chl and chl . unquenched chl triplet states are also produced by photo-excitation in the lhc-ii complex. putative sites of these triplet states are chl , chl , chla and chl , since they do not contribute to the formation of the observed carotenoid triplet states. fluorescence lifetime spectrum of the plant photosystem ii core complex the photosystem ii kinetic model (diffusion or trap-limited) is still much debated. there is discussion about whether energy transfer from the core antenna (cp and cp ) to the reaction center complex (d -d -cyt b ) is rate-limiting (transfer to trap-limited). this study investigates this problem in isolated core particles by exploiting the different optical properties of the core antenna and the reaction center complex near nm, due to p and an isoenergetic pheophytin. this was used as a marker feature for the reaction center complex. if the transfer to the trap-limited model were correct, assuming excited-state thermalization, the specific reaction center fluorescence decay lifetime should be shorter near nm, where there is reaction center complex specificity, than at the other emission wavelengths. such a selective reaction center feature was not observed in fluorescence decay measurements. at the experimental resolution used here, we conclude that the traplimited energy transfer to the reaction center could, at the most, be % limiting. thus, the transfer to the trap-limited model is not supported. photosynthetic apparatus response under heat stress n. pshybytko , i. divak , l. kabashnikova , e. lysenko institute of biophysics and cell engineering, national academy of sciences of belarus, belarus, institute of plant physiology, russian academy of sciences, russia the mechanisms of psii thermoinactivation and adaptation under high temperature impact and participation of hydrogen peroxide at these processes were studied. the twice suppression of oxygen evolving activity of thylakoids with simultaneous decrease in d protein content and the release of extrinsic kda polypeptide from woc after - min heating at o c were registered. using inhibitor analysis it was shown that thermoinduced degradation of d protein after min heating occurred by proteases. the participation of ftsh protease in thermoinduced d protein degradation was observed. level of transcription of psba gene in chloroplast was raised after min heating and was decreased through h. the content of hydrogen peroxide was increased three times after min of heating and was decreased to normal level through h and was raised after . h again. it is interesting that level of peroxidation lipids products was increased after . h heating only. received data indicated that hydrogen peroxide is signal molecular at the photosynthetic apparatus under heat stress. during heating the inactivation of woc and d protein is occurred. as result the h o is generated. hydrogen peroxide as signal molecule activates transcription of psba. turnover of psii is occurred. more long heating induces degradation of proteins and lipids and h o represents as the destructive agent. a. nurisso , m. a. morando , f. j. cañada , j. j. barbero , a. imberty cermav-cnrs, grenoble, france, centro de investigaciones biológicas, madrid, spain the mechanism of symbiosis between legume plants and rhizobial bacteria is a relevant topic of interest since it is at the basis of the nitrogen fixation process. this mechanism is strictly related to the production of lipochitooligosaccharides, nod factors (nf), which allow the bacterial invasion into the legume host roots. in medicago truncatula, the recognition of nf from the symbiont sinorhizobium meliloti requires the nfp gene, able to encode a lysm-motif receptor-like kinase. the extracellular part of this protein is characterized by three lysm motifs: only one of them seems to be involved in the nf recognition. herein, we report an in silico investigation of different structural aspects of this process. first of all, the conformational behaviors of a new generation of nf analogues were elucidated by mds simulation. then, a homology model of the lysm domain from m. truncatula was proposed and compared with a model of lysm domain from pisum sativum: docking calculations of natural nf identified a common binding site in which the carbohydrate portion is the main responsible of the binding. both studies provide support for the idea that the carbohydrate part of nf plays a key role in the interaction with lysm domains, while the lipid moiety modulates ligand specificity probably interacting with a potential second receptor. interaction between frutalin with biomembrane models and its correlation with cell adhesion property frutalin is a homotetrameric lectin d-galactose (d-gal) binding that activates natural killer cells in vitro and leukocyte migration in vivo, being a potent lymphocyte stimulator. in this study we investigated the interaction of frutalin with different phospho/glyco-lipids using langmuir monolayers as biomembrane mimetic system. the results attest the specificity of the protein for the carbohydrate d-gal when attached to the biomembrane model. the adsorption kinetics for frutalin to the mixed monolayers containing glycolipids showed that the interaction depends on the presence of charged groups and on the position of the d-gal on the polar head of the glycolipid. using brewster angle microscopy (bam), we investigated the morphology of the interface for the binary mixtures containing galcer, where small domains were formed at high lipid packing, suggesting that frutalin can induce the formation of likelipid rafts domains in vitro. the results obtained with the membrane models were associated with those from fibroblast adhesion induction. the cell adhesion promoted by frutalin is in accordance with the results observed in langmuir monolayers, which probes the specificity of the interaction between the lectin and d-gal on cell-membrane surfaces. based on these results, frutalin can be considered as a promise biotechnological tool to actuate in tissue engineering regeneration. supported by fapesp, cnpq and capes aggregation and glycation processes of proteins are of peculiar interest for several scientific fields. serum albumins are widely studied proteins for their ability to self-assemble in aggregates and also to undergo to non enzymatic glycosylation in cases of diabetes. in this work we report a study on thermal aggregation of glycated bovine serum albumin (bsa) prepared with different concentrations of glucose at ph . . increasing concentration of sugar modulates the effect of different glycation levels on the protein aggregation. fluorescence spectroscopy, ftir absorption, static and dynamic light scattering are used to follow the time evolution of the aggregation process and of protein conformational changes. conformational changes of secondary and tertiary structures are measured by ftir absorption; the kinetics of amide i, amide ii and amide ii' bands are monitored. the kinetics of tryptophans fluorescence give complementary information on the tertiary structure changes and on the polarity modification of the fluorophores environment. the aggregates growth is studied by dynamic light scattering measurements and rayleigh scattering peak. the results show that the partial unfolding of the protein is not affected by the glycation, while the presence of increasing amounts of glycated molecules progressively inhibits the aggregates formation. solvent occupancy analysis in ligand structure prediction: the case of galectin- s. di lella, m. a. martí, d. a. estrin inquimae-conicet, universidad de buenos aires, argentina formation of protein ligand complexes is a fundamental phenomenum in biochemistry. during the process, significant solvent reorganization is produced along the contact surface. using md simulations in explicit solvent combined with statistical mechanics analysis, thermodynamic properties of water molecules around proteins can be computed and analyzed in a comparative view. based on this idea, we developed a set of analysis tools to link solvation with ligand binding in a key carbohydrate binding protein, human galectin- (hgal- ). specifically, we defined water sites (ws) in terms of the thermodynamic properties of water molecules strongly bound to protein surfaces. we then succesfully extended the analysis of the role of the surface associated water molecules in the ligand binding and recognition process to many other carbohydrate binding proteins. our results show that the probability of finding water molecules inside the ws, p(v), with respect to the bulk density is directly correlated to the likeliness of finding an oxhydril group of the ligand in the protein-ligand complex. this information can be used to predict possible complex structures when unavailable, and suggest addition of ohcontaining functional groups to displace water from high p(v) ws to enhance drug, specially glycomimetic-drugs, protein affinity and/or specificity. glyconanoparticles: nano-biomaterials for application in biotechnology and biomedicine s. penadés laboratory of glyconanotecnology, cicbiomagune -ciber-bbn, san sebastian, spain to study and intervene in carbohydrate interactions our laboratory has developed a chemical strategy (glyconanotechnology) to produce sugar functionalized gold nanoclusters with multivalent carbohydrate display (glyconanoparticles, gnps). by combining these tools with biophysical and analytic surface techniques (itc, afm, tem, spr) we have demonstrated and evaluated ca + -mediated carbohydratecarbohydrate interactions involved in cell adhesion processes. the gnp system complements other currently available multivalent systems incorporating carbohydrates and presents some advantages as: ) exceptionally small core size; ) high stability in water and physiological buffers; and ) multivalency and multifunctionality with control over ligands number. specific gnps have been applied to the inhibition of melanoma metastasis in mice and as inhibitors of hiv-trans infection. magnetic gnps have also been used in cellular labelling and imaging as probes for mri. in this lecture, the glyconanotechnology strategy will be presented and some application will be highlighted. -glycobiophysics - expanded ataxin- induces membrane mechanical instability: evidences from model systems and cells c. canale , s. averaimo , d. pesci , d. paulis , v. fortunati , a. gliozzi , m. mazzanti , c. jodice , a. relini iit, genoa, italy, university of milan, italy, university of tor vergata, rome, italy, university of genoa, italy spinocerebellar ataxia type is a neurodegenerative disorder involving the expansion of a polyglutamine stretch beyond a threshold in the protein ataxin- , with intracellular deposition of amyloid aggregates. ataxin- variants with polyglutamine stretches of pathological length are not associated to disease when the stretch is interrupted by histidines. mounting evidence suggests that ataxin- aggregates interact with the nuclear membrane. to get insight into the mechanisms leading to neurological disorders, we studied model lipid membranes containing an expanded pathological form of ataxin- or expanded normal forms interrupted by histidines. electrical measurements on planar bilayers showed the occurrence of current steps, much larger for the pathological form, indicating the formation of pore-like structures. atomic force microscopy measurements showed that the longer the polyglutamine tract, the smaller was the force required to penetrate the bilayer with the afm tip. the smallest penetration force, and then the strongest membrane destabilization, was observed for membranes containing the expanded pathological form. experiments on cos cells provided evidence that pathological protein aggregates damage the nuclear membrane eventually causing cell autophagy. modification by dopamine adducts links αsynuclein to oxidative stress in parkinson disease m. bisaglia , e. greggio , l. tosatto , f. munari , i. tessari , p. polverino de laureto , s. mammi , m. r. cookson , l. bubacco university of padova, italy, nih, bethesda, md, usa oxidative stress has been proposed to be involved in the pathogenesis of parkinson disease (pd). a plausible source of oxidative stress in nigral dopaminergic neurons is the redox reactions that specifically involve dopamine (da) and produce various toxic molecules, i.e., free radicals and quinone species. α-synuclein (αsyn), a small protein found in lewy bodies characteristic of pd, is also thought to be involved in the pathogenesis of pd. to investigate the possibility of a synergistic role of oxidative stress and αsyn in pd, we analyzed the modulation of da toxicity by αsyn overexpression in dopaminergic human neuroblastoma cells. our results indicate that the increased expression of αsyn enhances the cellular toxicity induced by the accumulation of intracellular da. we then correlated our results with the structural modifications induced by the oxidation products of da on αsyn by studying two potential pathways for the oxidative chemistry associated with da. the first one is the auto-oxidation reaction which leads to the concomitant formation of radical and quinone species. the second is the enzymatic oxidation, mediated by tyrosinase, which leads only to the accumulation of quinones. our data suggest a link between da and αsyn in the progression of pd and provide novel insights on both the mechanisms involved in the oxidative chemistry and the aggregation properties of αsyn. dynamic light scattering aggregation studies of aβ ( - ) peptide s. bertini, r. beretta, d. gaudesi, a. naggi, g. torri ronzoni institute, milan, italy heparan sulfate (hs) proteoglycans play a role in formation of amyloid plaques by facilitating formation of amyloid aggregates. heparins and low-molecular weight heparins which mimic hs sequences could interact with amyloid peptides putatively involved in neurodegenerative processes. the aim of this work is to study the influence of chain length and structure of heparin oligosaccharides on the aggregation of amyloid. to this purpose, the interaction with amyloid peptide aβ − with a number of heparin/heparin oligosaccharides had been investigated using dynamic light scattering (dls) which permits to determine the size and the stability of molecular aggregates in solution. as indexes of aggregation in solution, two parameters were chosen, i.e., the area under the autocorrelation function (af) and the average hydrodynamic ratio (rh). we evaluated the aggregation kinetic of different preparations of aβ and the ph of solubilization. a clear indication was obtained for the trend of aggregation of the peptide aβ − alone and in the presence of oligosaccharides. the size of the aggregate in the presence of the tetrasaccharide is definitely lower than for the peptide alone. the size of the aggregates increases with increasing size of the oligosaccharides. in fact, the octasaccharide promotes further aggregation. it is crucial for pharmaceutical protein formulations to have low aggregate content. using the monoclonal igg antibody rituximab as a model system, we have studied the mechanisms by which antibodies aggregate at physiological ph when incubated at - • c. light scattering showed two coupled stages: an initial fast stage followed by several hours of exponential growth of the scattered intensity. data analysis showed the fast formation of a large species, which subsequently increased in size. the aggregate number density had a maximum suggesting that aggregates increase in size by coagulation. the analysis also predicted the actual underlying increase in aggregate mass to be linear and reach saturation. this was confirmed by size-exclusion chromatography of incubated samples. in an arrhenius plot the activation energy of the first stage was similar to the unfolding energy of the ch domain, suggesting a pivotal role of this domain in the aggregation process. cd and fluorescence showed only minor structural changes in the temperature interval studied. we conclude that coagulation is the main mechanism driving rituximab aggregation and that aggregation is due to small structural changes. -condensed colloidal phase in biology: from proteins crystals to amyloid fibrils -abstracts solid-state nmr structural studies of alzheimer's disease amyloid β( - ) in lipid bilayers j. d. gehman , a. k. mehta , f. separovic school of chemistry, bio institute, university of melbourne, vic, australia, department of chemistry, emory university, dickey dr., atlanta, ga, usa amyloid-β peptides are believed to cause loss of nerve cell function in individuals suffering from alzheimer's disease, where evidence suggests that interaction with the cell membrane correlates strongly with cytotoxicity. previous studies report a range of different but plausible structures, which depend on the molecular environment of the peptide. this sensitivity to sample preparation suggests that the structure relevant to disease is found in lipid bilayer membranes. while model membrane vesicles are too large to be studied by conventional solution nmr, solid-state nmr is one of the few technologies available to study such systems. we present recent measurements which suggest a novel peptide structure in a lipid bilayer environment: (i) rotational-echo double-resonance (redor) distance measurements between selectively enriched c-carbonyl and n-amide positions constrain dihedral angles of intervening residues and suggest that at least part of the aβ( - ) peptide folds into a β-sheet like conformation, in contrast to the helical and coiled structures in previous reports; and (ii) double quantum filtered draws measurements indicate that the extended strand does not assemble into an in-register parallel sheet as reported for amyloid fibrils. a mutation in app gene with dominant-negative effect on amyloidogenesis: a light scattering study e. del favero , g. di fede , f. tagliavini , m. salmona , l. cantù university of milan, segrate, italy, "carlo besta" national neurological institute, milan, italy, istituto di ricerche farmacologiche "mario negri", milan, italy we studied the effect of a single-point mutation (a v) on the aggregative properties of alzheimer's peptides aβ − . by laser light scattering it was possible to follow the early stages of aggregation of aβ − wt and aβ − mut (within hours after sample preparation), when intermediates in the aggregation pathway, rather than the mature insoluble fibrils, are formed. results show that both the kinetics and the extent of aggregation are strongly enhanced by the mutation. on the reverse, coincubation of mutated and wild-type peptides slows down the aggregation process and results in aβ aggregates that are much more unstable against dilution. it is evident that the interaction between mutant and wild-type aβ − interferes with nucleation or nucleation-dependent polymerization, hindering amyloidogenesis. these results account for the highly amyloidogenic effect of the mutation in vitro, if alone, reversely reduced by the mutated-wild type mix. also a clinical case exists that constitutes the in-vivo evidence of these two opposing effects. this finding may offer grounds for the development of therapeutic strategies, based on modified aβ peptides or peptido-mimetic compounds, for the potential treatment of alzheimer's disease. the collagen i is the major structural protein of the body and it is found organized in a hierarchical manner in the extra-cellular matrix of several tissues (bone, tendon, cornea and sclera, etc..). it is responsible for their specific architecture. it is already known that collagen i is capable of forming cholesteric liquid crystalline phases that once stabilized by a ph increase, lead to collagen matrices that mimic the organization of the organic matrix found in bone [ ] . in the present work, we analyze physico-chemical ways to modulate long range organizations obtained in this liquid state. we study the effect of collagen concentration, ph ( . / . ) and acids (hydrochloride acid / acetic acid) over the liquid crystalline order. in a original approach, correlation of collagen endofluorescence and shg signals has enabled us to quantify cholesteric pitch and phase transition as a function of collagen concentration within a gradient. cholesteric pitch have proved to be very responsive to physico-chemical conditions. indeed, the results allowed us to quantify a i / cholesteric transition as a subsequent transition from cholesteric to a phase hexagonal or colummar. prion diseases are deadly neurodegenerative diseases affecting human and mammalian species. according to the 'protein-only' hypothesis, the key event in the pathogenesis is the conversion of the α-helix-rich monomer (prp c ) into a polymeric β-sheet-rich pathogenic conformer (prp sc ). we have used a combination of biophysical techniques with molecular dynamics simulations (md) to elucidate the molecular mechanisms of prp c unfolding and polymerization. under well established conditions, three β-sheet-rich soluble oligomers were generated from the partial unfolding of the monomer, which were found to form in parallel. to obtain a deeper insight into the molecular events, doublecystein mutants were designed, thus 'locking' different regions of prp. single mutations were also performed, which affected dramatically and selectively the prp oligomerization pathway. furthermore, we have now identified the minimal region that leads to the same oligomerization profile as the full-length prp, namely, h h . the existence of at least three distinct oligomerization pathways and the effect of single mutations reveal the conformational diversity of prp and a possible relationship with prion strain phenomena. the identification of domains involved in the conversion process may lead to a better understanding of the effect of mutations or gene polymorphism on the evolution of prion pathology. investigating toxic protein nanoclusters and their interactions with living cells s. nag, b. sahoo, a. bandyopadhyay, c. muralidharan, r. abhyankar, s. gurav, s. maiti tata institute of fundamental research, homi bhabha road, colaba, mumbai , india amyloid protein aggregation is responsible for many neurodegenerative diseases, such as alzheimer's and parkinson's. it appears that quasi-stable small soluble aggregates are the key to amyloid toxicity. though amyloid aggregation appears to be a nucleation mediated process, simple nucleation theory is unable to explain the stability of these intermediate species. we investigate this issue with fluorescence correlation spectroscopy in solution and on cell membranes. our initial data, varying the ph and the ionic strength of the solution, show that a charged colloid model can explain the overall stability of these species. in addition, this understanding suggests possible ways of modulating the stability of these aggregates in solution. some of these strategies successfully decimate the stable aggregate population, and also reduce the toxicity of amyloid beta to cultured neurons. afm studies of artificial amyloid fibrils and filaments p. mesquida , a. kurtossy , c. macphee department of mechanical engineering, king's college london, london, u.k., school of physics, university of edinburgh, edinburgh, u.k. amyloid fibrils are beta-sheet-rich superstructures of peptides or proteins. although these aggregates have first been found in connection with protein-misfolding diseases, such as alzheimer's or parkinson's disease, there is evidence that the ability to form fibrils is a generic property of any polypeptide rather than a result of specific, disease-related amino-acid sequences. fibrils can easily be formed in vitro from nondisease-related proteins and even from synthetic peptides. these artificial fibrils can thus serve as model systems to investigate the biophysical properties of amyloid fibrils. the system presented here is built from the artificial peptide ttr − , which contains amino-acids and which forms well-defined nanorods of ca nm diameter. we present atomic force microscopy (afm) results of their inner morphology by chemically dissecting the rods into their constituent filaments without completely breaking up the aggregates. in contrast to the stiff rods, their constituent filaments of ca - nm diameter were much more flexible. this shows that the particular way filaments are arranged around each other can massively change the mechanical rigidity of the resulting structures in amyloid fibrils, which could be one cause of the different strains in amyloid diseases. the formation of insulin fibrils is characterized by an initial apparent lag-phase, related to the formation of oligomers, protofibrils and aggregation nuclei. afterwards, the aggregation proceeds via fibril elongation, thickening and/or flocculation, and eventual gelation. here, we focus on the formation of such a gel, made of insulin amyloid fibrils, upon incubation at high temperature and low ph. by light scattering and rheological techniques, we monitor the development of the structural, dynamical and mechanical properties of fibrillar aggregates, up to the dynamic arrest of the sample and to the appearance of a non-ergodic behaviour, which marks the onset of gelation. our experiments were able to reveal the structural details hidden in the apparent lag-phase, displaying the slow fibril nucleation and elongation. this initial stage is followed by the known exponential growth of structures of different sizes. these two kinetic stages of structural growth are mirrored by the kinetics of the viscoelastic properties and, in particular, by the growth of the elastic modulus. our results show that the appearance of a noteworthy elastic network, is associated with the initial fibril nucleation and elongation more than with the formation of large structures, which causes the eventual gelation. capturing the initial events of in-cubo crystallization of membrane proteins c. v. kulkarni , o. ces , s. iwata , r. h. templer chemical biology centre and department of chemistry, imperial college london, united kingdom, division of molecular biosciences, imperial college london, united kingdom, institute für chemie, heinrichstrasse- , university of graz, austria lipid based methods for the crystallization of membrane proteins including in-cubo crystallization are becoming popular in recent times. however, a complete understanding of the basic principles behind these methodologies is still elusive. the crystallization of membrane proteins in the lipid cubic phases involves following major steps: removal of membrane proteins from the native membrane using detergentlike molecules, followed by their insertion and equilibration into a lipid bilayer. the crystallization itself commences with precipitant induced osmotic dehydration which in turn stimulates the nucleation that subsequently leads to a crystal growth. using time-resolved x-ray scattering (saxs) and ultraviolet spectroscopy we have been able to gain new insights into the mechanism behind protein insertion into these complex three dimensional lipid structures including information on the timescales of protein folding relative to crystal growth and the effect of protein insertion on the morphology of the surrounding lipid matrix. several proteins can form amyloid fibrils under given environmental or thermodynamic conditions that affect their native conformation. lysozyme forms amyloid fibrils upon incubation of solutions at acid ph and at about • c for a few days. differential scanning calorimetry experiments confirm that lysozyme is mainly unfolded above • c at ph . we monitored the growth of aggregate size by dynamic light scattering for some days at different temperatures. our results show that the fibrillogenesis is characterized by an initial apparent lag phase and a subsequent growth with quadratic dependence upon time of the scattering intensity. this behaviour recalls a simple kinetic model of nucleation and elongation, with nuclei in equilibrium with monomers. at the end of the incubation at high temperature, we collected atomic force microscopy images which show fibrils with a diameter of a few tens of nanometers and a length of a few microns, characterized by a periodicity along the elongation axis. interestingly, the fibrils morphology exhibits no branching or thickening. this is consistent with the non-exponential growth observed in light scattering experiments. in order to elicit the role of repulsive electrostatic interaction in protein unfolding and self-assembly we extended our study at different ph. in this work we used afm to follow the amyloidogenesis pathway of transthyretin (ttr) to form protofilaments. single-molecule force spectroscopy (smfs) of native ttr and protofilaments were also compared in order to evaluate dynamic and structural differences. we observed that this pathway proceeds through the formation of transient amorphous aggregates, followed by the occurrence of annular oligomers. although implicated in cytoxicity, the role of such oligomers within the amyloidogenesis pathway is poorly understood. we show that the annular species display a tendency to stack, forming tubular-like structures that precede the formation of protofilaments. the protofilament height and pitch resemble those of ttr amyloid reported in previous structural studies. upon solvent exchange, we also observed protofilament disassembly that revealed structures reminiscent of the initial ttr annular oligomers. smfs of protofilaments showed a time-dependent increase in the length of the manipulated structures, suggesting that associations between monomers stabilize with time. force spectra of native ttr and protofilaments contained transitions spaced by ∼ nm, indicative of sequential unfolding of individual β-strands. based on these results a model of ttr protofilament assembly is proposed. ability to undergo amyloid aggregation is affected by protein size c. parrini , h. ramshini , f. chiti , a. relini physics department, university of genoa, italy, biochemical sciences department, university of florence, italy short polypeptide chains, typically between and residues, are generally involved with the conversion from the native state into insoluble fibrillar aggregates. the low prevalence of large proteins is disproportionate with their high occurrence in the human proteome. in order to explore the propensity of large proteins to form amyloid-like fibrils, the -residue hexokinase-b from saccharomyces cerevisiae (yhkb) has been induced to aggregate under two separate conditions, at low ph in the presence of salts and at ph . in the presence of trifluoroethanol. such conditions are among the most promising to form amyloid-like fibrils by normally globular proteins. under both conditions yhkb aggregates very rapidly into species with significant β-sheet structure, as detected by circular dichroism, and a weak thioflavin t and congo red binding. atomic force microscopy revealed globular aggregates eventually clustering into large amorphous aggregates at low ph, while in the presence of trifluoroethanol ribbon-like structures with distinct morphology from typical amyloid fibrils were observed. they had irregular width, no twist, and were connected by thinner fibril segments perpendicular to them. in general, yhkb aggregates displayed an unusual softness, as they were very easily perturbed by the afm tip. these results suggest that inability to form amyloid fibrils may prevent large proteins from being associated with protein deposition diseases. the initial stage of proteins aggregation leading to amyloid fibrils: a saxs study m. g. ortore , f. spinozzi , f. carsughi , t. narayanan , g. irace , s. vilasi , p. mariani dip. saifet, univ. pol., ancona, italy, esrf, grenoble, france, dip. biochim. biofis., ii univ., napoli, italy under some conditions, a protein converts from its soluble form into highly ordered aggregates called amyloid fibrils, which are associated with many human diseases. in order to tackle the prevention and treatment of these diseases, we need to understand the mechanism of the pathological aggregation of proteins. in vitro amyloid fibril formation is preceded by the formation of metastable non fibrillar forms, which are responsible for cytotoxicity underlying neurodegeneration. the molecular mechanisms leading proteins into prefibrillar aggregates are still unclear. we present a saxs study performed at id beamline of esrf on the apomyoglobin mutant w fw f, which at physiological ph, firstly aggregates in prefibrillar forms that are cytotoxic and then forms amyloid fibrils. the first stages of w fw f oligomerization are induced by a ph jump. data show that big changes in w fw f in solution happen in less than ms. singular value decomposition (svd) of the data yields a set of functions, from which all the scattering curves can be reproduced. the major result of our study is the determination of the presence of different oligomers in each step of the process. hence, time-resolved saxs experiments together with the estimation of different oligomers via svd method, can be a new and useful approach to investigate the first stages of amyloidogenesis. -condensed colloidal phase in biology: from proteins crystals to amyloid fibrils - study of structure/toxicity relationship of amyloids by infrared spectroscopy h. p. ta amyloid diseases (alzheimer, parkinson, type ii diabetes, prion) correlate with protein aggregation. all the proteins associated with these pathologies aggregate into amyloid fibrils with a common ß-cross structure. according to many in vitro studies, the toxicity of various amyloids seems to be linked to some intermediates formed during the aggregation pathway and to their interaction with membranes. our aim is to understand the link between structure and toxicity of amyloids by studying their interaction with model membranes. for this, we use as an amyloid model, the prion-forming domain pfd - (wild type wt) of het-s, a prion protein of the fungal p. anserina. when expressed in yeast, wt is not toxic whereas one of these mutants, m is toxic. in vitro, m forms very unusual short amyloid fibers contrary to wt which polymerizes as long fibers. furthermore, atr spectra have shown that m is essentially assembled into mixed parallel and anti-parallel ß-sheets whereas wt displays a predominant parallel organization. we are currently studying the interaction between wt or m with lipid langmuir film by polarized modulation-infrared reflexion absorption spectroscopy (pm-irras). this technique allows determining the secondary structure of proteins and their orientation in the membrane. the study of interactions with different charged phospholipids is following. a. stirpe, m. pantusa, r. bartucci, l. sportelli, r. guzzi dipartimento di fisica, laboratorio di biofisica molecolare & udr cnism, università della calabria, rende (cs), italy an increasing number of experimental studies is demonstrating that the propensity of forming amyloid fibrils is not limited to proteins related to neurodegenerative diseases but it is a more general phenomenon. we present data on human serum albumin (hsa) aggregation induced by a combination of thermal and metal ions effects investigated by optical density, fluorescence and electron paramagnetic resonance (epr). the turbidity experiments as a function of temperature show that the hsa aggregation starts at about • c which is higher than the denaturation temperature (∼ • c). in the presence of copper and zinc metal ions the onset temperature for protein aggregation is markedly reduced as the metal:protein molar ratio is increased from : to : . moreover, the copper is more effective in inducing protein aggregation compared to zinc ion. the hsa aggregation analyzed by tht fluorescence at different incubation temperatures lacks a lag phase and the kinetic traces can be fitted by double exponential functions. the tht fluorescence increase evidences the formation of protein aggregates with fibrillar features. epr experiments for the cu(ii)-hsa complex show the binding of cu(ii) in the protein native state in a square planar coordination with equatorial n atoms, and is not influenced by the heat treatment of the protein. the overall results suggest that hsa aggregation is compatible with a downhill process that does not require the formation of an aggregation nucleus. protein condensation diseases -a colloid physicists viewpoint p. schurtenberger adolphe merkle institute, university of fribourg, ch- marly , switzerland a broad class of diseases, such as cataract, alzheimer's disease and sickle-cell disease, involve protein association phenomena as an essential aspect. the basic element common to all members of this class of molecular condensation diseases is the subtle interplay between protein interactions that produces condensation into dense, frequently insoluble mesoscopic phases. among this class of diseases, cataract is particularly important as the world's leading cause of blindness. this disease is most often the consequence of an uncontrolled aggregation (or phase separation) of the proteins in the eye lens that results in a loss of its transparency. the high concentration protein mixtures present in the eye lens are normally stable and produce a high refractive index that aids the eye in adaptive focusing of light. moreover, the proteins themselves exhibit a rich variety of repulsive interactions, attractive interactions, sizes, phase transitions and self-association. these mixtures also exhibit the pathological aggregation and opacification of the cataract disease that has inspired their study. in my presentation i will illustrate how we can use a combination of small-angle neutron and xray scattering experiments combined with molecular dynamics computer simulations to identify, measure and model the molecular interactions and emergent optical and viscoelastic properties and the phase behavior of the relevant, complex cytoplasmic mixtures. fluorescence microscopy studies of iapp fibrillation at model and cellular membranes d. c. radovan , n. opitz , r. winter department of physical chemistry i -biophysical chemistry, tu dortmund university, dortmund, germany, max-planck institute for molecular physiology, dortmund, germany type diabetes mellitus (t dm) is characterized by islet amyloid deposition and beta cell death, the main culprit being a small a.a. peptide hormone, islet amyloid polypeptide (iapp), which forms fibrils under pathological conditions. we studied the interaction of iapp with giant (guvs) and large (luvs) unilamellar vesicles as well as with ins- e cells. by using confocal / two-photon excitation fluorescence microscopy and guvs, we tested the influence of charge (dopc:dopg : ) and model raft systems, displaying liquid-ordered (l o ) / liquid-disordered (l d ) phase coexistence (such as dopc:dppc:cholesterol : : ), respectively, on the kinetics of iapp fibril formation. a preferential partitioning into the l d phase was observed and fibrils grew along with lipid uptake. fluorescence spectroscopy leakage experiments with carboxyfluorescein-filled luvs and the corresponding tht kinetics of iapp fibril formation were carried out as well. moreover, using the wst- reduction assay and fluorescence microscopy, we could show that the red wine compound resveratrol is a potent inhibitor of iapp fibrillation and its cellular toxicity on ins- e cells, these findings highlighting the potential role of resveratrol in future clinical applications, i.e., in the treatment of t dm. a remarkably high viscosity has been induced in aqueous solutions of lysozyme by the addition of certain structurally related organic solvents, such as tetramethylurea (tmu), dimethylsulfoxide (dmso), dimethylformamide (dmf), and hexamethylphosphortriamide. dmso-induced gelation is observed in samples fulfilling the two following requirements: ( .) lysozyme concentration in excess of mm, and ( .) volume fractions of dmso exceeding . . based on spectroscopic data, the whole process was characterized as consisting of two mutually independent stages. the first involves an extensive transition of the polypeptide backbone, from a predominantly helical to increased random coiled and beta-sheet structures, with the occurrence of non-orthodox protein secondary structures at regions above the solvent critical point. the second stage consists of short-lived interchain contacts leading to an entanglement of the macromolecular system as a whole. here we present a set of scattering and microrheology experiments that investigate both the structural and dynamic properties of the gels under various experimental conditions. studying alpha-synuclein aggregation by fluorescence polarization based kinetics l. tosatto, f. munari, i. tessari, g. de franceschi, m. pivato, p. polverino de laureto, m. bisaglia, l. bubacco università degli studi di padova, padova, italy alpha-synuclein (syn) is linked to parkinson's disease (pd) by two evidences: the accumulation of amyloid fibrils of the protein and the autosomal dominant forms of the disease (a t, a p and e k mutants). protein oligomers seem to be the most toxic species, causing dopaminergic neuronal death, probably disrupting cell membrane (volles & lansbury, ) . the aggregation of syn follows a nucleation dependent mechanism, i. e., aggregation is favoured only after the formation of an oligomer composed of a critical number of monomers (wood et al., ) . as the constitution of the nucleus is a rare event, early aggregation stages are difficult to study. to explore the early stages of the aggregation process a fluorescence polarization (fp) based method (luk et al., ) was applied to study syn oligomerization. fp depends on the size of the fluorescent molecule, therefore it is suitable for the detection of oligomers formation. we measured aggregation kinetic properties under several different conditions. a to mixture of syn and oregon green labelled syn was used to analyze the aggregation behaviour of wild type (wt) and pd mutants. the wt protein shows the fastest aggregation rate. this aggregation process is slowed down by the presence of the chaperone - - eta in wt syn samples. finally, dimers formed by a disulfide bond at the n-terminus or c-terminus of syn, when tested for their aggregation behaviour, show a different propensity to aggregate. a. pardini , r. bizzarri , a. diaspro , p. bianchini , c. usai , p. ramoino , g. checcucci , g. colombetti istituto di biofisica, cnr, italy, a) nest, sns, iit udr, pisa, italy; b) nest, cnr-infm, pisa, italy, univiversity of genova, genova, italy the colored ciliates blepharisma japonicum and fabrea salina show photomotile responses triggered by endogenous pigments of the hypericin family. b. japonicum exists in two forms: the wild one contains red blepharismin; the other one, generated by irradiating the cells with dim visible light, blue blepharismin. b. japonicum shows step-up photophobic response, whereas f. salina, that contains fabrein, shows step-down photophobic responses and positive phototaxis. we showed by confocal microscopy that the pigments are localized not only in pigment granules, but also in the cilia. this fact implies that the structure of the pigment in the cilia differs from that in the cell body, (pigment granules are too big to fit in the axoneme) and suggests that ciliary pigments might play a decisive role in photoreception. fluorescence lifetime imaging microscopy (flim) shows that there is a spatial distribution of lifetimes, which are shorter for the pigment in the cilia. this might indicate a functional role for these pigments. furthermore, lifetimes for f. salina are always longer than those of blepharisma. in order to further characterize the structure of ciliary pigments by means of spectroscopic methods, we have also performed spatially resolved static and time-resolved fluorescence anisotropy measurements. biosensing applications of micro/nano structured silicon several topics related to porous silicon (ps) biosensing properties were carefully considered in this study. ps allows the increasing of the immobilised biomolecule number on its surface and the creation of stable covalent bonds due to its controllable chemistry. beside this, ps is suitable for electrical (conductance, impedance), electrochemical and optical amplification of the detected signal. the experimental results on the fabrication of the ps microstructures such as: (i) protein immobilization and detection using microarray technique; (ii) dna biomolecule detection by impedance or by fluorescence spectroscopy; (iii) very sensitive sers biosensors (raman signal of -mercaptoundecanoic acid); (iv) sensitive element for neurons in nutmix culture are in detail presented. various characterisation techniques have been used, optical and scanning electron microscopy (sem), x-ray diffraction, raman, laser fluorescence and impedance spectroscopy for investigation molecule attachment on the au/ps structures. we have demonstrated that different morphologies of ps as-prepared or coated with gold nanoparticles have an important role in biomolecule/cell detection, due to its large internal surface combined with specific optical properties, being in the same time sensing element/support for immobilization of sensing biomolecules as well as transducer for biochemical interactions. heterotrimeric g-proteins interact with their g-protein coupled receptors (gpcrs) via key binding elements comprising the c-terminal segment of the α-subunit and the two lipid anchors at the α− and γ−subunit. direct information about diffusion and interaction of gpcrs and their g-proteins is mandatory, as these properties will affect the timing of events in the complex signal transduction cascade. in the case of the photoreceptor rhodopsin, receptor packing in the membrane and the related diffusion coefficients are discussed controversially ( , ). by using single particle tracking we show that the encounters of rhodopsin with the fluorescently labeled cterminus of the α-subunit as well as with the holo-g-protein transducin change upon rhodopsin light-activation. our results indicate confined areas of interaction for the c-terminal segment of the α-subunit with inactive rhodopsin disk membranes and less restricted diffusion of the receptor-bound cterminal segment after light-activation. this suggests dynamic short-range order in rhodopsin packing and specific structures for efficient interaction ( ) in vivo, skeletal muscle actively shortens and also resists lengthening, for example when landing after a jump. we study the energy during shortening and during lengthening by measuring the rate of pi release which results from atp hydrolysis. we show here the rate of pi releases in an in vitro protocol that involves muscle stretched followed by muscle shortening. it is hypothesised that the magnitude of deceleration of pi release in the stretch phase is less than the acceleration during the release due the energy input of the motor that is used to apply the length changes. therefore the end point of pi release is similar to isometric conditions. pi release responses to a ramped stretch ( %) followed ms later by a symmetrical release at low velocities ( . l /s − ) were measured in permeabilised fibre bundles of rabbit psoas at • c. laser diffraction and high speed video were also recorded to confirm length change. we show that the rate of pi release drops during the stretch phase, returns to isometric levels during the length hold phase, and finally accelerates during the ramped release. tracking of sarcomere length change using video analysis demonstrated that laser diffraction is unreliable at times during stretches due to lack of uniformity of the sarcomere spacing. microbial rhodopsins: receptors, channels, and pumps from a single design j. l. spudich, e. negri-spudich center for membrane biology, university of texas, houston, texas, u.s.a. the microbial rhodopsin family is comprised of ∼ homologous proteins containing transmembrane helices forming a pocket for the chromophore retinal. most are lightdriven ion pumps ("transport rhodopsins") and others are photosensory receptors ("sensory rhodopsins"). phylogenetic analysis indicates frequent lateral gene transfer of proton pumps among prokaryotic and unicellular eukaryotic species, followed by coupling of the pump's mechanism to the cell's existing signal transduction machinery to create photosensors. this evolutionary path is strongly supported by our studies of sensory rhodopsins in various organisms which demonstrate remarkably diverse signaling mechanisms with diverse transducer partners. the best studied are the phototaxis receptors in haloarchaeal prokaryotes (sri and srii) and in eukaryotic algae (channelrhodopsins). sri and srii transmit signals by protein-protein interaction to control a phosphorylation cascade that modulates motility. channelrhodopsins are light-gated cation channels that depolarize the membrane mediating calcium ion influx into the flagellar axoneme. crystallography and molecular biophysics have begun to clarify how modifications of the same architecture enable the rhodopsins to carry out their distinctly different molecular functions. interconversions of their functions by mutation reveal the elegant simplicity by which evolution uses existing genes to create proteins with novel functions. gold colloids-fluorophore complexes for protein detection assay l. sironi , s. freddi , l. d'alfonso , m. collini , m. caccia , g. tallarida , s. caprioli , g. chirico dipartimento di fisica, università di milano-bicocca, italy, laboratorio nazionale mdm, agrate brianza (mi), italy noble metal nanoparticles (np) are endowed with peculiar optical properties related to the surface plasmon resonances (spr). the interaction of surface plasmons of gold nanoparticles with fluorophores a few nanometers away from the surface modifies their brightness and excited-state lifetime, and this effect can be exploited to obtain nanodevices for proteinprotein recognition. we studied different types of constructs based on gold nps on which derivatives of fluorescein were bound. the interaction of this fluorophore with the gold surface plasmon resonances, mainly occurring through quenching, affects its excited-state lifetime, that is measured by fluorescence burst analysis in standard solutions. the binding of proteins to the gold nps through antigen-antibody recognition further modifies the dye excited-state lifetime. this change can therefore be used to measure the protein concentration. streptavidin-functionalized gold nps of size - nm are used to bind biotin-fluorescein and biotin-antibodies for specific proteins. we have first tested the constructs for bovine serum albumine (bsa) detection. the data reported here indicate that one can measure the concentration of bsa in solution with an apparent limit of detection of ± pm. we have then extended the study to the antitumor protein p -p antibody interaction in standard solution and directly in cellular extracts. calcium transport and phototransduction in isolated rod outer segments g. rispoli dip. biologia ed evoluzione, università di ferrara, italy ca + concentration in photoreceptor rod outer segment (os) strongly affects the generator potential kinetics and light adaptation. light stimuli may produce voltage changes exceeding mv: since the os ca + extrusion is entirely controlled by the na + :ca + ,k + exchanger (nckx), it is important to assess how the nckx ion transport is affected by voltage and intracellular factors. the nkcx regulation was investigated in whole-cell recorded os, using ionic conditions that activated maximally forward and reverse exchange. in all species examined of amphibia and reptilia, the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. since hyperpolarization increases ca + extrusion rate, the recovery of the dark level of ca + (and of the generator potential) after light stimuli results accelerated. mg-atp doubled the size of forward and reverse exchange current without modifying their voltage dependence, indicating that mg-atp regulates the number of active exchanger sites and/or the nckx turnover number. ca + jumps achieved via photolysis of caged-ca + produced current transients, possibly originating from electrogenic partial reactions. no monovalent cation substituted for na + at the nckx binding sites, but rb + substituted for k + , while sr + , ba + , mg + substituted for ca + with an apparent permeability ratio of . , . , < . : , respectively. a. pfeifer , k. zikihara , s. tokutomi , j. heberle , t. kottke bielefeld university, bielefeld, germany, osaka prefecture university, sakai, japan the blue light receptor phototropin regulates the growth of plants towards the light. it contains two light-, oxygen-, or voltage-sensitive (lov) domains and a kinase domain. lov domains bind noncovalently flavin mononucleotide (fmn) as chromophore. upon illumination, the triplet excited state of flavin reacts within few microseconds with a nearby cysteine under formation of a photoadduct, which represents the signaling state. in response to adduct formation, a jα helix adjacent to the lov domain dissociates and allows for autophosphorylation by the kinase domain. the mechanism of the photoreaction and the signaling pathway from fmn to the jα helix are still unclear. we have investigated the lov domain of arabidopsis phototropin by microsecond ft-infrared spectroscopy. the difference spectrum recorded at µs provides evidence that the flavin is unprotonated in the triplet excited state. therefore, a previously proposed ionic mechanism of bond formation is disfavored. changes in secondary structure were detected concomitant with adduct formation that relax with a time constant of µs. this early adduct intermediate has not been previously characterized. the final adduct state is formed in milliseconds by further alterations in secondary structure. these findings raise the question of whether the early or late adduct intermediate propagate the signal to the jα helix. -photosensory biophysics - the psychostimulant amphetamine increased no generation measured by epr as well as amino acid release in the rat brain nitric oxide (no) is a novel messenger that modulates many functions of the nervous system. the involvement of no in brain damage was shown mainly by indirect evidence and the data are controversial. the short half-life of no makes its direct detection difficult. we measured no generation using epr spectroscopy based on determination of the amount of paramagnetic mononitrosyl-iron complexes. the aim was to elucidate whether psychostimulant drug amphetamine (amph) modulates formation of no and lipid peroxidation (lpo) products as well as the neurotransmitter release in rat brain. the output of glutamate, aspartate, gaba and acetylcholine (ach) was monitored in striatum by microdialysis with hplc detection. amph produced fold elevation of no generation and lpo formation in brain areas. while amph increased the aspartate, gaba and ach release, the glutamate output was not affected. pretreatment with the neuronal nos inhibitor was highly effective in abating the rise of no and neurotransmitter levels but failed to influence the lpo intensity elicited by amph. the findings suggest that activation of no synthesis is a potent factor in the amph-induced neurotransmitter release. light scattering study of dna over a wide chain length range: comparison with wormlike model p. baeri, m. zimbone dipartimento di fisica e astronomia, università di catania, italy this work reports light scattering measurements on dna in aqueous solutions ( mm nacl , mm edta and mm tris-hcl buffer, ph . ) over a wide range of molecular weights ( - base pairs) and shows that, in the above standard solvent, shorter chains ( < base pairs) behave as a "wormlike chain" and their diffusion coefficient as obtained by dynamic light scattering measurements, confirm the prediction of standard wormlike model, whilst longer chains ( > base pairs) behave in a different manner. dynamic and static light scattering and sem analysis indicate that dna molecules base pairs long, condense into compact structures in our solvent condition. calculations done using a wormlike model are also presented and discussed in comparison both to our experimental data and to other data reported in the literature. a. asandei, t. luchian 'alexandru i. cuza'university, faculty of physics, laboratory of biophysics and medical physics, blvd. carol i no. , iasi, romania amphotericin b (amb) is an antifungal antibiotic which, despite the severe side effects, is still used for the treatment of systemic fungal infections. in this study we investigated the influence of ph upon the selectivity and the transport properties of amb channels inserted in reconstituted, ergosterolcontaining zwitterionic lipid membranes. our electrophysiology experiments carried out on single and multiple amb channels prove that at ph= . these channels are anion selective, whereas at neutral and alkaline ph's (ph= and ph= ) they become cation selective. we attribute this to the ph-dependent ionization state of the carboxyl and amino groups present at the mouth of amb molecules. surprisingly, our data reveal that the single-molecule ionic conductance of amb channels varies in a non-monotonic fashion with ph changes, which we attribute to the ph-dependent variation of the surface and dipole membrane potential. we demonstrate that when added only from one side of the membrane, in symmetrical salt solutions across the membrane and low ph values, amb channels display a strong rectifying behavior, and their insertion is strongly favored when positive potentials are present on the side of their addition. we report a detection method for the redox state of proteins which combines fret-based fluorescence/confocal microscopy on dye-labeled protein with cyclic voltammetry. by using this combined method, electron transfer properties can be revealed from protein to electrode or from redox enzyme to substrate. we applied the fluorescent detection to azurin, a blue copper protein from the bacterium ps. aeruginosa, fluorescently labeled on the n-terminus for monitoring the redox state of the protein. the dye fluorescence is quenched by energy transfer to the copper in oxidized, but not in reduced azurin. fluorescence results demonstrated that cy -labeled wtazurin switched in fluorescence intensity by up to % by varying the applied potential. labeled zinc-azurin was used as a control sample and did not show any fluorescence switching. for single molecule studies wt-azurin was labeled with atto dye and a mixed sam was used, with -hydroxy- octanethiol as a blocking agent to prevent non-specific binding of protein on the surface, and , decanedithiol for the specific covalent binding to the protein. preliminary data show that single-molecule fluorescence switching with the potential is indeed possible. -single molecule biophysics - is there a specific erythrocyte membrane receptor for fibrinogen? an atomic force microscopy approach f. a. carvalho , s. connell , r. a. ariëns , n. c. santos instituto de medicina molecular, univ. lisbon, portugal, university of leeds, u.k. fibrinogen (fg) contributes to erythrocyte (rbc) hyperaggregation by an increase in. rbc-fb protein binding considered to be non-specific. glycoprotein α iib β is a specific integrin receptor for fibrinogen on platelets. we showed that there is a single molecule interaction between fg and an unknown receptor on rbc membrane, with a lower affinity when compared with platelet binding. we evaluated if rbc-fg binding is through an integrin-like receptor or not. interactions between fg and platelet/rbc receptors were studied by force spectroscopy. force curves were performed between fg-functionalized atomic force microscope tips and rbc or platelets. to evaluate if the fg-rbc binding is calciumdependent, similar studies were performed in the presence of ca + or edta. we also carried out studies in the presence of a α iib β inhibitor and of methyl-ß-cyclodextrin (to disrupt lipid rafts by cholesterol depletion). rbc-fg single forces were of - pn and of - pn for platelet-fg binding in presence of calcium. a significant decrease of the platelets-fg force-rupture was obtained in the presence of edta or α iib β inhibitor. significant lower fg-rbc force value was obtained in the presence of mβcd, but not in the presence of edta. conclusion: fg-rbc binding seems not to be calcium-dependent but the existence of cholesterol on rbc membrane is important. nanoscopic and spectroscopic investigation of p -based complexes at single molecule level a. r. bizzarri biophysics and nanoscience centre, cnism, facoltà di scienze, università della tuscia, largo dell'università - viterbo, italy p is a transcription factor that plays a widely recognized role in preventing cancer development in response to dna damage. the tumor suppressor activity of p involves the formation of several complexes whose detection and study, at single molecule level, could be extremely relevant to understand, in detail, the mechanisms governing the cancer defense processes, as well as to develop ultrasensitive biosensors. p -based complexes, are investigated at molecular level, by combining atomic force microscopy (afm), atomic force spectroscopy (afs) and surface enhanced raman spectroscopy (sers) with the support of computational docking. our attention is mainly devoted to study complexes between p and the electron transfer azurin which has been demonstrated to interact with p , by promoting its stabilization. a possible competition between azurin and the cellular oncogene mdm is also investigated. unwinding the dna helix in force clamp condition p. bianco , l. bongini , m. dolfi , l. vincenzo physiolab, dbe, university of florence, italy, university of florence and centro interdipartimentale studio dinamiche complesse, firenze, italy we use a dual-laser optical-tweezers (dlot, smith et al., science, ) to define the highly cooperative conformational transition in the molecule of dna, where the natural b-dna has converted into a new overstretched conformation called s-dna (bensimon et al., phys. rev. lett., ; cluzel et al., science, ) . single molecules of double stranded λ-phage dna (in a solution with mm naci, mm tris-hcl, mm edta, ph . . and • c) are stretched either in length clamp or in force clamp mode. when the dna molecule is stretched in length clamp mode with a ramp lengthening, it shows the previously described highly cooperative overstretching transition at ∼ pn, attributed to unwinding from the b-form to the . times longer s-form. stretching the molecule in force clamp mode with a staircase of force steps at s intervals (step size - pn, rise time - ms) shows, for any given clamped force f in the region of the overstretching transition, different amounts of dna elongation (∆l) with exponential time courses. the analysis of the elongation rates allows to recover all the necessary parameters for an effective two-state model able to reproduce the out-of-equilibrium properties of the system. the results imply an unwinding cooperativity of bps. this value is significantly lower than that obtained assuming force independent rate constants. supported by miur, ente cassa di risparmio di firenze and itb-cnr (milano). c. m. becker , a. benedix , b. l. de groot , a. cafisch , r. a. böckmann saarland university, saarbrücken, germany, mpi for biophysical chemistry, göttingen, germany, university of zürich, zürich, switzerland modifying the stability or the binding behavior of the involved proteins by mutation can influence the activity of cellular processes. for an efficient identification of possible mutation-sites a fast calculation of the free energy of proteins is crucial. here we developed a fast and reliable method (cc/pbsa) [ ] for the prediction of the change in stability of proteins and binding affinity of protein-protein complexes upon mutation. the energy function of cc/pbsa is based on gas phase energies, solvation free energies and entropic contributions. the protein flexibility is taken into account by generating random conformations based on geometrical constraints only applying the concoord [ ] program. we applied cc/pbsa on the tem -blip complex, which is important in bacterial antibiotic resistance. the results of single-and double-point alanine scanning are used to detect hot spots, cooperative effects, and the corresponding energy distribution. cc/pbsa is freely accessible on our web-server: http://ccpbsa.bioinformatik.uni-saarland.de the human recombinase hrad is a key protein for the maintenance of genome integrity and for cancer development. this protein plays a central role is the dna strand exchange occurring during homologous recombination. here we report the polymerization and depolymerization of hrad on duplex dna observed with a new generation of magnetic tweezers, allowing the measurement of dna twist with a resolution of • in real time. at odds with earlier claims, we show that, after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition of single proteins, involving dna twisting steps of ± • . simple numerical simulations support that this mechanism is an efficient way to minimize nucleoprotein filament defects. this behavior, consisting of different stoichiometry for nucleation and growth phases, may be instrumental in vivo. fast growth would permit efficient continuation of strand exchange by rad alone while the limited nucleation would require additional proteins such as rad , thus keeping this initiation step under the strict control of regulatory pathways. besides, our results combined with earlier structural information, suggest that dna is somewhat less extended ( . versus . Å per bp) and more untwisted ( . versus • per bp) by hrad than by reca, and confirm a stoichiometry of - bp per protein in the hrad -dsdna nucleoprotein filament. biofunctional micropatterned surfaces to study the spatio-temporal organisation of lfa- r. diez-ahedo , d. normanno , c. g. figdor , a. cambi , m. f. garcia-parajo bionanophotonics, ciber-bbn and ibec, barcelona, spain, tumor immunology, nijmegen center for molecular life sciences, the netherlands lymphocyte function associated antigen- (lfa- ) adhesion depends on receptor occupancy and lateral organization on the cell membrane. however, the signals and mechanisms which dynamically reorganize lfa- into high avidity clusters are still a subject of many studies. to obtain deeper insight on the mechanisms that control and regulate lfa- clustering, patterned surfaces of immobilized lfa- ligand areas were fabricated using microcontact printing. the diffusion of lfa- expressed by monocytes stretched over patterned surfaces was followed in time using single molecule tirf microscopy. single lfa- nanocluster trajectories on individual cells showed an increase of immobile lfa- fraction and a slow-down of diffusing lfa- on the ligand areas compared to the non-ligand areas. moreover, single-cluster intensity analysis indicated a reorganization of lfa- nanoclusters in microclusters upon ligand binding. finally, single particle motion analysis of lfa- trajectories in close neighborhood to the ligand areas showed no assisted diffusion of lfa- towards the adhesive regions, consistent with random ligand-encountering and binding. we are currently investigating the effect of cell membrane organizers to regulate the spatio-temporal organization of lfa- . r. diez-ahedo et al, small, in press. optical and electrophysiological detection of single phages across a lipid membrane n. chiaruttini , p. boulanger , m. de frutos , l. letellier , u. bockelmann , v. viasnoff nanobiophysique, espci paristech, cnrs, paris, france, ibbmc, université paris xi, cnrs, orsay, france, lps, université paris xi, cnrs, orsay, france we present an investigation study of the ejection of single t bacteriophages. in vivo studies of dna ejections from the bacteriophage capsid show that the t genome is introduced in the bacterial host in two steps. first % of the genome is ejected then after a pause of a few minutes the rest is internalized. bulk in vitro studies showed that various mutants of t eject their genome in solution following a single or a multistep process. by immobilizing single bacteriophages on a surface and following their ejection by fluorescence microscopy we showed that in all cases the ejection occurs in one step, but some mutants seem to have a subpopulation for which the triggering signal of the ejection is transmitted more slowly to the capsid entrance. we then reconstituted the phage receptor fhua into giant liposomes and followed the ejection of the dna into the liposome by fluorescence. finally we incorporated fhua in a suspended bilayer and followed the infection of the phages through the bilayer both by fluorescence labeling and electrophysiological measurements. we will discuss the influence of the cross membrane potential on the ejection speed of the dna. helixlike pili is a prerequisite of uropathogenic e. coli to adhere to host and withstand urine flow the gram-negative uropathogenic escherichia coli (upec) bacteria, invades the urinary tract region and cause in some cases severe infections, pyelonephritis, if they can withstand the rinsing action of urine and ascend to the kidney, via the bladder and ureters. to mediate adhesion, upec express quaternary surface organelles that are assembled from ∼ identical subunits into a helix-like coil, with a single adhesin located at the tip. it is believed that the single adhesin mediate attachment to host cells while the helix-like structures act as shock absorbers to dampen the irregularly shear forces induced by urine flow. to unravel the biomechanical properties of such quaternary structures, in particular in terms of their force-elongation and kinetic behavior, force-measuring optical tweezers (fmot) have been used. a plethora of different types of pili have been identified in the literature and we show, using fmot, that those dissimilarities might reflect the host environment. for example, we have found differences among pili expressed at diverse environment inside the urinary tract, which imply that pili presumably have evolved to resist specific forces under in vivo conditions. it is thus worth striving for understanding bacterial adhesion in order to figure out alternative to the over-abundance of antibiotics worldwide. single molecules studies have probed protein conformational fluctuations and monitored the oscillating activity of enzymes that remained masked in ensemble measurements. various statistical models have attempted to connect the conformational motions to the fluctuating activity of enzymes and suggested that they adopt inter-converting conformations each one of them exhibiting different catalytic activity. the main drawback of these studies is the non specific interactions that may bias the observed catalytic behavior. we have developed new innovating tools to study the behavior of single enzymes. as a first step we utilized liposomes as scaffold to confine enzymes while keeping them under physiological conditions. keeping them in their native conformation allowed us to monitor the inherent properties of their behavior. as a second step we developed a new model that accurately connects enzyme activity behavior to conformational motions. using this approach we accurately predicted the behavior of single lipases in a highly controlled environment. we modulated the enzyme's conformational mobility and activity by systematically varying its accessibility to liposomes. as we anticipated, that resulted in altering the time the enzyme spends on active conformations.our results provide new insights on interpreting the behavior of enzymes. simulations of single-molecule fret experiments on ribozymes m. hajdziona, a. molski adam mickiewicz university laboratory for dynamics of physicochemical processes, poznan, poland rnazymes are important biological molecules and that can catalyze the cleavage of their own nucleotide chains. this precess is called self splicing [ ] . there are several methods to to study kinetic properties of ribozymes at the singlemolecule level. in this work we focus on fret (förster resonance energy transfer) of single, immobilized molecules. single-molecule fluorescence spectroscopy gives an insight into the behavior of individual molecules rather than the average behavior of an ensemble of molecules, which makes it possible to observe the kinetic heterogeneity. we simulated fret trajectories for single immobilized ribozymes. in our computer simulations we consider two-and three-state kinetic models motivated by actual experiments, published in [ ] and [ ] . we fitted the histograms of on and off times to recover the kinetic parameters of the models. we investigated the bias and standard deviation of the recovered parameters when one or two thresholds are applied to fret trajectories between adjacent states. we found that two thresholds give better parameter estimates than one-threshold analysis. references frap probes the average dynamical properties of a population of fluorescently labeled molecules whereas spt obtains these properties from observations of the trajectories of individual molecules tagged with a colloidal particle (the "bead"). when results of frap are compared with those of spt, frap yields significantly higher diffusion constants than spt. to understand the origin of this difference, we have developed and tested a model system to evaluate the influence of the bead on the dynamics of a diffusing molecule. we use a dsdna tether to attach a bead to a supported lipid bilayer (slb). the dna tether is modified at one end by cholesterol for anchoring to and diffusion in the membrane, and at the other end by biotin for tagging with a bead. with this system, we can vary several parameters: distance between slb and bead, size and nature of the bead, lipid composition of the slb, external force. we can also model the brownian motion of the bead and the hydrodynamic flow around it. we will present results using φ = nm neutravidin-coated latex particles attached to an eggpc slb via a bp to bp dna tether. elucidation of the mechanism of the lambda bacteriophage epigenetic switch l. finzi physics department, emory university, dowman dr, atlanta, ga , usa the lambda bacteriophage epigenetic switch determines the growth lifestyle of the virus after infection of its host (e. coli ). it is now clear that the switch consists of a ∼ . kbplong dna loop mediated by the lambda repressor protein. using tethered particle microscopy (tpm), magnetic tweezers and afm, our laboratory has novel, direct evidence of loop formation and breakdown by the repressor, the first characterization of the thermodynamics and kinetics of the looping reaction and its dependence on repressor non-specific binding and dna supercoiling. these in vitro data provide insight into the different possible nucleoprotein complexes and into the lambda repressor-mediated looping mechanism which leads to predictions for that in vivo. the significance of this work consists not only of the new insight into a paradigmatic epigenetic switch that governs lysogeny vs. lysis, but also the detailed mechanics of regulatory dna loops mediated by proteins bound to multipartite operators and capable of different levels of oligomerization. mechanical force at the molecular level is involved in the action of many enzymes. for example, the phi dna polymerase mechanically unwinds the dna helix as it moves processively along the dna replicating one strand of the dna molecule. using optical tweezers we have developed a single molecule mechanical assay to elucidate the physical mechanism of dna unwinding by the phi dna polymerase as the protein replicates the dna. a single dna hairpin is hold between an optical trap and a mobile surface. as a single polymerase works on the dna hairpin, its replication and unwinding reactions can be measured in real time by measuring the change in extension in the dna polymer, revealing the fluctuations of its rate in response to the dna sequence. moreover, by gradually pulling on the opposite strands of the dna hairpin we can promote the controlled mechanical unwinding of the dna helix and determine the effect of increasing unwinding forces on the polymerization and unwinding rates. the effect of force and dna sequence on the activities of the wild type and an unwinding-deficient polymerase mutant will allow us to determine the inner workings of this molecular motor. single centrosome manipulation reveals its electric charge and associated dynamic structure we demonstrate laser manipulation of individual early drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively-charged organelle with a very low isoelectric region ( . ± . ). from this single-organelle electrophoresis, we infer an effective charge smaller or of the order of electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. by investigating the centrosome hydrodynamic behavior, we show that its charge has a remarkable influence over its own structure. specifically, we find that the electric field which drains to the centrosome expands its structure to a physiological size a % larger than previous electron microscopy determinations, a self-effect which modulates its structural behavior via environmental ph. this methodology further proves useful to study the action of different environmental conditions, such as the presence of ca + , over the thermally-induced dynamic structure of the centrosome. magnetic contrast neutron reflectivity delivers significant improvements in resolution for membrane structure analysis neutron reflection (nr), with its high resolution and use of contrast variation, is a unique tool to gain information on the orientation and structure of lipid bilayers in the z-axis perpendicular to the surface. to improve the use of nr we have used a highly oriented and stable layer of membrane proteins and lipids made possible by self-assembly upon a gold surface. this also provides a model of the bacterial outer membrane. as the dimensions of the layer can be predicted with accuracy, the system provides a molecular ruler for improvements in methodology and the complexity of the layer structure adds significantly to the modelling challenge. early improvements in resolution were obtained through sample preparation and gold smoothness. further improvement however required clearer discrimination between similar models. this was achieved using the new approach of magnetic contrast variation which uses a magnetic layer to provide two different scattering length densities for oppositely polarised neutrons. during this data collection the sample is unchanged. we show that this approach delivers significant improvements in data analysis and resolution of the protein, lipid and solvent structures. the method will provide a new level of understanding of membrane structure and dynamics. single molecule force spectroscopy and recognition imaging p. hinterdorfer institute for biophysics, johannes kepler university of linz, altenbergerstr. , a- linz, austria in single molecule force spectroscopy, interaction forces of ligand-containing tips with receptors on probe surfaces are quantified. here he attachment of human rhino virus (hrv ) to the cell surface, the first step in infection, was characterized. sequential binding of multiple receptors was evident from recordings of characteristic quantized force spectra. this suggests that multiple receptors bound to the virus in a timely manner. unbinding forces required to detach the virus from the cell membrane increased within a time frame of several ms. the number of receptors involved in virus binding was determined and estimates for on-rate, off-rate, and equilibrium binding constant were obtained. furthermore, we show that accurate free energy values of membrane protein unfolding can be obtained from single molecule force measurements. by applying a statistical theorem developed by jarzynski, we derived equilibrium unfolding free energies of from unfolding force data acquired at different force loading-rates and temperatures. finally, we present a method for the localization of specific binding sites and epitopes with nm positional accuracy. a magnetically driven afm tip containing a ligand covalently bound via a tether molecule is oscillated at a few nm amplitude, during scanning along the surface. in this way, topography and recognition images on membranes and cell surfaces were obtained simultaneously. mapping of the forces acting on biomolecules in cell membranes has spurred the development of effective labels, e.g. organic fluorophores and nanoparticles, to track trajectories of single biomolecules [ ] . standard methods use particular statistical observables, namely the mean square displacement (msd), to extract cues on the underlying dynamics. yet, msd is not an appropriate tool to access force fields and becomes easily a biased estimator in the presence of positioning noise. here, we introduce general inference methods [ ] to fully exploit information hidden in the experimental trajectories, providing sharp estimates for the forces and the diffusion coefficients within membrane microdomains. rapid and reliable convergence of the inference scheme is demonstrated on trajectories generated numerically. the inference method is then applied to infer forces and potentials acting on the receptor of the ε-toxin labelled by lanthanide-ion nanoparticles. results show a constant diffusivity inside a complex force field confining the receptor inside a specific domain. our scheme is applicable to any labelled biomolecule, and results presented here show its general relevance to the issue of membrane compartmentation and protein motion. large scale domain motions are structural rearrangements often adopted by enzymes to achieve their full functionality. understanding the mechanism responsible for such movements by means of computational approaches is of great interest especially in rational drug design, since induced fit or population shift effects are closely related aspects of the problem. unfortunately, the simulation time required to sample these events by conventional techniques such as plain molecular dynamics, is unfeasible. here, the domain motion required by adenosine kinase (ak) to achieve its (pre-)catalytic conformation was studied using well-tempered metadynamics [barducci, a. et al. phys rev lett. ( ) , : ] with path collective variables (pcvs) [branduardi, d. et al. j chem phys. ( ) , : ]. first, a low energy path for the apo form of the enzyme was obtained, and the potential of mean force along the pcvs was reconstructed. then, the large scale movement was simulated in the presence of two inhibitors known to bind to the enzyme in different conformational states. the adopted approach was proven to be successful both to understand the mechanistic features of the ak domain motion and to provide a picture of the population shift effects upon ligand binding. force measurement study of the interaction between s rrna and the ribosomal protein l single molecule force measurements enable to probe the complex structure and folding dynamics of rna molecules and furthermore to investigate the numerous interactions with proteins that affect rna folding in vivo. we use a double optical tweezers setup to study a region of the ribosomal rna s from e.coli and its interaction with the essential ribosomal protein l . the apparatus permits us to probe the dynamics of the rna structure with milisecond time-resolution. first, we pull the rna and show that it mechanically unfolds in several reproducible steps. we use these results in combination with structure prediction tools to evaluate the possible structures of the rna fragment in vitro. the most probable structure exhibits a unique binding site for l . then, force measurements are done on the rna in presence of l . we show that the protein specifically binds the rna and stabilizes it. the binding site is recognized with a resolution of a few bases. finally, two bases are mutated on the rna fragment. in presence of these mutations, in vivo and in vitro binding of l to s rna is abolished. the single molecule approach gives an explanation of this result: the force measurements show that the mutated rna folds differently from the natural one and that it does not bind l . control of the translocation of single dna molecules through alpha-hemolysin nanopores g. maglia, h. bayley department of chemistry, university of oxford, ox ta, oxford, uk the analysis of single nucleic acid molecules by electrophoretic threading through nanopores is under intense investigation as a rapid, low cost platform for dna sequencing. biological nanopores such as staphylococcal alpha-hemolysin (hl) have an added advantage over solid-state nanopores because they can be modified by genetic engineering with atomistic precision. although we showed that all four dna bases can be identified in an immobilized ssdna molecule ( ), the translocation of free dna is too quick to observe single bases. here we show that by a small increase of the net internal charge of the hl nanopore (e.g. by introducing a ring of arginine residues), we have augmented the frequency of dna translocation events through the pore and dramatically lowered the voltage threshold required for dna translocation ( ) . by further increasing the net positive charge of the transmembrane barrel region of the pore (e.g. by introducing extra positive charges) we have also reduced the speed at which dna translocate the pore by more than two orders of magnitude. these experiments provide a means of controlling dna translocation with protein nanopores, which might be translated to solid-state nanopores by using chemical surface modification. the atomic force microscope (afm) is a tool for imaging, measuring and manipulating matter at the nanoscale. single-molecule pulling experiments give information on the thermodynamics and kinetics of biomolecules. the purpose if this work was to develop software to simulate single-molecule pulling experiments and to analyze singlemolecule pulling data. the long term objective is to asses the accuracy and precision of the parameters recovered from single-molecule pulling data.we carried out simulations for two different models [ , , ] , using a wide range of loading rates. from the simulated force-extension curves we extracted the kinetic parameters and compared them with the values used for simulations. the kinetic parameters were the intrinsic rate coefficient (k kramers rate), the location of transition state (x ) and the free energy of activation (∆g). we have found that the loading rates have a small effect on the recovery of the free energy of activation, but have a significant effect on the recovery of the kramers rate. we report the tracking of single myosin v molecules in their natural environment, the cell. myosin v molecules, labeled with quantum dots, are introduced into the cytoplasm of living hela cells and their motion is recorded at the single molecule level with high spatial and temporal resolution. we perform intracellular measurements of key parameters of this molecular transporter: velocity, processivity, step size and dwell time. our experiments bridge the gap between in vitro single molecule assays and the indirect measurements of the motor features deduced from the tracking of organelles in live cells. a mathematical model of neurotransmission at the input stage of the cerebellum t. nieus , s. solinas , l. mapelli , e. d'angelo dept. neuroscience and brain technologies, iit, italian institute of technology, genova, italy, dept. of biomolecular sciences and biotechnology, milan, italy, dept. physiological and pharmacological sciences and cnism, university of pavia, italy the granule cell (gc) of the cerebellum has some peculiar properties compared to other cells of the vertebrate brain. the gc has a small soma (diam= microm) and just a few ( to ) excitatory and inhibitory inputs (e&i). the e&i gc synaptic inputs are formed inside the cerebellar glomerulus, which favors neurotransmitter diffusion between neighboring sites ( ) protracting postsynaptic receptor activation and ( ) causing cross-tall between e&i synapses through presynaptic receptors. neurotransmitter release probability (p) can be regulated by long-term plasticity (ltp and ltd at e synapses) and by gaba b and mglu presynaptic receptors (both at e&i synapses). the p change in turn modifies shortterm plasticity, affecting the first response in a train much more than the followings. to gain insight into the role that p changes might have in computations performed at the cerebellum input stage, we have built detailed biophysical models of the e&i synapses. the model has allowed to investigate glomerular processing of high frequency inputs reaching the cerebellum. optimal synaptic transmission through the mfs inputs resulted when gos inhibited synchronously the gcs. the role of feed-forward inhibition onto synaptic transmission is under investigation. unzipping dna with a nanopore j. muzard, n. chiaruttini, u. bockelmann, v. viasnoff cnrs/espci nanobiophysique, rue vauquelin, paris, france the use of proteinaceous or artificial nanometer size pores has become a promising approach for sensing biomolecules at the single molecule level. it was shown that alphahemolysin, a toxin from staphylococcus aureus, can be employed to sense the translocation of dna strands through a lipid bilayer. the pore dimension allows the electrophoretically driven passage of single stranded dna whereas double stranded structures need to open prior their translocation. we study the unzipping process of the double stranded part of dna both experimentally and theoretically. we show that in a first approximation the duplex opens progressively in a sequence dependent manner. the experimental results can be accounted for by modeling the unzipping process as a free diffusion of the unzipping fork in the energy landscape defined by the sequence of the basepaires. we then discuss the effect of the pore/dna interaction on the translocation process. we further characterize the effects of the pore geometry, showing that the pre-confinement of the dna in the pore vestibule is essential to the unzipping process. we eventually discuss the possibility to use this nanopore approach to sequentially probe rna secondary structures. we report on the interaction forces in the range -- pn determined between pairs of the lectin soybean agglutinin (sba) and a modified porcine submaxillary mucin (tn-psm) using a single-molecule approach. lectins are carbohydrate-binding proteins with biological activities related to i.e. cellular recognition, adhesion, growth and metastasis. here, dynamic force spectroscopy is used to investigate pairs of sba and tn-psm with the aim to understand the mechanisms of binding and cross-linking of multivalent lectins. the unbinding force increased from pn to pn with increasing force loading rate and the lifetime of the complex in the absence of applied force was . - . s. published kinetic parameters describing the rate of dissociation of other sugar lectin interactions are in the range . x − - . x − s. the long lifetime of the sba -tnpsm complex is compatible with a previously proposed "bind and jump" mechanism. this mechanism has also been suggested for lectins binding to multivalent carbohydrates and globular glycoprotein. the bind and jump mechanism is also similar to that observed for binding of proteins to dna, and suggest a common conserved binding mechanism of ligands to the two biopolymers and possibly between ligands and all biopolymers, as recently suggested. the rate of topoisomerase ii activity correlates with persistence length of dna q. shao , l. finzi , d. dunlap dept. of physics, emory university, atlanta, georgia, u.s.a., dept. of cell biology, emory univ. med. school, atlanta, georgia, u.s.a. type ii topoisomerases catalyze the transection of one double helical segment by another to modify the topological state of dna. reversible ' linkages to the phosphate-sugar backbones are established on each strand of the "gate" segment to create an opening through which the enzyme drives the "transfer" segment. a recent crystal structure shows that the "gate" segment bends approximately • upon binding to the enzyme. bending a stiff polymer like dna requires considerable energy and could represent the rate limiting step in the catalytic (topological) cycle. using modified deoxyribonucleotides in pcr reactions, more rigid dna fragments have been produced and used as substrates for topoisomerase ii-mediated relaxation of plectonemes introduced in single molecules using magnetic tweezers. before relaxation the persistence lengths were measured by fitting force extension data with the worm-like chain model. topoisomerase ii was found to release mechanically introduced supercoils more slowly in stiffer dna suggesting that dna bending might be a rate limiting step in topoisomerase ii activity. h. seidel , t. klose , h. lilie , c. g. hübner institute of physics, ratzeburger allee , lübeck, germany, institute of biochemistry and biotechnology, kurt-mothes-str. , halle, germany one essential element of a virus is its protein shell, the viral capsid, which encloses the viral genome. the murine polyomavirus is a non-enveloped dna tumor virus with an icosahedral t= d structure. besides the knowledge of the structure, it is of utter importance to understand the process of viral assembly [ ] . the assembly reaction of polyoma vp does not show the typical sigmoidal kinetics in light scattering experiments. the apparent kinetics is of fourth order, which appears rather unrealistic. in order to gain knowledge on capsid composition during assembly beyond ensemble average, we apply methods of single molecule fluorescence, namely fluorescence correlation spectroscopy (fcs), fluorescence-intensity-distribution-analysis (fida), and single-particle-imaging (spi). the molecular brightness and the diffusion time reported by fcs are in agreement with the results from light scattering. fida as well as spi, moreover, point to a polymerization of subunits to complete capsids along the assembly pathway without pronounced intermediates. mixing experiments show that already early in the course of the assembly reaction no exchange of pentamers between capsids occurs, and that the effect of breathing [ ] can be excluded for polyoma vp . genetic information coded in dna provides complete instructions to cells regarding metabolism and proper functioning. a number of endogenous and exogenous sources can damage the genomic dna. unrepaired dna damage can give rise to mutations and may cause cell death. cells have evolved different mechanisms to repair damaged dna and to protect genetic integrity. uracil in dna occurs as a result of incorporation of dump in the place of dtmp during replication and deamination of cytocine, resulting in u:a and u:g base pairs, respectively. uracil-dna glycosylase (ung) is a dna repair protein that searches and removes uracil from dna. ung removes mismatched base by flipping it out from the base stack into the active site. the exact mechanism by which ung searches dna for uracil is unknown. therefore, in our study we use atomic force microscopy (afm) to investigate the interaction between single ung molecules with dna carrying the u:a or u:g mismatches. furthermore, we study the complexation of dna and ung protein. afm images of ung bound to dna reveal the structural changes at the level of single complex, e.g. dna kinking that may occur upon binding of ung to dna. imaging of dnaprotein complexes can provide a a new level of understanding of ung-dna interaction. atomic force microscopy (afm) has been a useful device to visualize cellular and molecular structures at a single-molecule resolution. especially, afm imaging under the trec t m (topography and recognition) mode (recognition imaging) can map a specific protein of interest within an afm image. in this study, we employed an antibody-coupled afm cantilever in recognition imaging, and dissected the structural/functional domains of α actinin- , an actin binding protein that cross-bridges actin filaments and anchors it to integrin via tailin-vinculin-α actinin adaptor-interaction. a use of monoclonal anti-α actinin- antibody enabled us to map its epitope in the amino-terminal actin-binding domain within a single molecule afm image of α actinin- . counting fluorescent molecules by photonantibunching h. ta, m. schwering, d. p. herten bioquant, heidelberg university, germany information on the stoichiometry of labelled biomolecules is highly desirable. a method called photo-antibunching has successfully been used to determine the number of fluorescence emitters in multichromophoric systems. a statistical model to estimate the number of fluorescent molecules in a confocal observation volume is established based on photonantibunching in time-correlated single-photon counting (tcspc) scheme with avalanche photon diodes (apds) for detection of individual photons. numerical simulations based on realistic experimental conditions show that the model is able to estimate the number of molecules with moderate errors. experiments on immobilized double-strand dna oligonucleotides with photon stabilizing agents show promising results even when the number of molecules is ∼ . the proposed method allows us to monitor labelled single molecules and in the near future we plan to implement it in complex biological systems (cell extracts/live cells). single molecule afm force spectroscopy and steered molecular dynamics of contactin- protein j. w. strzelecki, k. mikulska, a. balter, w. nowak institute of physics, nicolaus copernicus university, grudziadzka , - torun, poland contactin- (cntn ) is an axonal cell adhesion protein that contains six igc and four fniii domains and is responsible for creating neural outgrowths. recent research shows that mutation of cntn gene may be responsible for autism, while its absence in transgenic mice results in lack of smell sense. we use a homemade afm single molecule puller and steered molecular dynamics simulation to test its elastic properties through force spectroscopy. stretching experiments show unfolding of fniii domains while igc domains stay coiled as they are stabilized by disulfide bonds. unfolding of contactin- molecule appears to play role of buffer that helps to protect neural contacts from damage when neural cells are subjected to shock or tumor. single molecule studies of the thermophilic bacillus ps f -atpase have revealed kinetic and structural information that cannot be discerned using other methods, including the presence of and degree physical substeps (yasuda et al. ) and the order and kinetics of chemical substeps. we are interested in using single molecule techniques to observe the effects of mgi mutations on enzyme kinetics and torque production in f from the yeast saccharomyces cerevisiae. using a high speed imaging camera, we have captured the rotation of wild-type and mutant forms of yeast f -atpase. rotation data for the wild-type and preliminary data for some mgi strains will be presented. we show for the first time that at saturating atp, wild-type yeast f rotates approximately four times faster than the thermophilic f . kinetic and substepping behaviour in yeast appears to be similar to that observed in bacterial f . -single molecule biophysics - biophysical characterization of a dna gquadruplex formed in the promoter of human mef d gene w. zhou , l. ying molecular medicine, national heart and lung institute, imperial college london, london, sw az, uk, chemical biology center, imperial college london, sw az, uk g-quadruplex is believed to be involved in many crucial biological processes, such as the gene regulation and the maintenance of human telomere. mef d, a member of mef (myocyte enhancer factor- ) family of transcription factors, regulates the response of heart to cardiac stress signals. we found that a g-rich sequence starting at - bp of mef d promoter can form a very stable intramolecular g-quadruplex. here we present a detailed biophysical characterization of this quadruplex. we also found that this quadruplex is more stable than its duplex form under physiological conditions by cd melting and single molecular fret. we observed subpopulations in smfret measurements possibly due to different conformations of the quadruplex. we characterized its unfolding process by monitoring the change in fret when it hybridizes to its c-rich complimentary strand. finally, we proposed several possible structures of this quadruplex based on the smfret and fluorescence dms footprinting results. this research is supported by national heart and lung institute foundation. by stretching a polymer in solution using single molecule techniques it is possible to infer about its physical properties. afm stretching experiments allow for a full characterization of the elasto-mechanical properties of the sample under study. in the presented work, single molecule afm force spectroscopy experiments were used to determine mechanical properties of a peptide obtained from exon (ex ) of the human elastin gene. elastin is a protein with important mechanical properties and, in particular, it shows quasi ideal elastic behavior associated to the presence of many hydrophobic unstructured domains (such as ex ) into the protein structure. ex coded polymer was used as a starting point to obtain bio-materials with specialized elastomechanical functions. in particular, a mutated polypeptide based on the ex sequence was synthesisized with the aim of obtaining a new polymer with the same mechanical and physical properties of the native molecule but with increased aggregation properties, induced by a cross-linking reaction. afm stretching experiments were used to verify the mechanical properties of the engineered proteins at a single molecule level. the obtained results allowed not only to address this question, but also to give some insight into the first aggregation steps of the polymer towards the formation of reticulated structures. luminescent lanthanide-ion doped nanoparticles (nps) are attractive single-biomolecule labels. they are synthesized directly in water, are highly photostable, and display narrow emission without intermittency. we coupled y . eu . vo nps to ε-toxins produced by clostridium perfringens, which bind to a specific receptor on mdck cells. single-molecule tracking experiments using these labels produce long ( min) uninterrupted trajectories with temporal resolution down to ms or localization precision down to nm. we found that the toxin receptor exhibits confined motion in cell membrane microdomains. to analyze the receptor trajectories, we used a novel approach based on an inference method [ ] . this method fully exploits the information of the ensemble of the trajectory, in contrast to the usual mean square displacement analysis. applying both techniques to recorded trajectories, we highlight the difference in extracted parameters. from the shape of the confining potential, obtained by mapping the forces inside the domain, we can deduce information about the mechanism of confinement. in combination with experiments on cholesterol depletion and cytoskeleton destruction, this technique will shed light into the nature of the membrane micropatterning. background: implantable cardioverter defibrillators (icds) are save-life device for patients at risk of sudden cardiac death, and help cardiac patients to avoid slow beat-rate by means of anti-bradichardia pacing (abp) feature. however, recent literature evidenced the presence of ventricular tachyarrhythmias (vts) immediately following abp, leading to the hypothesis that icd devices might be pro-arrhythmic. aim: study differences between pacing-associated tachyarrhythmias (pat) and other spontaneous vts. signals and methods: spontaneous vt episodes are retrieved from patients with icd. vts are examined and pat episodes, classified. characterization of vts is based on the analysis of seconds immediately preceding vt-onset quantified by: heart cycle (hc-prevt), prevalence of premature ventricular contraction (pvcprev) and prevalence of paced beats (pprev). significant differences are evaluated by student-t or chi-square tests with p< . . results: vt episodes from patients are pat episodes ( %). cardiac activity preceding pats vs. non-pats differs: pat episodes have higher hc-prevt (p< . ), greater pprev (p< . ), and greater pvcprev (p< . ). analysis also shows that pat episode often occur when the paced beat immediately follows a premature contraction. conclusion: the study indicates that new icd generations should avoid abp after premature ventricular contraction. towards mechanistical understanding of adsorption: combining technologies in situ and in real time p. h. bjöörn q-sense ab, västra frolunda, sweden a growing number of researchers in different surface related fields present evidence from more than one analytical technique when detailing their findings. thus a logic and useful development is to combine technologies for simultaneous measurements on a single sensor surface. to develop a biosensor platform such as an assay fast and accurately, mechanistical understanding of why the platform works is essential. quartz crystal microbalance-with dissipation (qcm-d) technology and instrumentation provides an open platform and enable easy and precise quantification of mass, thickness and viscoelastic properties of surface bound molecules. these parameters provide both a reference tool in assay development, but also detecting and identifying your target molecules as the combination of mass and material property info in many cases provide a unique response for a specific molecule. by combining qcm-d with other technologies, a range of useful info is put within easy reach of the researcher. recent advances will be presented where simultaneous real time and in situ measurements using qcm-d together with electrochemistry, ellipsometry and fluorescence microscopy enables manipulation of films as well as quantification of the variation of water content as a result of conformational changes in immobilized molecules. examples will include new data from the formation of protein films, polyelectrolyte multilayers and polymer brushes. intrinsic gravity versus metabolically inert infrastructure and basal metabolic rate in living mass i. r. bhattacharjee , r. kashyap , b. shaptadvipa assam agricultural university, jorhat- , india, international institute of intrinsic gravitation biology (i gb), jorhat- , india 'self gravitation bio' is an emerging concept in biophysics. intrinsic or 'self' gravitational force might exert when mass increases beyond critical level in biological particle pyramid. (http://en.wikipedia.org/wiki/biomechanics of intrinsic gravity) 'metabolically inert infrastructure' (mii) consists of total body mass (body water, dissolved substances, mineral and organic deposits) and serves as storage of nutrients, transport and distribution of these materials. to act independently as living body, mii is suggested also to provide structural support to the organism with density-gradient buoyant force against intrinsic and extrinsic gravitational attraction for the biological mass. it is shown that 'amniotic fluid', 'isotonic saline to ailing patient', 'growth factors', 'cultural medium' and other 'medium matrices' act as counter-gravity mechanism for micro to macro living organisms under center-of-biomass reference frame. controversy over relationship between bmr (basal metabolic rate), rmr (resting metabolic rate), pal (physical activity level), on one hand, and mass of the living organism, on the other, (as described in rubner's / law, kleiber's / law that continued to be contested by many) can be favorably resolved substituting the concept of self gravitation bio, considering 'mass' as synonym of gravitational force under center-of-biomass reference frame. bioenergetics would be an unequal but opposite entity of self-gravity reinforced by extrinsic gravity. platelet arrest on von willebrand factor (vwf) occurs transiently via the platelet receptor gpib. depending on the combination of shear stress and surface density of vwf binding sites the platelets either only adhere, pull tethers or generate microparticles. these processes are influenced by the properties of the platelet membrane and the cytoskeleton. under shear flow conditions these platelet characteristics were examined with reflection interference contrast microscopy and a viscosimeter. in addition we quantified platelet adhesion energy and tether pulling forces. disrupting platelet f-actin had several effects. the tethers were shorter, the membrane contact area was larger, the receptor αiibβ density of the microparticles was depleted and no platelet spreading occurred. breaking up the microtubular system had different implications. the number of severed tethers tripled, the contact area was smaller, microparticle formation was increased and the area of spread platelets was reduced. however changes in the membrane elasticity had no effect on the platelets. our results suggest that platelet attachment and adhesion is not only determined by the platelet adhesion receptors and their cytoplasmic linker proteins, but that cytoskeletal structures have also a crucial influence on how platelets interact with thrombogenic surfaces. gene expression is orchestrated by a host of regulatory proteins that coordinate the transcription of dna to rna. regulatory proteins function by locating specific binding sequences of dna and binding to these sequences to form the transcription initiation complex. in many instances, these regulatory proteins only have several hundred copies that must efficiently locate target sequences on the genome-length dna strand. the non-specific binding of regulatory proteins to random sequences of dna is believed to permit the protein to slide along the dna in a stochastic manner. periodically, a thermal kick or an interaction with another bound protein will disengage the regulatory protein from the dna surface, leading to three-dimensional diffusion. eventually, the protein will reattach to the dna at some new location that is dictated by both the diffusivity of the protein and the dna configuration. cycling through these random events constitutes a search strategy for the target site. we build a reaction-diffusion theory of this search process in order to predict the optimal strategy for target site localization. the statistical behavior of the dna strand acts as a necessary input into the theory, and we consider several governing behaviors for the dna strand. we explore the impact of dna configuration and protein occlusion on target site localization in order to predict how protein expression will vary under different experimental conditions. a. di garbo , s. alloisio , s. chillemi , m. nobile istituto di biofisica cnr, via g. moruzzi , pisa, italy, istituto di biofisica cnr, via de marini , genova, italy in the nervous system of vertebrates there are more glial cells than neurons: from to times, depending on the animal type. glial cells are not able to generate action potentials but, nevertheless they play an important role for the functioning of the different brain's area. the astrocytes are the more abundant cells of the macroglia and through their processes they modulate synaptic activity. in this contribution a biophysical neural network model consisting of a pyramidal neuron, an interneuron and the astrocyte is studied. the corresponding dynamical properties are mainly investigated by using numerical simulations. the results show that the presence of the atp and of the interneuron strongly impacts the neural activity. moreover, it is shown that the fluxes of calcium through the cellular membrane strongly affect the modulation of the neural activity arising from the astrocyte. microscopic origin of the very-low energy vibrational dynamics in proteins g. d'angelo , v. conti nibali , c. crupi , a. paciaroni , u. wanderlingh department of physics, faculty of science, messina university, italy, department of physics, faculty of science, perugia university, italy important functions of biological processes require large atomic rearrangements and conformational fluctuations. for proteins, atoms are mostly displaced along the soft directions identified by the delocalized, low frequency vibrations. the study of very low energy vibrational spectrum of proteins is therefore of particular interest. as a step toward understanding their functional role, we have investigated the low frequency vibrational motions ( . ÷ mev) in different proteins by performing inelastic neutron scattering and low temperature ( . ÷ k) specific heat measurements. for the first time for biological systems, the well-known boson peak found in neutron scattering spectra at ∼ mev is put in relationship with a clear anomaly of the measured specific heat located at around k. this departure from the debye-like behavior further expands the analogy of proteins with glassy systems. quite interestingly, in the very low sub-mev energy range and below ∼ k, we observe an additional anomalous behaviour, which could be ascribed to the existence of twolevel-systems states. increasing the hydration degree, the low energy vibrational region is drastically altered, revealing that the addition or removal of the hydrogen bond network around the protein deeply modifies the interatomic forces, affecting the character of the vibrational modes. a. cupane , m. levantino , m. cammarata , g. schirò department of physical and astronomical sciences, university of palermo, via archirafi , i palermo, italy, european synchrotron radiation facility, grenoble, france our efforts in recent years have been to study in great detail the way hemoglobin works in confined geometries [ ] [ ] [ ] . to this aim we have contributed to the development of a new experimental technique, time-resolved wide-angle x-ray scattering (tr-waxs), that enables one to watch proteins at work in solution [ , ] . a very recent and challenging application of this technique is the study of hemoglobin functioning inside intact red blood cells (rbc). our preliminary results show that, by using laser pulses of about ns, it is possible to photolyse hemoglobin inside rbcs obtaining about % - % photolysis. good structural signals from hemoglobin are obtained, with limited radiation damage to the cells: this opens the possibility of studying the conformational transitions of hemoglobin in its "natural" environment. preliminary results concerning the timing of the r->t quaternary transition inside the rbc and comparison with the behaviour in dilute solution will be discussed. physical review letters and physical review e invite submission of your best work in biological physics. submissions must present significant new results in physics; topics may range from the microscopic to the macroscopic. we will provide information on the different types of articles published in the journals, on authors and referees, and on the review process. for publication in physical review letters, the work should be important and of broad interest. for rapid communications in physical review e, the work should be important for the field. biological physics papers published in physical review are indexed in medline. in an effort to bring important research to the attention of a wider community, the physical review journals have recently begun highlighting important work with viewpoints in the online publication physics. all physical review journals may be accessed through the website http://publish.aps.org/. modeling of fibrin gels using confocal microscopy, light scattering and turbidimetry f. ferri , d. magatti , b. cardinali , a. profumo , m. rocco dipartimento di fisica e matematica, università dell'insubria, como, italy, istituto nazionale per la ricerca sul cancro (ist), genova, italy fibrin gels are biological networks playing a fundamental role in blood coagulation. confocal microscopy of fibrin gels shows a collection of straight fibers, not uniformly distributed in space, connected together at low-order ( ) ( ) branching points. although each fiber is quite straight (mass fractal dimension d m = ), for the overall system d m > . based on the confocal images, we generated threedimensional ( d) synthetic gels made of cylindrical sticks of diameter d, joined together at randomly distributed nodal points. the resulting d network is no more random but ordered on length scales of the order the average fiber length, and exhibits a fractal morphology with d m ∼ . - . . the gel structure is analyzed by means of its d correlation function g d (r)=<n(x) n(x+r)> x , where n(x) is the gel local density. since the gel is isotropic, g d depends on the modulus r=|r| and can be obtained by averaging d correlation functions evaluated at different heights of the gel volume. from this analysis we recover the fiber diameter d (fwhm of g d ), the fractal dimension d m (power-law decay of g d ) and the gel mesh-size ξ (minimum in g d ). furthermore, the d-fourier transform of g d (r) gives the gel power spectrum i(q), which compares quite well with elastic light and multi-wavelength turbidimetry data taken on real gels. a model coupling vibrational and rotational motion for the dna molecule e. drigo filho, j. r. ruggiero, r. a. d. s. silva unesp -sao paulo state university, brazil a largely used mechanical model for vibrational motion of dna has been proposed by peyrard and bishop (pb). in this model, dna is represented by a pair of harmonic chains coupled by a nonlinear potential. the most frequently used nonlinear potential for this purpose is the morse potential. some extensions of the original model are proposed considering, for example, the helicoidal structure of dna. an important objective for this kind of model is to understand the phenomenon of thermal denaturation and, through this, get some knowledge about other processes such as the genetic transcription and drug intercalation. it is possible to obtain from this type of model interesting properties, such as the average stretching between base pairs as a function of the temperature using the transfer integral operator. dynamical properties of this model were also explored and several phenomenological quantities are studied, such as energy localization. in this work, we extend the original pb model by introducing rotational motions for the nucleotides. in this way, both the vibrational and rotational motion for each nucleotide are considered. the stretch of the base pairs is given by the morse potential in the same way as in the original pb model. however, the coupling between the two kinds of motion, rotation and vibration, is obtained through a nonlinear combination of them in the morse potential. a. dobovišek , m. brumen , p.Županović , d. juretić university of maribor, faculty of natural sciences and mathematics, maribor, slovenia, jožef stefan institute, ljubljana, slovenia, faculty of science, university of split, split, croatia mepp is a physical principle, widely used for quantitative explanation of non equilibrium phenomena in physics, chemistry and biology [ ] [ ] [ ] . here, we applied mepp to study two and three state reversible michaelis-menten kinetic schemes of enzymatic reactions. by applying constraints as a diffusional limit for kinetic constants, constant free energy differences between enzymatic states and constant thermodynamic force, we calculated shannon information entropy and entropy production of the entire reaction system as a function of forward rate constants. we found maxima in the net steady-state metabolic flux, total entropy production and shannon entropy for equal values of forward rate constants. moreover, for these values an analytical expression derived gives a relation between substrate and product concentrations at which enzymes operate in the optimal way. in conclusion, we demonstrated that mepp is an appropriate selection principle for evolutionary optimization of enzymes. attractive interaction between like-charged lipid surfaces mediated by spherical nanoparticles with spatially distributed charge is theoretically described by using functional density theory and mc simulations. the spatial distribution of charge within a single nanoparticle is considered by two effective charges at a finite distance. the minimization of the free energy is performed to obtain the equilibrium configuration of the system. both, the rigorous solution of the variational problem and the mc simulations show that orientational ordering of nanoparticles subject to the gradient of the electric field gives rise to an attractive interaction between charged lipid surfaces for high enough charge densities of the interacting surfaces and large enough separations between charges within the nanoparticle. the attraction is explained by orientational ordering of dimeric charges in the electric field which lowers free energy. viral genomes encode for a series of membrane proteins which are embedded or attached to the lipid membrane of the host, or penetrate them during the very first step of viral invasion. we are focussing especially to those of the former which are known to assemble and from channels or pores for small ions or substrates. with these channel forming proteins the virus manipulates the host cell interior for the benefit of its replication. strategies are developed to answer the question about the assembly process of these proteins based on the 'two stage model' and the substrate flux. only few experimental data are available to answer these questions, consequently we stress computational methods to derive the adequate answers. the methods applied are ab inito molecular dynamics (md) simulations based on density functional theory (dft) and conventional md simulations. potential routes for assembly of the three transmembrane domains of a protein from sars-cov will be outlined and compared with computational data from other viral membrane proteins. with a novel pore lining motif suggested for a, the dynamics of ions at selected positions within the putative pore will be assessed. probing the molecular mechanism of antibiotics diffusion through the ompf channel e. hajjar , a. kumar , m. winterhalter , p. ruggerone , m. ceccarelli department of physics, university of cagliari, italy, jacobs university, bremen, germany in gram-negative bacteria, the outer membrane porin-f (ompf) is the preferred entry point of antibiotics. bacteria can resist to antibiotics by altering the expression and the structures of ompf. a key feature in the structure of ompf is the presence of a constriction zone, characterized by both spatial and electrostatics restrictions. to study the process of antibiotics translocation at a molecular scale, we performed molecular dynamic simulations accelerated with the metadynamics algorithm. we studied the diffusion of antibiotics with different structural and chemical properties through ompf wild-type and variants. the free energy surface suggests faster translocation for the cephalosporins compared to the penicillins antibiotics, and also for ompf mutants compared to the wild type. further, the conservation of favored orientation and affinity sites of antibiotics inside the ompf channel reveal which specific interactions govern translocation. the calculated energy barriers and rate determining interactions for translocations compared well with the electrophysiology measurements and liposome swelling assays from our collaborators. this study demonstrates how theory and experiments can be combined to reveal the structural determinants and mechanism of ompf permeation. this will benefit to the design of antibiotics with improved transport properties. m. g. gauthier, j. bechhoefer dept. of physics, simon fraser univ., burnaby, bc, canada dna replication requires two distinct processes: the initiation of pre-licensed replication origins and the propagation of replication forks away from the fired origins. experiments indicate that these origins are triggered over the whole genome at a rate i(t) (the number of initiations per unreplicated length per time) that increases throughout most of the synthesis (s) phase, before rapidly decreasing to zero at the end of the replication process. we propose a simple model for the control of dna replication in which the rate of initiation of replication origins is controlled by the interaction with a population of ratelimiting proteins. we find the time set by reaction-diffusion kinetics for such proteins to find, bind to, and trigger a potential origin. the replication itself is modeled using a formalism resembling that used to study the kinetics of first-order phase transitions. analyzing data from xenopus frog embryos, we find that the initiation rate is reaction limited until nearly the end of replication, when it becomes diffusion limited. initiation of origins and hence i(t) is suppressed when the diffusionlimited search time dominates. we find that, in order to fit the experimental data, the interaction between dna and the rate-limiting protein must be subdiffusive. we also find that using a constant nuclear import of the limiting proteins leads to a more accurate description of the experimental data. the microsoft research -university of trento centre for computational and sisytems biology, trento, italy we propose a new method for inferring the structure of a biochemical network from the time-series of the reactant species. it consists of two parts: the first is the quantification of the correlation between the time-series profiles. correlation in time series data can be used to reveal dependencies between variables and to infer the graph of connectivity among species. the second part consists in the elimination from the connectivity graph of the relationships that have a non-null correlation coefficient, but that are not biochemically plausible. the cutting of false correlations from the graph is performed through the estimation of the parameters ("calibration") of the network. to calibrate the network for detecting null dynamics correlations, we developed the software tool kinfer (knowledge inference). based on a new probabilistic model of the variations in reaction concentrations, kinfer infers the values of the kinetic rate constants, their confidence regions and the level of noise in the input data by maximizing the likelihood to obtain the observed variations given the network model. the a priori knowledge required as input is minimal, as it consists only in the time-series of reactant concentrations. the minimal a priori knowledge and the probabilistic formulation of the calibration method make the accuracy of the predictions strong against experimental, biological and stochastic noise, and allows to use it to cut the null-dynamics edges of the connectivity graph. a. kuzmanic, b. zagrovic mediterranean institute for life sciences, split, croatia root-mean-square deviation (rmsd) is a measure used to give information on the global structure of macromolecules. for example, pairwise rmsd (prmsd) is used to assess similarity of the lowest energy nmr structures or for clustering large ensembles of structures. on the other hand, to obtain information on the local structure of a macromolecule and its dynamics, root-mean-square fluctuation (rmsf) is often used. rmsf can be calculated from md simulations, but also from experimental x-ray b-factors. since prmsd and rmsf report on different features, it is interesting to ask what the relationship between them is. first, we provide a mathematical derivation showing that, given a set of conservative assumptions, the <prmsd> rms is directly related to the <rmsf> rms and, consequently, experimental b-factors. second, we demonstrate this on structures taken from distributed-computing md simulations of the native and unfolded state of villin headpiece. both our analytical and computational results suggest a strong correlation: <rmsf> rms = [(s- )/ s] / <prmsd> rms , where s is the number of compared structures. furthermore, if <prmsd> rms is defined as a generalized radius of gyration in the space of d structures and using rmsd as a measure of distance, the following identity holds: <rmsf> rms = <prmsd> rms. our results provide a basis for determining the level of structural diversity of molecular ensembles, as captured by <prmsd> rms , directly from experiment. charge migration along dna helices may be biologically important because extended electronic states could play a role in the processes of sensing of dna damage and/or dna repair via long-range charge transfer. we measured conductivity and other physical characteristics of several models of natural and diversely damaged molecules of dna. dna polymers were mimicked by various sequences of nucleotide-long double helices with fully watson-crick (wc) paired bases, with several central bases mismatched, and also with chemical modifications that included removal of bases from a few central nucleotides (abasic dna), and neutralization of phosphate charges by their derivatization. the model dnas were investigated by scanning tunneling microscopy, time-resolved thz spectroscopy, raman spectroscopy, circular dichroism, and modeled by molecular dynamics simulations. dna has the highest conductivity in its biologically most relevant double helical form with wc base pairs and negatively charged phosphates equilibrated with counterions. mismatches and all chemical modifications always lower the conductivity. the mechanism of charge transfer is consistent with electron or hole hopping between parallel stacked bases. these observations and data showing that the natural dna has also the most regular double helical form suggest that the continuous base stacking is critical for charge transfer. to control the passage across the bacterial cell wall nature created a large number of "nano"-channels which may act as selective gates for water soluble molecules. here we focus on porins from e. coli which control permeation through interactions with the channel surface. comparing single channel temperature dependent conductance measurements with all atom modeling allow conclusions on the mode of permeation. for example, surprisingly modeling ompf-conductance revealed not only a good agreement with the experiment over a broad range of temperature but also the selectivity for ions. the primary task of porins is to provide facilitated diffusion. we investigated facilitated diffusion of maltooligosaccharide or antibiotics. time resolved conductance measurements allows conclusion on the flux and molecular modeling identifies the limiting interactions with the surface, reveal potential barriers and pathways. exploiting the selectivity of natural or bioengineered channels has promising applications for detecting molecules, characterizing molecular interaction, sequencing dna, protein folding etc. traditionally, studies of diffusion-controlled reaction of biological macromolecules have been made in diluted solutions. however, the high concentration of macromolecules in intracellular environments results into non-specific interactions (macromolecular crowding), which have a great importance on the kinetics and thermodynamics of possible reactions that occur in these systems. in the literature there are studies concerning monte carlo (mc) simulations, giving results that are satisfactory agreement with experimental data, showing, for example, that the protein diffusion in cell cytoplasm is reduced considerably. in addition, there are mc studies about enzymatic reaction, which predict a temporal dependency of the velocity constant in macromolecular crowding. in this work, we try to compare the predicted behavior by mc simulation with the results obtained from the study of the diffusion and reaction of a model protein (alpha-chymotrypsin) using spectroscopic techniques in highly confined media in order to study experimentally the temporal dependence of its diffusion and reaction coefficients. a. paciaroni , a. orecchini , c. petrillo , a. de francesco , f. sacchetti university of perugia, italy, cnr-infm, genova, italy the single-particle and collective dynamical properties of protein hydration water have been studied by neutron scattering experiments in a wide temperature and hydration range. an unprecedented accuracy has been achieved thanks to the availability of a large amount of fully deuterated protein powder and the use of the high-flux spectrometers in and brisp. the protein under investigation was the maltose binding protein (mbp), which is a well-known and widely studied model of biosensor systems. we found that the low-temperature single particle dynamics of mbp hydration water shows clear features that can be traced back to amorphous systems. more in detail, its vibrational density of states is simply described as the superposition of the contributions of low-density and high-density amorphous ice. the quasielastic signal, which appears at the higher temperatures, can be excellently described with a fractional power law which may put in relationship with the peculiarities of fractal systems. quite strikingly, there is a strong similarity, on both the qualitative and quantitative point of view, with the behaviour of hydrated proteins. the collective dynamics of protein hydration water is characterised by the presence of two modes, whose dispersion curves are reminiscent of those of bulk water. however, the relevant damping factors suggest a strong similarity with glassy systems. m. malferrari , f. francia , s. sacquin-mora , g. venturoli università di bologna, bologna, italy, cnrs upr , paris, france the coupling between electron transfer and protein dynamics has been compared in reaction centers (rc) from the wild type (wt) and the carotenoid-less mutant r , by combining brownian dynamics simulations and the kinetic analysis of charge recombination. upon incorporation of the rc into a progressively dehydrated trehalose matrix the electron transfer between the primary photoreduced quinone and the photoxidized donor accelerates progressively and becomes broadly distributed. this behaviour reflects the hindrance of protein relaxation following charge separation and the inhibition of interconversion between conformational substates. in extensively dehydrated matrices the recombination kinetics is two-times faster and three-times more distributed in the wt rc, indicating a larger inhibition of the internal protein dynamics. in line with this findings brownian dynamics simulations reveal a larger rigidity of the carotenoid-containing structure, in which a cluster of residues close to the quinone acceptors is stiffened as compared to the r rc. the in silico and experimental results indicate that the introduction of an internal void in the rc structure has long-range effects on the protein dynamics and that the coupling between the glassy matrix and the rc interior depends markedly on the local mechanical properties of the protein. n. maghelli , v. krstic , n. pavin , f. julicher , i. tolic-norrelykke mpi-cbg, dresden, germany, mpi-pks, dresden, germany in the fission yeast schizosaccharomyces pombe, the nucleus is positioned at the cell center. since the nucleus determines the cell division site, keeping the nucleus at the center is crucial for ensuring symmetrical cell division ( ) . microtubules push against the cell ends and exert force on the nucleus ( ), but how the cell regulates these forces in order to center the nucleus remains unknown. here we tackle this problem by using a combination of live cell imaging, cell manipulations by optical tweezers, and a theoretical model. we show that microtubule pushing forces can center the nucleus because of a larger number of contacts between the microtubules and the proximal cell end than the distal one. moreover, kinesin- motors (klp / ) increase the rate of microtubule catastrophe (transition from growth to shrinkage) in a microtubule length-and contact-dependent manner. thus, the motor behavior results in a longer contact between a microtubule and the proximal than the distal cell end. taken together, our experimental and theoretical results provide a novel centering mechanism, where kinesin- motors increase the efficiency of nuclear centering. electropermeabilization is a commonly used physical method which can induce a transient permeabilization of the cell membrane allowing the entry of therapeutic molecules into the cell and is thus of great interest in the fields of cancer treatment and gene therapy [ ] . however, very little is known about the mechanisms occurring at molecular level. there is clearly some microscopic reorganization of the membrane which is responsible for this change in its transverse transport properties. rather than studying the change of these transport properties, we adopt a simple strategy based on the use of giant unilamellar vesicles and videomicroscopy, as described below. we apply a series of permeabilizing electric pulses to the liposomes, and we observe a size decrease down to a critical radius beyond which their size no longer changes. this decrease in size points to the fact that during the physical processes leading to electropermeabilization, lipids are lost from the vesicles. our results published in [ ] suggest different possible modes for lipid loss, which can be small vesicles, pores, or tubules formation. imaging of brain activation using core techniques as fmri, pet and synchrotron radiation in parallel c. poitry-yamate, g. margaritondo, r. gruetter ecole polytechnique fédérale de lausanne, switzerland functional magnetic resonance imaging (fmri), magnetic resonance spectroscopy (mrs), positron emission tomography (pet) and synchrotron x-ray emission imaging form a highly complementary set of imaging core technologies for studying brain function and energy metabolism. owing to differences in the information each conveys and the temporal and spatial scales on which they measure labeled substances, a single working hypothesis can be tested from different angles to provide a cross-validated, consistent and coherent explanation. the cibm is a unique research facility in europe for advancing our understanding of biomedical processes in health and disease. the housing of a -tesla human magnet, . and . -tesla animal magnets, an animal pet imaging facility and fully-equipped neurochemistry and rf laboratories has enabled us to develop and perform well-targeted experiments around one research theme. in parallel, a longterm collaborative project using synchrotron x-ray transmission and emission imaging at elettra enables us to combine these core technologies towards understanding brain function in vitro and in vivo, from tissue to cells. a brief presentation of these methods will be followed by their application in studying the visual system from man to mouse. p. picone , r. carrotta , d. giacomazza , m. di carlo dip. chimica e tecnologie farmaceutiche, università di palermo, italia, ibf -cnr, palermo, italia, ibim -cnr, palermo, italia diabetes and alzheimer's disease are connected in a way that still is not completely known. diabetes has been implicated as a risk factor for developing alzheimer's disease. some diabetes drugs appear to decrease the cognitive decline associated with alzheimer's disease. it has been recently demonstrated that extracellular injection of insulin is able to protect neurons against a-beta amyloid cell death. one of the proposed theories to explain such an effect is that the hematic glucose levels affect the metabolism of the hippocampus, a part of the brain (associated with memory, emotion and motor skills), which is strongly damaged in alzheimer's patients. the aim of this study is to investigate the effect of insulin on the a-beta induced degeneration and oxidative stress on the neuroblastoma lan cell line. in particular, the present study looks into the role of insulin in the inhibition of abeta specific degenerative apoptotic pathways. preliminary results indicate that insulin dissolved in culture medium in its hexameric form (as tested by absolute scale light scattering) is able to reduce neurodegeneration induced by a-beta amyloid in a dose dependent manner. the link between diabetes and alzheimer's disease may provide new targets for future alzheimer's treatments. moreover, due to the increased incidence of diabetes in western countries, a deeper understanding of such a link is relevant in order to control the escalation in the number of people dealing with dementia. a. pelizzola dipartimento di fisica, politecnico di torino, torino, italy many features of protein folding have been shown to be described by an ising-like model (one-dimensional, with longrange, multispin interactions) whose equilibrium thermodynamics is exactly solvable [ ] [ ] [ ] . we have generalized such a model to the problem of mechanical unfolding. the equilibrium thermodynamics is still exactly solvable, and the characteristic kinetic responses found in force ramp and force clamp experiments are well reproduced [ , ] . unfolding and refolding pathways and intermediates can also be studied, again with good agreement with experiments [ ]. applications to various proteins and rna fragments will be discussed. [ the key role of water in living systems has been widely studied in literature, along with its anomalies, consequence of the extensive three-dimensional hydrogen bonding of water molecules. moreover protein-water interactions take place at protein surface where cell water has been recognized to behave differently from bulky water. the two-states theory of water assumes that water is a mixture of microdomains of different structure and density, the low-density water (ldw) and the high-density water (hdw) domains, and ions partition selectively into ldw or hdw domains. the idea developed in this work was to explore the ordered water structure by measuring delayed luminescence (dl) from salt aqueous solutions in which water structuring is anticipated. it appeared that dl signal from salt solutions is significantly relevant when prevalence of ldw domains is foreseen, with a decay time probability distribution function characterized by a broad maximum in the microsecond range. the obtained results support the ability of dl to reveal the different properties of ldw and hdw domains induced by salt molecules. moreover, the results reveal the existence of clusters, whose characteristics strongly depend on the specific ion effects, of surprisingly long lifetimes not observed till now. this could give new insight into biological water properties. self assembly of patchy particles and dnafunctionalized dendrimers f. sciortino dipartimento di fisica e infm-cnr-soft università 'la sapienza', roma, italy i will report numerical results on the phase behavior of very simple models of patchy particles with the aim of understanding the interplay between phase separation and selfassembly and how the fraction of surface allowing for attractive interactions controls the collective behavior of the system. the case of janus particles, particles characterized by a surface divided evenly into two areas of different chemical composition, will be discussed. i will also discuss the self-assembly of a simple model for four single strands of dna tethered to a central core, and show that the model exhibits a rich phase diagram that includes at least four thermodynamically distinct amorphous phases (polyamorphism) in a one-component system. the dense phases consist of a hierarchy of interpenetrating networks, reminiscent of a woven cloth. peptide dimer motifs in the phospholipid environment -structure, interaction and molecular design p. e. schneggenburger , a. beerlink , t. salditt , u. diederichsen iobc, universität göttingen, germany., irp, universität göttingen, germany. based on recently reported homodimeric peptide pores with a d,l-alternating configuration a novel double helical hairpin-motif of a membrane active gramicidin a analog was designed. [ , ] the cd spectroscopic analyses of the peptide-lipid complexes revealed the structural preservation and elucidated the importance of a zwitterionic interaction of the peptide termini. [ ] the peptide design was enhanced regarding the versatile functionalization with analytical probes as well as molecular recognition moieties like peptide nucleic acids (pnas) to observe the effects of aggregation and specific organization within model lipid membranes even at high peptide-to-lipid ratios. [ ] x-ray reflectivity on lipid bilayer stacks in combination with heavy atom labeling and spectroscopic studies of vesicle systems provides information about the peptide structure and interaction in the native fluid state of the membrane system. [ , ] for this, the fmoc-diiodo-allylglycine building block was created to serve as a novel iodine label pinpointing at a certain position with respect to the membrane normal. we study thermal undulations of giant unilamellar vesicles (guvs) of lipids by flickering spectroscopy. getting values for the mechanical parameters of lipid bilayers requires the experimental fluctuation spectra to be scrutinized in view of the classical helfrich's theory. pure bending modes are revealed unable in predicting the large fluctuations systematically found at high wavevectors. hybrid curvature-dilational modes have been invoked as a more efficient mode of motion in producing high curvatures. a bimodal spectrum of the thermal undulations has been theoretically developed for the shell-like topology. from this new description, two important consequences emerge a priori, the dependence of the fluctuation dynamics on either vesicle size and on bending/compression parameters. for popc and dopc vesicles containing cholesterol the experimental fluctuation spectra are well described by the new spectrum. reconciliation between experiments and theory is achieved when this bimodal spectrum is considered. the new theory opens enormous possibilities for better exploring membrane mechanics in guv models. under normal conditions, platelets circulate in the vascular system having very low interaction with each other and with other cells. the platelets become activated when the biological system is disturbed, for instance by vascular damage in which blood gets in contact with collagen. upon activation, different types of receptors/molecules are exposed on the cell membrane to support adhesion, spreading and aggregation of the platelets onto the damaged vessel. the measurement of altered platelet function is particularly important in cardiovascular diseases such as thrombosis. we are investigating biosensor technologies for the detection of functional properties of platelets. an important study is the specific and non-specific stimulation of platelets in a biosensor cartridge. we will present experimental results on biosensor platelet activation using the platelet-specific membrane markers p-selectin and gp b. multi-joint analysis of locomotion in the first neonatal rats flown in space d. sulica, j. vinersan "carol davila" university of medicine and pharmacy, bucharest, romania the first mammalian neonatal animals in space were the rats flown on the space shuttle endeavor during a -day mission, sts- . the development of locomotion in weightlessness was evaluated using two litters of neonatal rats, launched at postnatal days and . age-and cage-matched animals were used as ground controls. free walking was videotaped from the landing day. although preliminary analysis of walking showed differences in both hindlimb and forelimb joint angles and a hyperextension of the hindlimbs was apparent, the numerical values reached the significance level only for the ankle angle measured at specific moments of the step cycle: foot contact, maximum loading with weight, foot lift and maximum flexion during swing. we report here on the behavior of all the joints during the whole step cycle, by computing the integral of these angles over the step cycle. the results were affected by the differences in the walking speed (the young animals walked faster than the controls), so we scaled the integrals by the step cycle duration. we found that, besides the ankle, the knee was also more extended throughout the whole step cycle in both groups of animals. moreover, all the joints (including the toe and the hip) were affected in the same way (hyperextended), since the differences were still significant when we added together these angles. the animals recovered slowly, with significant differences remaining after days of readaptation. the effect of dextran concentration on red blood cell deposit formation j. strzelecka, b. grzegorzewski department of biophysics, collegium medicum in bydgoszcz, nicolaus copernicus university - bydgoszcz, poland red blood cell (rbc) deposit formation was examined by means of an optical method. blood was obtained from healthy donors and measurements were performed at initial hematocrit %. the intensity of scattered light was measured during sedimentation of rbcs suspended in saline -dextran solutions at different polymer concentrations ( - g/dl). the changes in the intensity of the scattered light manifest rbc aggregate formation, their sedimentation and the process of deposit formation. the deposit formation curve was determined. it is shown that the concentration of dextran affects the deposit formation. an empirical model has been used to describe the experimental data. the parameters of the deposit formation curve as a function of dextran concentration are analyzed. water is essential to life and a major scientific interest lies in a detailed understanding of how it interacts with biological macromolecules in cells. we studied water dynamics in whole cells with neutron scattering [ , , ] . the cellular environment is extremely crowded with macromolecules and water molecules are permanently in close contact to biological interfaces. we measured water dynamics in e. coli and human red blood cells with neutron scattering [ , ] . the data revealed two populations of water in the cells: a major fraction which has dynamical properties similar to those of bulk water (relaxation times ∼ps) and a minor fraction in the order of ∼ % which is interpreted as bound hydration water with significantly slower dynamics (relaxation times > ps). in this contribution we report on investigation of model membrane dynamics by means of quasi elastic and inelastic incoherent neutron scattering and on the effect of membrane inserted pore forming peptide gramicidin. model membrane are realized by highly oriented, hydrated phospholipid bilayer stacks of dmpc ( , -dimyristoyl-snglycero- -phoshatidylcholine) hydrated with d o in excess of solvent condition. the bilayer were supported on mica substrates and prepared at different concentrations of gramicidin, a -residue oligopeptide showing antimicrobial activity by forming pores on the membrane surfaces which allow water and small ions to permeate across the membrane. incoherent qens and ins spectra, measured on in and in spectrometer at ill, allows to obtaining information on the mean dynamics of the hydrogen atoms in the system. moreover, by proper orientations of the membrane plane respect to the scattering wave vector q, we were able to derive information on in plane and out of plane motions of the phospholipids. the using of media products for the creating attracted bioenergetic brain rhythmus advertisement v. i. vlastopulo, v. g. nikolajev str. gen. petrova , app. , odessa, ukraine the technical efficiency of bioenergetical influence of advertisement is present with assistance of consciousness of memory at revision and hearing of advertisement on tv, radio stations, mobile phones, at supermarkets and other places. in a basis of useful model it is put a task to improve the method of creating the attracted advertisement, in which the creation of bioenergetical influence by the oscillation of not less electromagnetic fields or video-images is introduced with their creating as the base on the spatial or flat structure formative macro matrix or matrixes with repeatable structure with the brain α-rhythm frequency of extreme attention and δ-rhythm frequency of meditation. the point of the patent on the device is in the bioenergetical influence increases in addition to the information influence by advertisement of pictures and audio oscillations the bioenergetical influence increases at the consciousness contribution of human memory at the moment of watching or hearing of television, radio stations, working in the internet, music in the supermarkets, banks, clubs, metro and other places. cell adhesion and motility are processes involved in fundamental biological phenomena. they imply multimolecular scaffolds as anchorage points and actin cytoskeleton filaments to build internal stress and eventually crawl onto the substrate. these processes, very dynamic by nature are out of equilibrium. we study cell adhesion on micro-patterned substrates where the introduction of a finite distance between the possible anchorage points of the cell modifies drastically the organization of the cytoskeleton and the anchorage point's distribution. because statistical quantification shows that some shapes are more likely than other, we believe they represent particular organizations of the system which should minimize the energy dissipation. we checked this hypothesis by using the cellular potts model. shapes obtained by simulation are in excellent qualitative agreement with experimental shapes. they depend on phenomenological parameters such as interaction between cells and the extra cellular matrix, a line tension and an elastic modulus. the aim of this work is to link model parameters to physico-chemical properties of cells and to establish phenomenological relations between relevant biochemical regulators controlling the cytoskeleton organization. c. canale, d. ferrera, f. benfenati, l. gasparini the italian institute of technology, genova, italy alzheimer disease (ad) is characterized by cerebral extracellular deposits of β-amyloid (aβ) fibrils. aβ aggregation is a multi-step process involving the formation of various conformational species including soluble intermediate species (i.e. aβ oligomers), protofibrils and fibrils. such aggregates may have various effects on neuronal and glial function and differentially contribute to ad neurodegeneration. aim of this study was to investigate the structural properties of distinct aβ aggregated species and dissect out their effects on neuronal viability. recombinant aβ and aβ peptides were aggregated in vitro in conditions differing by ionic strength, temperature and ph and were analyzed by gel electrophoresis, thioflavin t binding assay and atomic force microscopy (afm). afm analysis was performed using both hydrophilic and hydrophobic substrates, to analyze the full spectrum of structural species. stable low molecular weight oligomers were obtained when aβ was incubated at • c for days in low salt concentration buffer. doughnut-shaped conformational species were detected by afm alongside globular aggregates ( . - . nm height range). acidic ph promoted aggregation of aβ into thioflavin-positive fibrils and protofibrils. protofibrils appeared as beaded chains having a mean height of . ± . nm. effects of aβ on viability of mouse hypppocampal neurons were assessed and correlated with their conformational features. a. bellova , e. bystrenova , m. koneracka , p. kopcansky , f. valle , j. bagelova , f. biscarini , z. gazova institute of experimental physics, slovak academy of sciences, kosice, slovakia, ismn cnr, bologna, italy peptide amyloid aggregation is a hallmark of amyloid diseases including azheimer's disease or type ii diabetes. recent works have addressed the potential of nanoparticles to affect amyloid aggregation. the experimental data are very controversial suggesting that particle characteristics markedly influence the final effect of nanoparticles on the amyloid aggregation (initiation, acceleration or inhibition of amyloid aggregation). we investigate the ability of electrostatically stabilized magnetic nanoparticles of fe o to affect the amyloid aggregation of lysozyme, as a prototype amyloidogenic protein. we have used a combination of spectroscopic (tht fluorescence) and local microscopic techniques (afm). we found, that the ability of magnetic nanoparticles to inhibit formation of amyloid aggregates or destroy pre-formed amyloids exhibit concentrationdependence. the values of inhibition ic and depolymerization dc were determined suggesting that nanoparticles interfere with lysozyme aggregation at stoichiometric concentrations. the observed features make magnetic nanoparticles of potential interest as a therapeutical agent against amyloid diseases. (this work was supported by project of esf and by slovak academy of sciences in frame of cex nanofluid, vega grants , and and eu-strp biodot.). a. bellova , l. balogova , b. chelli , e. bystrenova , f. valle , j. imrich , p. kristian , l. drajna , j. bagelova , f. biscarini , z. gazova institute of experimental physics, sas, kosice, slovakia, faculty of sciences, p. j. safarik university, kosice, slovakia, ismn cnr, bologna, italy numerous diseases have been linked to a common pathogenic process called amyloidosis, whereby proteins or peptide clump together to form amyloid aggregates in the body. an attractive strategy to develop therapies for these diseases seems to be reduction of polypeptide aggregation. we have tested several acridine derivatives characterized by various glycosyl groups for their potential to affect the lysozyme amyloid aggregation in vitro. the ability of glycosyl acridines to interfere with lysozyme aggregation was investigated by tht assay. we found that structure of acridine side chain is factor affecting their anti-aggregation activity significantly. for the most effective compounds the values of ic and dc were obtained. the reduction of protein aggregation was confirmed by afm. to investigate influence of the glycosyl acridines on the cell processes we examined effect of compounds on cell viability. we performed glycosyl acridines characterization by high anti-aggregation activity and low toxicity suggesting their possible application for therapeutical purpose. (this work was supported by project of esf and by slovak academy of sciences in frame of cex nanofluid and vega grants , and and eu-strp biodot.). a. j. beevers, a. m. dixon department of chemistry, university of warwick, uk due to the immense medical importance of proteins which span the membrane of cells, detailed molecular structural information of these systems is essential. practical difficulties in employing high-resolution structural elucidation techniques have resulted in a relative paucity of fully resolved membrane protein structures. therefore a variety of lower-resolution techniques are used to determine structural information of the transmembrane (tm) domains of proteins. one example of such a membrane protein is erbb- , a receptor tyrosine kinase responsible for triggering cell division and which is prone to a mutation in its transmembrane domain resulting in permanent activation and oncogenic effects. we have predicted an interface for the mutated tm domain dimer using site-specific infrared spectroscopy containing a repeating sequence of ile, val and leu . applying the in vivo toxcat assay to the tm domain sequence and to specific mutants of it, confirms this proposed interface whilst another proposed interface is discounted. current studies are focussing on the effect of the tm mutation to the activation of the erbb- receptor and to any possible change in this interface. bacteriophages are complex molecular assemblies which multiplication relies on bacteria infection. the process starts with the binding of the phage on its specific host receptor and the injection of its genome into the host cytoplasm. our work aims to determine the physical mechanisms and forces driving the dna transfer from the phage capsid. the in vitro dna ejection has been analyzed by using light scattering and gel electrophoresis measurements for three phages (t , spp and lambda) belonging to the same family (syphoviridae). our results reveal two forces contributing to drive the dna transfer: the first one is originated from the pressurization due to the strong confinement of dna into the capsid; the second one comes from a pulling mechanism originated by the presence of condensed dna outside the capsid. these two contributions were characterized in in vitro conditions but they likely play a role in the in vivo transfer. the ejection kinetics was also analysed and the characteristic time of the mechanism was studied as a function of the temperature. it appears to follow an arrhenius law, allowing the determination of the activation energy that governs the transfer. the energy values are close for the different phages, suggesting that the mechanism regulating the ejection is common for a given phage family. below these general features, our studies also reveal differences between the three phages. the effect of &beta-amyloid peptide on polymer cushioned membranes s. dante , r. steitz , t. hauss , c. canale , n. a. dencher istituto italiano di tecnologia, genova, italy, bensc, helmholtz center berlin, germany, tu-darmstadt germany beta-amyloid (aβ) is a peptide implicated in the neurodegenerative process characteristic of the alzheimer's disease (ad). to clarify its mechanism of action it is crucial to elucidate the interaction of aβ with the neural membrane. in previous work we demonstrated the capability of aβ to penetrate and perturb stacked lipid bilayers. in this study we considered polymer cushioned lipid bilayers as a model for neural membranes. the polymer cushion is aimed to preserve the membrane natural fluidity; it is obtained depositing charged polyelectrolites layer-by-layer; the lipid membrane is built on the top of the polymer film by fusion of unilamellar vesicles. the floating membranes were kept always in contact to the subphase. the kinetics of adsorption of the lipid double layer at the polymer/water interface was monitored by neutron reflectivity; different experimental conditions to obtain the best surface coverage were exploited. after administration of aβ to the subphase the lipid membrane still adhered to the polymer cushion, but its structure was modified by the interaction with aβ. neutron reflectivity showed a change of the scattering density profile in the direction perpendicular to the membrane plane, suggesting a penetration of aβ inside the double layer. a change in the surface morphology was detected by afm imaging; afm film-rupture experiments showed that aβ weakens the lipid packing. x. cheng , r. pacheco-gomez , a. rodger , h. matthew , d. i. roper university of warwick, u.k., university of birmingham, u.k. ftsz, the ancestral homolog of eukaryotic tubulins, is a gt-pase that assembles into a cytokinetic ring structure (z ring) essential for cell division in prokaryotic cells. the z ring also recruits other proteins (e.g. zapa, ygfe, zipa) to the division site, where they participate in formation of the septum that separates the two daughter cells. we have studied ftsz polymerization and its dynamic behaviour in real time by right angle light scattering. similar to tubulin, ftsz polymerizes into dynamic protofilaments in the presence of gtp; polymer assembly is accompanied by gtp hydrolysis. the kinetics of inorganic phosphate (p i ) released from the gtp hydrolysis have been studied as well, employing a fast and sensitive colourimetric assay. at ph . , approximate % of the p i was released into the media within minutes of gtp addition. the effects of gtp, ph, k + , and mg + were studied in both cases, and the results were used to build up a model for the mechanism of fibre assembly and disassembly. ygfe, a ftsz accessory protein, is identified as a functional zapa orthologue. finally, we have studied the ygfe bundling to ftsz polymers. it strongly promotes ftsz bundling and is an inhibitor of the gtpase activity. many genomes of viruses encode small membrane spanning proteins which are proposed to modify membrane permeability for ions and small molecules. these channel or pore forming proteins are getting into the focus for antiviral therapy since they are essential for some of the viruses. one of the general themes of the mechanism of function of the proteins is to self-assemble to form the functional form. we present a study on the open reading frame (orf) a membrane protein encoded in structural region of human severe acute respiratory syndrome coronavirus (sars-covs). the full length orf a protein is residues long and contains a single transmembrane (tm) domain. full length protein is synthesized using solid phase peptide synthesis and reconstituted into artificial lipid bilayers forms cation-selective ion channels. the bilayer recordings show cation selection channel activity with a major conductance level of around . ps also at elevated temperatures ( . • c). in silico studies with a amino acid tm domain are done to assess conformational space of the monomeric orf a helix. with this monomeric helix homooligomeric helical bundle models are built and embedded in a fully hydrated -palmitoyl- -oleoyl-sn-glycerol- phosphatidylcholine (popc) bilayer. results of both experimental channel recordings and computational modeling show sars orf a to act as a channel forming protein. -biomolecular self-assembly - microtubules are involved in many vital processes. their rigid structure can resist high forces while their intrinsic ability to switch stochastically between growth and shrinkage phases allows them dynamically to reorganise. in cells, a sizeable network of microtubule binding proteins control and regulate microtubule dynamics. alp and dis are members of the dis / xmap family that are major players in s.pombe. the deletion phenotypes of alp and dis are similar, but nonetheless distinct. both are involved in the formation of spindles but alp is also involved in the maintenance of cytosolic microtubules in interphase. the restrictive temperatures of alp -deletion and dis -deletion mutants are different. alp interacts with alp , a potential member of the tacc protein family. i am working to reconstitute alp /dis -dependent microtubule dynamics in vitro, using purified s. pombe tubulin. both alp and dis express well in insect cells and can be readily purified. preliminary data show that both proteins bind tubulin at low salt concentrations and that both influence the dynamics of pig brain microtubules. my goal is a complete functional analysis of alp and dis , individually and in combination, to test candidate molecular mechanisms for alp /dis -catalysis of s. pombe microtubule dynamics. the syphoviridae coliphage t is a well-suited model to study the assembly of large viral capsids. biochemical and biophysical approaches were used to reconstitute in vitro the assembly pathway of its capsid. the t structure was recently solved from cryo-em and image reconstruction. its icosahedral capsid (t = ) is built from the major head protein (pb , copies) forming both the pentons and hexons and from the portal protein (pb , copies) located at one vertex. its assembly proceeds by steps. pb and pb first assemble into a precursor structure called prohead i, which is converted to prohead ii by proteolysis of pb and pb by a head maturation protease. packaging of the kbp dsdna is then driven through the portal pore by a molecular motor, the terminase. this promotes expansion of prohead ii leading to the mature capsid. the different assembly steps and the conformational changes accompanying capsid maturation were characterized using proheads i either self-assembled from the overproduced and purified capsid proteins or isolated from a phage mutant. these precursor capsid structures were analysed by small angle x-ray scattering. the d structure of prohead ii and of the expanded capsid were solved from cryo-em. our data show that the assembly process of a large icosahedral capsid can be efficiently reconstituted in vitro. amyloid beta peptide fibril formation modulated by phospholipid membranes e. hellstrand , e. sparr , s. linse lund univ., department of biophysical chemistry, sweden, lund univ., department of physical chemistry, sweden disease-causing amyloid fibril formation can be modulated by many factors including interactions with biological lipid membranes. an increasing amount of evidence suggests that the process of fibril formation in vivo and the mechanism of toxicity both involve membrane interactions. alzheimer's is probably the most well-known amyloid disease and the associated amyloid beta peptide originates from the membrane incorporated amyloid precursor protein (app). we use recombinant abeta m - and abeta m - produced in escherichia coli, which allows us to perform large scale kinetics assays with good statistics where the amyloid formation process is followed in means of thioflavin t fluorescence. the lipid membranes are introduced in the system as large unilamellar vesicles composed of dopc, dppc and sphingomyelin, with and without incorporation of cholesterol. we find that the phase behanviour of the membrane in the vesicles has a large effect on the lag time of the amyloid formation process for both abeta m - and m - . all membranes increase the lagtime to some degree but dppc has the largest effect. by comparing different phases we can conclude that the translational diffusion in the membrane seems to be more important than the acyl chain ordering. furthermore, electrostatics, concentration dependence and membrane addition at different time points in the amyloid formation process have been investigated. equilibrium/non-equilibrium transitions in macromolecule interactions p. dumas , g. gibrat , s. bernacchi , e. ennifar ibmc-cnrs, strasbourg, france, llb (cea/cnrs), saclay, france usually, so called 'relaxation phenomena' occur on a fast time-scale and 'p-jump' or 't-jump' techniques are required to follow such events lasting (much) less than ms. we report that, during stability studies of proteins or nucleic acids, such relaxation events can be observed on astonishing long time-scales. we first performed 'melting studies' with nucleic-acid duplexes by using linear variations of temperature (t) with time (t). we observed that even very low rates dt/dt could lead to a frozen state for temperature values below a sharp temperature range, and relaxation to equilibrium beyond that range. this allows defining a 'relaxation temperature' t r separating the two regimes. numerical simulations very accurately described the related hysteresis phenomenon observed upon a heating-cooling cycle, which is the hallmark of departure from equilibrium. analogous observations were made with protein oligomers submitted to either a variable pressure, or variable concentration in denaturant. importantly, a single theoretical frame predicts that the critical relaxation value x r (x standing indifferently for temperature, pressure or denaturant concentration) depends on ln(dx/dt). one may ask whether some thermosensor rnas known for switching on or off genetic expression by 'feeling' a temperature variation, might also 'feel' dt/dt. if true, the exact switching temperature would depend on dt/dt and faster temperature changes would increase t r . -biomolecular self-assembly - the major component of amyloid plaques in the gerstmann-sträussler-scheinker disease is a prion peptide fragment from - to - residues. here, we present a structural study of prp - in form of oligomers and fibrils by fourier transform infrared spectroscopy (ftir) and atomic force microscopy (afm). after incubation at • c, the unfolded peptide was found to aggregate into oligomers characterized by intermolecular β-sheet infrared bands and by a wide distribution of oligomer volumes. after a lag phase, a conformational rearrangement of oligomers into fibrils, with a parallel orientation of the cross β-sheet structures, was observed. by afm, different morphologies were also detected for fibrils that displayed high heterogeneity in their twisting periodicity and a complex hierarchical assembly. in addition, we also studied thermal and random aggregation. the prp - peptide was found to undergo several aggregation pathways, whose end products display different structural properties and intermolecular interactions. these findings underline the high plasticity of the prion peptide, a peculiar feature of prion proteins to overcome species barriers (natalello et al. j.mol.biol. ; : - ). the role of proline isomerisation in the aggregation process and fibril formation of alpha-synuclein j. meuvis , m. gerard , v. baekelandt , y. engelborghs lab.of biomolecular dynamics, ku leuven, belgium, lab.of biochemistry, campus kortrijk, belgium, lab.for neurobiology & gene therapy, ku leuven, belgium alpha-synuclein (α-syn) plays a central role in parkinson's disease. the aggregation of this protein, which contains five proline residues (p ,p ,p ,p ,p ), is accelerated in vitro by fk binding proteins (fkbps), a family of enzymes with a peptidyl-prolyl cis-trans isomerase activity (ppiase). fkbps catalyze the cis-trans conformational change of proline, often a rate limiting step in protein folding. to elucidate the role of the proline residues in aggregation, we constructed a mutant p( , , , , )a α-syn . the kinetics of the aggregation of the mutant were studied with turbidity and thioflavin t fluorescence (tht). turbidity measurements show the formation of early, tht negative aggregates which is as fast for both wt and mutant. fibril formation however is faster for the proline-deficient mutant. we also studied the effect of fkbp on the aggregation of the mutant. although wt α-syn early aggregate formation is accelerated by the addition of µm fkbp , this effect disappears in the mutant. addition of ( pm- µm) fkbp accelerates the fiber formation of wt α-syn, which is abolished in the mutant. we can conclude that α-syn fiber formation is accelerated for the proline-deficient mutant, which suggests a role for the proline residues in fiber formation. furthermore all accelerating effects of fkbp are abolished in the mutant which suggests that the ppiase activity of fkbp is responsible for the accelerating effect on the aggregation of wt α-syn. materials used as gene delivery vehicles must be able to condense dna into small sizes to facilitate transport and crossing various barriers. one of the polycations investigated for dna compaction is chitosan, which has the advantage of being safe and biodegradable. as a step towards reducing the aggregation behaviour of dna-chitosan complexes, chitosans were modified by grafting peg-chains on the backbone. it is known that the transfection efficacy depends on the chitosan chain length. additionally, the degree of pegylation might influence the condensation process. here a systematic biophysical study of pegylated chitosans and how the interplay between chitosan chain length and degree of pegylation affect the compaction of dna in terms of particle size and structure, stability in pbs and when exposed to serum, and transfection efficacy is presented. three different chain lengths of chitosans are employed, and for each sample three pegylation degrees are investigated and the properties of the dna-pegchitosan complexes compared to complexes formed when employing the original, chitosan for dna compaction. it is found pegylation of chitosans can be used to increase both the stability of the dna-chitosan complexes when exposed to serum as well as increase their transfection efficacy in hek cells. max-planck institute for polymer research, mainz, germany model membranes mimic the essential function of a natural membrane. however, the complexity is reduced in order to allow the study of fundamental processes. tethered membranes consist in principle of a lipid bilayer that is covalently linked to a solid support through a spacer group. this architecture allows the characterization of the membrane itself as well as of incorporated membrane proteins using surface analytical techniques. we have established a versatile system of various anchor lipids, which allow membrane formation on different surfaces. the architectures have been characterized by surface plasmon techniques, neutron reflectivity and electrochemical methods. the membranes are electrically insulating and allow for the functional incorporation of ion channel proteins. polymerizable lipids allow to pattern the membrane and to study lateral diffusion processes. furthermore, the membranes can be used as a sensing platform, where embedded membrane proteins act as actual sensing units. a. perico, s. pietronave, l. arcesi, c. d'arrigo consiglio nazionale delle ricerche (cnr), institute for macromolecular studies (ismac) the electrostatic free energy (fe) of two parallel rigid likecharged polyelectrolytes (pes) is given as a function of the separation distance. for high linear charge density, z , the fe shows a minimum due to the increasing of the counterion condensation and condensation volume as the two pes approach. the interaction fe is governed by a critical linear charge density, z c , inversely proportional to the counterion valence. for highly charged pes (z > z c , like dna), the pes attract the stronger the smaller is the counterion valence, because the fe is dominated by the entropic term due to condensation of counterions in a volume displaying a maximum at short distances. for weakly charged pes (z < z c / ) the pes remain undercritical in the whole separation range and therefore repel. for moderately charged pes (z c / < z < z c ), the infinitely separated pes are undercritical but become supercritical as they approach a critical distance and charge condensation and condensation volume expansion start: in these circumstances the pes may attract if the counterion valence is sufficiently large. in the case of many dna rods, hexagonal clusters may be formed. upon interaction with hydrophobic surfaces, proteins show a tendency to expose regions that are normally buried in the hydrophobic core. unfolding is generally perceived as an undesired process in studies aimed to anchor functional proteins at surfaces. upon an upset of perspective the fine control of the unfolding/re-assembly process could be regarded as a strategy to build up molecular nanostructures for the development of organic-inorganic assemblies. we show that molecular layers patterned at the nanoscale, with longrange order properties extending over the microscopic scale, can be obtained upon adsorption of proteins onto the hydrophobic and ordered surface of pyrolytic graphite. upon adsorption, proteins lose their native folding and polypeptide chains re-assemble on the surface in a layered fashion, forming a molecular bilayer. the first layer, in contact with the substrate, and the second molecular layer show corduroy-like nanopatterns of different periodicity, with a relative orientation between the first and second layer patterns of • . surface-induced protein unfolding and polypeptide chain reassembly according to a layered ordered structure is a rather general phenomenon since it is observed for different proteins irrespectively of their specific structural properties. the possibility of using these ordered molecular structures as templates for the subsequent patterned deposition of supramolecular aggregates will be discussed. understanding protein-protein interactions and assemblies to control the hierarchical building of well-ordered supramolecular structures is highly relevant to new tailormade biomaterials. we previously evidenced that contrary to native calcium-loaded α-lactalbumin (holo α-la), calcium-depleted form (apo α-la) has the ability to selfassemble with lysozyme (lys) to form different supramolecular structures in temperature-dependent manner. in the present work, the events occurring at molecular scale were explored using fluorescence techniques. fluorescence anisotropy and fluorescence lifetime measurements provide a powerful and sensitive mean to measure intermolecular interactions. we showed that lys interacts with both apo α-la and holo α-la to form oligomers, assumed to be heterodimers, at • c and • c. the dissociation constants for dimerization, found to be in the µm range, were sensitive to the ionic strength. correlation time calculations suggest that formed heterodimers holo α-la/lys and apo α−la/lys differed in their shape and/or conformation. such conformation differences could explain why holo α-la/lys complexes are trapped as heterodimers while the apo α-la/lys complexes have the ability to further self-assemble into previously reported various supramolecular structures. polyglutamine aggregation and neurodegeneration g. nicastro, l. masino, a. pastore national institute for medical research, the ridgeway, nw aa london, u.k. polyglutamine (polyq) diseases are rare but dominant misfolding diseases linked to neurodegeneration. they are caused by the expansion of cag codon repeats, which encode polyq tracts in the corresponding gene products. aggregation of polyq proteins is thought to be triggered by polyq expansion but be strongly modulated by the protein context. in the attempt of understanding the molecular bases of polyq diseases, we are studying the structures, interactomes and aggregation properties of selected polyq proteins. here, we present recent work on ataxin- , taken as a representative example of the whole family. ataxin- is a ubiquitin specific cysteine protease, involved in the ubiquitinproteasome pathway and known to bind poly-ubiquitin chains of four or more subunits. the enzymatic site resides in the n-terminal josephin domain of ataxin- . we have characterized, using different biophysical techniques, the structure in solution and the aggregation properties of josephin both in isolation and in a ubiquitin complex. we demonstrate that interaction with ubiquitin strongly modulates the aggregation properties of ataxin- and suggest the importance of protein-protein interactions in preventing aggregation. our study also provides new insights into the molecular mechanisms which determine ataxin- specificity for poly-ubiquitin chains of the correct length and cross-linking. förster resonant energy transfer (fret) from an optically excited to a non-excited molecule has been widely used to probe molecular interactions in living cells. changes in the molecular makeup of a cellular region occurring during the acquisition of fluorescence images place tight constraints on the fret technology and data analysis, which could not be addressed satisfactorily until recently. we will describe a method for imaging protein complex distributions in living cells with sub-cellular spatial resolution, which relies on a spectrally resolved two-photon microscope (raicu et al, , nature photon. : - ) and a simple theory of fret in oligomeric complexes (v. raicu, , j. biol. phys. : - ) . then, we will overview recent results on the determination of the supra-molecular structure and distributions in living cells of oligomeric complexes of some g protein-coupled receptors. observing protein aggregates on surfaces m. rabe, d. verdes, s. seeger institute of physical chemistry, university of zurich, switzerland protein aggregation is an important topic of current protein research as it is associated with several human diseases including alzheimer's disease, parkinson's disease, and type ii diabetes. although protein aggregation mechanisms and conditions have been comprehensively investigated, studies on the formation and the fate of protein aggregates in contact with solid interfaces are scarce. we have comprehensively investigated the structure of protein assemblies that form spontaneously upon protein adsorption on solid interfaces using a surface sensitive fluorescence imaging technique based on super critical angle fluorescence (saf) detection. combining this technique with fret we not only succeeded to detect protein aggregates deposited on surfaces but also to characterize their behavior in real time, i.e., their emergence, growth, or spreading. the model protein bsa, for instance, was found to exhibit a certain tendency for aggregation in the buffer solution. these protein clusters can deposit onto solid surfaces and spread resulting in a large, flat structure after some time. a different possibility how protein aggregates emerge on surfaces consists of a direct deposition of protein monomers to pre existing aggregates. such a growth of protein aggregates on the surface has been observed in a model system using the protein α-synuclein, which is tightly associated with the parkinson's syndrome. we present the results of a spectroscopic ellipsometry (se) study of the adsorption process of yeast cytochrome c (ycc) on gold and graphite substrates, according to methods already applied to study the growth dynamics of organosulphur sams on gold [ ] . se investigation was carried out both in situ, at room temperature during protein deposition, and ex situ. on gold, se data demonstrate the formation of an about - nm thick layer, consistent with the formation of a dense monolayer of ycc molecules, confirmed by afm inspection. both in situ and ex situ measurements were characterized by well defined spectral features related to the soret band. analysis of the fine position of this feature allowed to obtain information on the oxidation state of the iron ion of the heme group. se data suggest that proteins have preserved their native structure. a completely different adsorption mechanism was observed on highly oriented pyrolytic graphite (hopg) [ ] . ex-situ se data on ycc/hopg, supported by afm observations, indicate the formation of an ultrathin molecular layer (∼ . nm) related to complete protein unfolding at the hydrophobic surface. the role of phospholipid anisotropy in the stability of inverted hexagonal phase was considered. the equilibrium configuration of the system was determined by the minimum of the free energy involving the contribution of the isotropic and deviatoric bending and the interstitial energy of phospholipid monolayers. the shape and local interactions of a single lipid molecule were taken into account. the minimization with respect to the configuration of the lipid layers was performed by the monte carlo simulated annealing method. at high enough temperature the lipid molecules attain a shape exhibiting higher intrinsic mean and deviatoric curvatures which fits better into the inverted hexagonal phase than into the lamellar phase. for the mathematical model the advanced geometry with non-spherical cross-section of inverted hexagonal phase was calculated, resulting in lower energy in non-spherical cross-section. theoretical results are in a good agreement with the small angle x-ray scattering experimental data. for a long while the conventional view has been that alzheimer disease is brought about by the beta-amyloid fibrils found in the senile plaques, but more recently it has been suggested that the main neurotoxic species would be the soluble oligomeric species, apparently prone to interact with cell structures and macromolecules potentially inducing neuronal dysfunction. peptide-peptide interactions resulting in self-assembly phenomena of beta-amyloid yielding fibrils can be modulated and influenced by small organic molecules that might also be effective therapeutic tools to ideally target both oligomeric and fibrillar species. in this perspective, polycyclic aromatic molecules are of special interest because they might disrupt the molecular architectures precursors of beta-amyloid fibrils by means of weak, non-covalent aromatic interactions, like stacking interactions. we have performed an in vitro spectroscopic study (light scattering, circular dichroism, ftir and fluorescence) of the effects on beta-amyloid fibrillogenesis of the natural pigment hypericin extracted from hypericum perforatum. our results show that, thanks to its structural characteristics and peculiar spectroscopic features, hypericin can be easily used to in vitro monitor the appearance of initial aggregation states of beta-amyloid peptides and, more importantly, that hypericin can interfere with the early stages of polymerization process, playing the role of an aggregation inhibitor. peptaibols are peculiar peptides produced by fungi associated to plants. they are composed by to amminoacidic residues and exhibit antibiotic and antifungal properties. due to their amphypatic nature, they can form ion channels in biological membranes. by making use of experimental models of biological membranes (biomimetic membranes) currently employed in the laboratory of bioelectrochemistry, and models of plant membranes (corn seed root), that are used in the international laboratory of plant neurobiology (linv), we characterized synthetic peptides such as trichogin gaiv and its shorter homologues ( and residues). we studied these peptaibols in a dioleoylphosphatidylcholine monolayer supported by hg using different electrochemical techniques (ac,vc,eis). the experimental technique employed in the linv (clark microelectrode coupled to mife system) allows to measure oxygen flux in the solution contacting plant cell membranes, after treatment with different peptide concentrations. preliminary results might indicate that short peptides can influence the whole metabolism of the plant and can therefore be used as "elicitors" in order to induce an acquired systemic resistance. supported biomimetic membranes (sbm) were developed for protein-membrane interactions studies. phospholipid vesicles were chemically linked onto amine grafted gold or glass surfaces; after an osmotic choc and liposomes fusion a continuous membrane bilayer was formed. the anchoring phospholipid molecule (dspe-peg-nhs) incorporated into the vesicles allowed the formation of a water-filled compartment between the surface and the bilayer. this first sbm model was used to monitor the membranes binding properties (dependent of calcium) of the adenylate cyclase toxin (cyaa) from bordetella pertussis. the sbm model was improved in order to study the translocation of the catalytic domain of cyaa across the bilayer. naturally, the cyaa catalytic domain, when it reaches the target cell cytosol, associates with intracellular calmodulin (cam) an activator of the adenylate cyclase activity of cyaa. to mimic this biological phenomenon, cam was first immobilized on the surface (gold or glass) and in a second step membrane construction was performed over the cam layer. the formation of the biomimetic membrane onto the cam layer was monitored by spr while membrane fluidity and continuity were analysed by fluorescence. our results demonstrated the potentialities of sbm for the study of protein insertion into and translocation across biological membranes. a multi-resolution approach to the structure and function of integrin αiibβ m. rocco , c. rosano , j. w. weisel , d. horita , r. r. hantgan istituto nazionale per la ricerca sul cancro (ist), genova, italy, university of pennsylvania, philadelphia, pa, usa, wake forest university, winston-salem, nc, usa. integrins are heterodimeric transmembrane receptors involved in mechanical anchoring and two-way signaling. each α and β subunit has a modular structure with a large extracellular portion, a single transmembrane region, and a cytoplasmic domain. integrins activation mechanism is regulated by controversial conformational changes: while crystallography revealed similar bent shapes for resting and primed extracellular region constructs, ligand binding-induced large structural rearrangements in smaller constructs suggested extension, "opening" and tails separation. in a multiresolution approach, we used experimental and computed hydrodynamics to discriminate among αiibβ integrin models built on x-ray, nmr, and em data. in contrast with xray data and d em maps, an extension is needed to match the hydrodynamics of full-length, solubilized αiibβ ; an electron tomography-based model fares better. consistent with that, and with our averaged d em images, a conformational change in the head region (β hybrid domain swingout) coupled to a simple transmembrane helices shift matches priming agents-induced frictional changes in full-length αiibβ . our multi-resolution study thus suggests that in integrins extension and immediate tail separation are uncoupled from head domain rearrangements following activation. -biomolecular self-assembly - stabilizing effects of α s -casein, a natively unfolded protein, on the aggregation of biomolecules a. trapani, r. carrotta, p. l. san biagio, d. bulone ibf-cnr palermo, italy α s -casein is one of the four types of caseins, a group of calcium phosphate-binding proteins that, in the form of micellar aggregates, makes up the largest protein component of bovine milk. the structure of α s -casein is that of a triblock polymer with a hydrophilic tract interposed between two hydrophobic blocks. due to the lack of a compact folded conformation, this protein can be classified as one of the intrinsically disordered (or natively unfolded) proteins. this class of proteins is known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces that partially unfolded molecules may expose to the solvent. here we present results on α s -casein effects on the thermally induced aggregation of gluthathione stransferase, a ligand-binding anzyme, and - β-amyloid peptide involved in alzheimer's disease. by means of light scattering and circular dichroism experiments, we attempt to reveal the molecular details of α s -biomolecules interaction. bacterial protein self-assembly on surfaces of well-defined chemistry s-layers are one of the most common cell envelope components of prokaryotic organisms and represent the simplest biological membrane developed during evolution. these (glyco)proteins, which can self-assemble into -d crystalline nanostructures on lipid films, liposomes, and polymers, play already an important role in nanobiotechnology. in this work, we present new findings concerning the recrystallization of bacterial proteins on substrates with defined chemistry. manipulation of the protein-sample interaction was carried out by changing the relative height of oh and ch terminated moieties in self-assembled monolayers (sams). we have found that differences in chain length lead to: i) protein bilayer-protein monolayer transition, ii) preferential protein side adsorption, and iii) increase of the crystal lattice parameters. further manipulation of the protein-sample interaction was achieved by using silane chemistry. we will show that sample hidrophobicity speeds up recrystallization kinetics and reduces the crystalline domain size (and layer compliance). the protein-adsorbed mass per unit area on these substrates is reported for the first time. self-assembly of phenylalanine oligopeptides: insights from experimental and computational studies p. tamamis , l. adler-abramovich , m. reches , k. marshall , p. sikorski , l. serpell , e. gazit , g. archontis dpt. of physics, univ. of cyprus, cyprus, dpt. of molecular microbiology and biotechnology, tel aviv univ., israel, dpt. of biochemistry, univ. of sussex, u.k. the diphenylalanine peptide (ff) forms well ordered nanotubes and its derivatives form nano-assemblies of various morphologies with promising material applications. we demonstrate for the first time by electron and laser microscopy and ftir spectroscopy that the related, triphenylalanine peptide (fff) assembles into rather planar nanostructures, rich in β-sheet. in addition, we conduct . -µs replica exchange m.d. simulations of aqueous ff and fff solutions in implicit solvent. the peptides coalesce into aggregates and participate frequently in open or ring-like linear networks, as well as elementary and network-containing structures with β-sheet characteristics. polar and nonpolar interactions, as well as the surrounding aggregate medium contribute to the network stabilities. within a network, consecutive peptides are linked by head-to-tail interactions; the aromatic sidechains of neighbor peptides assume approximately "t-shaped" orientations. these features are observed in ff crystals and could characterize early formations, or stabilize the mature nanostructures. the fff aggregates acquire higher stability and peptide-network propensity compared to the ff aggregates due to energetic contributions , . chlorophyll biosynthesis is light-dependent in angiosperms because the reduction of protochlorophyllide (pchlide) into chlorophyllide is driven by a photoenzyme, nadph:pchlide oxidoreductase (por). the unique properties of por are due to its ability to assemble into dimers and oligomers within the prolamellar body (plb) membranes of etioplasts studied mainly in leaves of dark-grown seedlings under laboratory conditions. we extended these studies to plant organs developed in the nature: cabbage heads, leaf primordia inside buds, pericarp-covered regions of sunflower cotyledons, potato tubers and seedlings germinating under the soil. in electron microscopic and fluorescence spectroscopic studies we found in many of these organs poorly developed plbs in which por was mainly in monomer state. as a consequence, the chlorophyll accumulation was slow and photo-oxidation processes occurred at illumination. in vitro we artificially induced the aggregation of por monomers into oligomers in glycerol and sucrose containing buffers. this resulted in the increase of the photoreduction rate at the expense of photo-oxidation. these results underline the importance of the self-assembly of por and the plbs in chloroplast development and chlorophyll synthesis in nature. v. vetri , g. ossato , v. militello , m. a. digman , m. leone , e. gratton dsfa, university of palermo, palermo, italy, lfd, university of california, irvine, ca, usa we report an experimental study on concanavalina (cona) aggregation in live cells. in vitro, close to physiological temperature, cona readily forms fibrils involving secondary structure changes leading to β-aggregate structures. the effect of cona on cell cultures and formation of protein aggregates were measured by confocal fluorescence microscopy. in particular, we monitored protein aggregation in live cells by means n&b analysis, cross-n&b and rics. n&b showed the aggregation kinetic and the progressive formation of cona oligomers at cell surface. this suggests that, at cell membrane where local concentration is higher, nucleation sites for aggregation are provided. in parallel, the morphology of the cells changes indicating the progressive cell compaction and death. aggregation and binding of small aggregates to the cell surface were assessed by rics: it is possible to distinguish regions where small aggregates are diffusing and regions where they are bound to the cell. oligomers formation may stimulate non-specific cellular responses due to the exposure of reactive regions of protein structure and of progressive formation of cross−β structures. moreover, aggregates stoichiometry was measured during the kinetic by cross-variance n&b. the two conductive pathways of p x purinergic receptor: different modulation and selectivity r. barbieri , s. alloisio , a. di garbo , m. nobile institute of biophysics, cnr, via de marini , genoa, italy, institute of biophysics, cnr, via g. moruzzi , pisa, italy the p x purinoceptor (p x r) is an atp-gated cation channel that is able to activate a cell permeabilizing pore. p x r cytosolic c-terminal tail is thought to modulate this function. this study was aimed to characterise the biophysical properties of p x r compared to those of the variant lacking the entire c-terminus tail (trp x r) by measuring whole-cell currents and intracellular ca + variations. a mathematical model is used to describe the experimental results. in p x r expressing hek- cells, the potent agonist '-o-( -benzoyl)benzoyl adenosine '-triphosphate (bzatp) -evoked ionic currents depending on concentration and frequency of agonist applications. the currents were strongly inhibited by extracellular mg + in a noncompetitive way. by contrast, in trp x r cells, only high bzatp concentrations elicited small currents not affected by mg + . interestingly, bzatp-induced ca + influx was present both in p x r and in trp x r cells, albeit in the latter the intracellular ca + elevation was smaller. importantly, in trp x r the intracellular ca + rise maintained a competitive mechanism of mg + inhibition similar to that observed in p x r. the experimental data and the modelling findings support the tenet of a functional structure of p x r possessing two distinct conductive pathways. the review of our data on the effects of physical and chemical weak signals on physicochemical properties of water, cell volume, activity and the number of membrane proteins (receptors, ionic channels and enzymes, na + /k + pump and na + /ca + exchanger), intracellular signal systems in norm and pathology (cancer and nerve disorders) would be presented. light microscopic, cell voltage-and patch-clamp, isotope, standard biochemical and genetic engineering methods were used. weak signal-induced effects on cell functional activity (intracellular enzymes activity, the number of functionally active membrane proteins) are realized by changing the physicochemical properties of extra-and intracellular aqua medium. the latter induces the modulation of na + /k + pump-induced cell hydration, which serves as a primary mechanism through which the autoregulation of pump and regulation of membrane excitability and chemosensitivity are realized. by genetic engineering method in oocytes it was shown that the correlation between na + /k + pump and na + /ca + exchanger, which is realized through intracellular messenger systems, plays a crucial role in weak signals transduction in cells and determination of aging-induced increase of cell pathology. listeriolysin pore forming ability in planar lipid membranes at different ph listeriolysin o (llo) is a cholesterol-dependent cytolysin secreted by the intracellular pathogen listeria monocytogenes. its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. llo exhibits optimal cytolytic activity at low ph but it is still able to bind membranes at physiological or even slightly basic ph values in a cholesterol-dependent fashion. high cholesterol concentrations in the membrane restore the low activity of llo at high ph values. based on this broad ph activity we investigated the electrophysiological properties of pores formed by llo at room temperature and at different phs using planar lipid bilayer technique. llo is able to form pores both at ph . and . with a similar permeabilizing ability and similar heterogeneous conductances in the range of picosiemes to nanosiemens. cholesterol content directly correlates with llo activity but it does not change the pore characteristics. collectively, our results demonstrate that llo activity at physiological ph cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for listeria pathogenesis. s. aimon, g. toombes, p. bassereau institut curie, paris, france the physics of membrane/channel and channel/channel interactions is difficult to investigate in cells where it is nearly impossible to modify relevant parameters to deduce physics laws. to overcome these difficulties we built a model system in which voltage gated ion channels were reconstituted in giant unilamellar vesicles (guvs) for the first time. as a first step, we successfully expressed kvap (a voltage gated potassium channel) in e-coli. the channel was purified, fluorescently labelled and reconstituted in small liposomes. its functionality was checked with electrophysiology via fusion of these liposomes into black lipid membranes (blm). as a second step, guvs were formed from these small proteoliposomes using electroformation in a buffer containing mm kcl salt. the proper incorporation of proteins into guvs was controlled using confocal microscopy. functional proteins were detected using the patch clamp technique. with our protocol, we are thus able to prepare guvs containing functional voltage-gated ion channels. one goal is now to study the effect of channel activity on its spatial distribution in these guvs. ryr activation in cultured shr cardiomyocytes at the end of the prehypertensive period the rate of [ca + ] i elevation after the ryanodine receptor (ryr) activation by -chloro-m-cresol ( -cmc) and l-type ca + channels (dhpr) activation by bay k was studied in cultured ( days) cardiomyocytes of spontaneously hypertensive (shr) and normotensive rats (wky, wistar) during weeks of postnatal development. the differences in dh-prs and ryrs activities began to be evident after weeks age when cicr formation has finished and became more expressed at the end of prehypertensive period ( weeks). in response to -cmc ( mm), a drastic increase in the rate of [ca + ] i accumulation ( . ± . times) in shr myocytes was registered after days age versus a decrease in the rates of ca + efflux from the sarcoplasmic reticulum of wistar and wky rat cardiomyocytes. bayk ( µm) also induced more sharp [ca + ] i elevation in shr myocytes ( . ± . times) as compared with wistar ( . ± . times) and wky ( . ± . times) ones of the same age. our results argue that in shr and wky cardiomyocytes, as opposed to normotensive wistar rats, gradual growth of dhpr activity is observed, which follows in parallel with cicr formation in the excitation-contraction coupling during early postnatal ontogenesis, and drastic activation of ryr in shr myocytes after the process termination. cytochrome ba from thermus thermophilus belongs to the large family of structurally related heme-copper oxidases. it accepts electrons from cytochrome c at the p-side of the membrane and uses them to reduce oxygen to water. the energy released in this reaction is used for proton pumping across the membrane to form of an electrochemical proton gradient, used by the cell for formation of atp. in this work we followed the kinetics of single-electron injection into the oxidized nonrelaxed state (o h →e h ) of cytochrome ba by time-resolved optical spectroscopy. two main phases of electron transfer were resolved. the first (τ∼ µs) includes oxidation of cu a and simultaneous reduction of both low and high spin hemes. the second (τ∼ µs) reflects reoxidation of both hemes by cu b . this is in significant contrast to the o h →e h transition of aa -type oxidases, where the fastest phase is due to transient reduction of the low-spin heme a only. on the other hand, the single-electron reduction of the relaxed o state in ba oxidase consisted of only rapid electron transfer from cu a to heme b, which is similar to that in aa oxidase. this indicates a functional difference between the relaxed o and the pulsed o h states of cytochrome ba . as opposed to the phospholamban pentamer, sarcolipin forms anion-selective channels in biomembranes l. becucci , c. b. karim , d. d. thomas , g. veglia , r. guidelli chemistry department, florence university, florence,italy, chemistry department, university of minnesota, minneapolis, mn , department of biochemistry, molecular biology, and biophysics, university of minnesota, minneapolis, mn sarcolipin (sln) and the phospholamban pentamer (pln) are two membrane proteins that inhibit ca-atpase of the sarcoplasmic reticulum at low concentrations. in contrast to pln, sln stimulates maximal ca + uptake rates. sln and pln were incorporated in a bilayer lipid membrane (tblm) tethered to a mercury electrode through a hydrophilic spacer. electrochemical impedance spectroscopy measurements show that sln forms channels permeable to chloride ion, weakly permeable to phosphate ion and impermeable to inorganic cations such as na + and k + . a relationship between this property of sln and its regulatory function on ca-atpase of sarcoplasmic reticulum is proposed. atp increases the permeability of a tblm incorporating sln to phosphate ion by associating to sln with an association constant of . µm. an explanation for this behavior is provided. sln can be identified with the " p i transporter" described by a.g. lee et al. conversely, both electrochemical impedance spectroscopy measurements and molecular dynamics simulations provide strong evidence that the pore of the pentameric form of pln does not act as a chloride channel. "social" domain organization and dynamics of nicotinic acetylcholine receptor at the cell membrane f. j. barrantes unesco chair biophys. & mol. neurobiology. univ. nac. sur, b. blanca, argentina a combination of experimental techniques (patch-clamp, confocal frap and fcs, single-particle tracking, highresolution fluorescence microscopy) has been used to analyze the supramolecular organization of the acetylcholine receptor (achr), the dynamics of the receptor at the cell surface, and the kinetics of receptor internalization. changes in cholesterol (chol) content affected muscle and neuronal-type achr organization and dynamics at the cell surface. chol depletion produced gain-of-function of single-channel dwell time. submicron-sized (∼ nm) domains, stable over a period of hours at the cell membrane, could be resolved into achr "nano-clusters" with a peak size distribution of ∼ nm by sted microscopy. chol depletion reduced the number of nanoclusters, increasing their size, and changed their supramolecular "social" organization on larger scales ( . - . microns). frap, fcs and spt experiments provided information on the dynamics of achr nanoclusters, disclosing the dependence of their mobility on chol content and cortical cytoskeleton. chol content at the plasmalemma may thus modulate cell-surface organization and dynamics of receptor domains, and fine-tune receptor channel function to temporarily compensate for acute achr losses from the cell surface. m. czaplinska, k. gwozdzinski, a. koceva-chyla division of research of structure of biopolymers university of lodz, lodz, poland doxorubicin (dox) and paclitaxel (ptx) are anticancer drugs commonly used in chemotherapy of breast cancer therapy, however, the use of these drugs is limited by the risk of developing heart failure. generation of reactive oxygen species contributes to the cardiotoxicity of doxorubicin. nitroxides are low molecular weight, stable free radicals reacting with ros and they present antioxidant properties. the aim of this study was to analyze the effects of pirolid (pd) on the oxidative stress induced by dox and taxane in mcf- breast cancer cell line. results from mtt test revealed that ptx is more cytotoxic towards mcf- cells than dox. the ic was . µm and µm, respectively. pd alone does not influence cell viability. pd in combination with both drugs did not change viability of cells. both drugs increased the level of carbonyl groups in cells. the highest level of peroxide was observed in cells incubated with dox (approx. -fold). nitroxide alone did not influence the level of peroxide in the whole range of concentrations. combination of pd with dox and ptx reduced the level of carbonyls depending on its concentration. pd did not affected on the level of peroxide in cells suspension. dox and ptx increased ( -fold) the level of carbonyls. pd decreased the level of peroxides in cells treated with dox and ptx. the lowest concentration of peroxide was observed at µm of pd. these results show that pd protect mcf- cells against oxidative stress induced by drugs. open channel structure of mscl from fret microscopy and simulation mechanosensitive channels open in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing and osmoregulation. here, we have determined the likely structure of the open state of the mechanosensitive channel of large conductance from e. coli (mscl) in a natural environment using a combination of patch-clamp studies, fret spectroscopy, epr data, molecular and brownian dynamics simulation. structural rearrangements of the protein are measured while controlling the state of the pore by modifying lipid bilayer morphology. fret efficiency changes can be related to distance changes using a monte carlo analysis program in conjunction with detailed orientational analysis. these measurements are used as restraints in all atom molecular simulations in order to determine the likely structure of the open state, whose probable conductance is derived from brownian dynamics simulations. transition to the open state occurs via large rearrangements throughout the protein that create a wide pore nearly a in diameter. both transmembrane helices are found to line part of the pore. the n terminal helix is found to lie along the face of the membrane where it can act to sense membrane tension and directly transfer this to the pore lining helices. the method of coupling spectroscopic data with simulations is likely to be of great value for studying conformational changes in a range of membrane proteins. putative potassium channels in synechocystis sp. pcc v. checchetto, m. zanetti, g. m. giacometti, i. szabò, e. bergantino department of biology, university of padova, italy we are interested in the identification and characterization of potassium channels in the cyanobacterium synechocystis sp. pcc , an organism which is considered the ancestor of plant chloroplasts. a bioinformatic screening of synechocystis proteome identified, among others, two proteins on which we focused our attention. the first one (syncak) displays sequence homology to mthk, a ca + -dependent potassium channel from m . thermoautotrophicum. the second one (synk) is predicted to contain six transmembrane regions and the typical selectivity filter of all potassium channels. our goal is to understand their roles in the physiology of cyanobacteria. we cloned their coding sequences in fusion with gfp and the hybrid proteins were expressed in chinese hamster ovary cells. we evaluated the presence of both proteins in plasma membrane by fluorescence microscopy and then we proceeded to their functional characterization using patch clamp technique.this analysis will allow us to gain information about channel activity, regulation and pharmacology. we also plan to evaluate the importance of syncak and synk channels in photosynthesis. to test the hypothesis that they could be involved in regulating this process, we will produce deletion and site-specific mutants in synechocystis. finally we would also identify the homologous of these channels in the higher plant a. thaliana and obtain some information about their localization and function. j. braunagel max-planck institute for polymer research, ackermannweg , mainz, germany the cyclododecadepsipeptide valinomycin is composed of two amino acids (l-valine and dvaline) and two hydroxyl acids (d-α-hydroxy-isovaleric acid and l-lactic acid). they form a membered ring of alternating amino and hydroxyl acids. in the cyclic structure, the polar groups are oriented towards the central cavity, whereas the rest of the molecule is relatively nonpolar. this enables the complexation of ions and valinomycin acts as a selective ion transporter (k + ) in lipid membranes. when one of the amino acids is exchanged, e.g one l-valine by an l-lysine, selected functionality can be engineered into the depsipeptide while maintaining its ion conducting properties. we induced several modifications into valinomycin, i.e. a biotin binding unit or a ferrocene group to induce an electrochemical active center. the ion conducting properties of the modified ion carriers have been probed in planar lipid bilayers as well as in solid supported membranes. the role of the membrane dipole potential (ϕ d ) is of a particular interest due to a powerful impact of this potential on the membrane permeability and lipid-protein interactions. channel forming activity of gramicidin a, alamethicin, syringomycin e, hpa peptide, and ompf porin are influenced by ϕ d . we have studied the effect of the membrane dipole modifier, phloretin, on the properties of single channels formed by a wild-type alpha-hemolysin in planar lipid bilayers. the single channel of a ∼ ps conductance exhibits transitions into a number of low-conductance states as the transmembrane voltage exceeds ∼ mv (regardless of the voltage polarity). the phloretin addition to the bathing solutions ( µm) (after the hemolysin channel was formed in the membrane) shifts dramatically the channel voltagedependence. transitions to the low-conductance states are observed at ∼ mv. the effect of the phloretin addition was not observed in the case when it was introduced into the bathing solution before alpha-toxin. since phloretin reduces ϕ d , the data may report on the influence of the electric potential profile on the energy of the low-conductance state of the alpha-hemolysin channel. the alternative explanation of this effect consists in a specific interaction between the phloretin and toxin channel. the work is supported in part by rfbr (# - - ), ss (# . . ) , and the program of the ras «molecular and cell biology». atypical mechanism of conduction in potassium channels c. domene , s. furini physical and theoretical chemistry laboratory, department of chemistry, university of oxford, oxford, u.k., department of electronics, computer science and systems, university of bologna, bologna, italy potassium channels can conduct k + ions with rates of up to ∼ ions per second at physiological conditions, and they are selective to these species by a factor of over na + ions. ion conduction has been proposed to involve transitions between two main states, with two or three k + ions occupying the selectivity filter separated by an intervening water molecule. the largest free energy barrier of such a process was reported to be of the order of - kcal mol − . here, we present an alternative mechanism for conduction of k + in k + channels where site vacancies are involved, and we propose that coexistence of several ion permeation mechanisms is energetically possible. conduction can be described as a more anarchic phenomenon than previously characterized by the concerted translocations of k + -water-k + . we exploited the au-deposited self-assembled monolayers of the type: [−s−(ch ) n −ch ] (where n = , and ) with hydrophobically adsorbed redox protein -azurin to verify intrinsic electron transfer mechanisms according to the charge-transfer theory. the enthalpies and volumes of activation were determined through the variation of temperature ( - o c) and pressure ( - mpa) and experimental values were compared with those expected on the theoretical grounds. for the case of n = the activation enthalpy definitely contains a large contribution originated from frictional-like dynamics of protein, and the activation volume has a small positive value. for n = the value of activation enthalpy directly matches / of that for the reorganization energy, and the activation volume attains substantially negative value. for n = we observed the intermediary performance. the whole kinetic pattern is consistent with the smooth changeover between adiabatic and nonadiabatic mechanisms of electron transfer. two gating modalities in the pore of the miniature k + channel kcv kcv is a viral protein that forms functional k + channel in heterologous systems. because of its miniature size ( amino acids) we use kcv as a model system to study and manipulate basic properties of the k + channel pore. by analysing single-channel recordings we highlighted two voltage-dependent modalities of gating in kcv: a slow and a fast gating. the presence of a slow gating is revealed by the very low (in the order of - %) mean open probability. slow gating is not related to the presence of a bundle crossing, as shown by accessibility of the cavity to mts reagents. channel opening might involve the transient formation of salt bridges between residues at the n and c termini of the channel, as suggested by mutational experiments inspired by molecular dynamics simulations of kcv. fast gating, analyzed by beta distributions, is responsible for the negative slope conductance in the single-channel i/v curve at extreme potentials and can be explained by depletion-aggravated instability of the filter region. aca is a type b caatpase with a regulatory n-terminus whose autoinhibitory action can be suppressed by binding of calmodulin (cam). aca n-terminus is able to bind a region of the small cytoplasmic loop connecting transmembrane domains and . to define the role of this interaction in autoinhibition we have analysed a number of single point mutants produced by mutagenesis of aca e -n sequence. mutation to ala of any of acidic residues (e , d , d , d , e , d ) originates an enzyme with normal activity in the presence of cam, but less camstimulated. these results highlight the relevance of a negative charge of the surface area of the small cytoplasmic loop in aca autoinhibition. the most deregulated mutant is d a aca , which is less activated also by controlled proteolysis or by acidic phospholipids; moreover, the phenotype of the d a mutant is stronger than that of d n aca suggesting a more direct involvement of this residue in autoinhibition. of the other mutants (i a, n a, p a, p a, v a, n a), only p a aca has a basal activity higher than that of the wt. these results provide the first evidence that the small cytoplasmic loop of a type b caatpase plays a role in the attainment of the autoinhibited state. complex i, the first member of the respiratory chain, serves as a proton pump catalyzing transfer of two electrons from nadh to ubiquinone coupled with the translocation of four protons across the membrane. so far the mechanism of energy transduction by complex i is unknown. the nadh-binding cavity of complex i has a very prominent feature -the presence of two invariant amino acid residues, glutamate and tyrosine, that are exposed to the solvent and located in the vicinity of the fmn, the primary electron acceptor in the enzyme. it was suggested that they might be involved in the binding of nadh through interaction with its nicotinamide moiety. in this work we assessed the function of corresponding glu from the nuof subunit of e. coli complex i by mutation for glutamine. we showed that the negative charge of glutamate in the catalytic site is needed for the electrostatic repulsion of negatively charged phosphates of nucleotides. this process facilitates release of the product nad + and, as a result, accelerates turnover of complex i. we also found that glutamate, as one of the four negatively charged amino acid residues surrounding the isoalloxazine ring of the fmn at a distance of - Å, has a share of mv of the overall mv depression of the midpoint potential of this redox cofactor. l. erokhova , p. kügler , p. pohl institute of biophysics, johannes kepler university, linz, austria, ricam, austrian academy of sciences, linz, austria the mechanism of water transport through epithelia is still under debate. in the present work we tested the hypothesis of isosmolal water transport using mdck cells stably expressing the human sodium-glucose cotransporter (hsglt ). tagging hsglt with egfp enabled determination of its abundance in the plasma membrane by fluorescence correlation spectroscopy (fcs). by monitoring tiny shifts in the concentration of water-soluble dyes in the vicinity of epithelia, fcs also allowed assessment of the water fluxes through confluent cell monolayers grown on permeable supports. fitting the set of differential equations for the osmotic drift and for the back diffusion to the experimentally determined dye distribution permitted calculation of water flow both in the presence and in the absence of an osmotic gradient. from the calorimetric measurements of glucose transported across the cell monolayer, the water:glucose stoichiometry was derived. dividing the increment in osmotic water flux due to hsglt expression by the number of hsglt copies in the plasma membrane resulted in a single transporter water permeability p f of . x − cm /sec. thus, p f is close to the single channel water permeability of aquaporin- . consequently, even small osmolyte concentration differences between the cytoplasm and the basolateral buffer solution are sufficient to drive a substantial water flux. m. emre, s. kavak cukurova university school of medicine, department of biophysics, balcali-adana, turkey experimental studies have shown that the at -receptor antagonists telmisartan (tel) has a ppar-activating property, but there does not appear to be a class effect. to test telmisartan's importance, we investigated its effect on electrical activities (ea) in diabetic (d) rats. the purpose of this study was to investigate the effects of the tel on ea of diabetic papillary muscle (dpm) with stz-induced. in this study, we used four groups: ( ) nondiabetic control (ndc) group (c), ( ) tel-treated ndc group (c+tel), ( ) diabetic group (d), and ( ) tel-treated d group (d+tel). diabetes was induced by a single i.v injection of stz. in the study, membrane potential (mp) and action potential (ap) recorded after the establishment of diabetes. ) mp was decreased significantly in both tel-treated c and d rats (from - , ± , to - , ± , mv and from - , ± , to - , ± , mv). ) ap unchanged in d group, whereas c+tel and d+tel groups showed increase in ap compared with c and d groups. ) repolarization time was prolonged in diabetic rats. ) in c+tel and d+tel groups depolarization rate values increased significantly. on the other hand, in d group repolarization rate values descreased increased significantly compared to baseline values in tel solution. as a result, our data suggest that the beneficial effects of tel-treatment on the ea of the dpm appear to be due to the diminished k + currents. cytochrome c oxidase is a terminal complex (cco, complex iv) of a respiratory chain that is located in an internal membrane of mitochondria or plasma membrane of bacteria. cco is an electron transfer enzyme that reduces o and uses the redox energy of the o reduction for the proton translocation across the membrane. the electron-and proton-transfer generate a transmembrane electrochemical gradient (∆µh + ) that is used for atp synthesis and all other kinds of work for the cell needs. the proton translocation mechanism of cco requires 'channels' for the h + uptake and expulsion within the enzyme. the proton transfer occurs on a time-scale of micro-to-milliseconds. in order to study the proton transfer in cco, a flow-flash approach based on a time-resolved ftir spectroscopy was developed and applied. our ftir flow-flash approach (the measurement of the reaction of cco with o ) allows to reach a time resolution up to tens milliseconds. with this approach and site-specific mutants of cco where the catalysis is slowed down, separate steps of the proton transfer were studied. the results showed that a unique cross-linked tyr- (in a combination with a time-resolved visible spectroscopy and electrometry) serves as a proton donor for the dioxygen bond cleavage during the o reduction by cco. furthermore, the protolytic transitions of glu- -a key amino acid in the proton transfer mechanism in cco -were shown for the first time. role of calcium ions in nickel potentiation of nmda currents p. gavazzo, i. zanardi, p. guida, c. marchetti biophysics institute ,cnr, genova, italy nmda receptors are glutamate-gated channels distributed throughout the brain in the excitatory synapses and are critical for the nervous system function. they are assembled from two types of subunits, the essential nr and at least one nr (a,b,c,d). nickel (ni) modulates the current flowing through nmda receptors in a different way depending on the nr subunit present. we have recently identified several domains of the channel involved in ni interaction, but many aspects of this modulation remain elusive. in this work we intended to determine the role of calcium (ca) ions in the potentiation induced by ni on the current through nr /nr b recombinant nmda receptors. when ni was applied in the presence of the physiological concentration of ca ( . mm) , a voltage-independent potentiation of the current was observed with a kp of . µm. this effect was progressively reduced by decreasing ca concentrations and it was no more detectable with . mm ca or in the presence of barium (ba, . mm). in this last case the effect of ni on nr /nr b receptors was mainly inhibitory (ki(- mv)= µm). therefore a physiological concentration of ca is necessary to induce ni amplification of the current. many data in the literature indicate a correlation between ca ion entrance through the channel, nmda current facilitation and cytoskeleton; however, in our experiments, the application of the actin perturbing agent cytochalasin-d did not produce major modifications in ni effect. heteromerization properties of voltage dependent potassium channels f. gambale , l. pedemonte , a. naso , i. testa , c. usai , a. diaspro , c. picco istituto di biofisica, cnr, genova, italy, lambs-microscobio, department of physics, genova, italy voltage-gated potassium channels are either homomeric or heteromeric tetramers composed of four α-subunits. in order to bring a contribution to the comprehension of channel heteromerization we have been investigating the properties of two plant voltage-gated k + -channels by using electrophysiological and fluorescence techniques. experiments were focussed on kdc and kdc , coexpressed in xenopus laevis oocytes. kdc , the first potassium channel cloned from daucus carota, belongs to the subfamily of α-modulatory silent channels as it doesn't form functional homomeric channels by itself. on the contrary kdc forms functional heteromeric channels when coexpressed with homologous subunits. kdc , the last k + channel cloned from d. carota, belongs to the kat family and shares an overall identity of % with kat . to correlate kdc functional properties with its localization in oocytes, kdc and/or kdc subunits were labelled with gfp and their properties investigated by confocal microscopy and voltage-clamp. we found that the kdc -egfp fusion protein is not targeted to the plasma membrane unless it is coexpressed with kdc . moreover electrophysiological experiments demonstrated that the heteromeric kdc -kdc channel has altered selectivity and activation properties with respect to homomeric kdc channel. circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome (ards) [ ] . we present a microfluidic investigation of the roles of actin organization and myosin ii activity during the different stages of leukocyte trafficking through narrow capillaries using specific drugs. the deformation rate during entry reveals that cell stiffness depends strongly on f-actin organization and hardly on myosin ii activity, supporting microfilament role in leukocyte sequestration. in the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. conversely, membrane unfolding was independent of leukocyte stiffness. the surface area of sequestered leukocytes increased by up to % in absence of myosin ii activity, showing the major role of molecular motors on microvilli wrinkling and zipping. finally, cell shape relaxation was largely independent of both actin organization and myosin ii activity, whereas a deformed state was required for normal trafficking through capillary segments [ ] . [ ] g.s. worthen et al., science, , - ( ) excitation-contraction coupling in skeletal and cardiac muscle is tightly regulated by the calcium release channel of the sarcoplasmic reticulum, the ryanodine receptor (ryr). we could previously show that suramin is a potent activator of the ryr via the calmodulin binding site. calmodulin shows dualistic action, i.e. activation or inhibition of the ryanodine receptor, depending on the absence or presence of ca + . screening of suramin analogues identified nf as a use-dependent inhibitor of the skeletal muscle ryr (ryr ). here we show that nf inhibits high affinity [ h]ryanodine binding and single channel recordings of the purified ryr . nf induced a reduction of open probabilities in a concentration dependent manner, with no effect on current amplitude and unitary conductance. importantly, nf triggers flickering episodes of channel openings and closings before the ryr is frozen in a complete non-conducting state, which is fully reactivated by the ryr agonist atp. moreover, zwitterionic behaviour of nf facilitates plasma membrane permeation, which prevented caffeine induced ca + transients in skeletal muscle cells and cardiomyocytes. conversely, ip mediated ca + signals were not altered by nf . this work was supported by herzfelder'sche familienstiftung and fwf . beyond steady-state protein dynamics t. hauß , j. pieper , a. buchsteiner , r. e. lechner , n. a. dencher helmholtz-zentrum berlin für materialien und energie, berlin, germany, technische universität darmstadt, germany, technische universität berlin, germany to study protein dynamics beyond steady-state experiments we have developed a novel laser-pump:neutron-probe experiment which allows us to monitor temporal changes in protein dynamics during a working cycle of a protein. protein dynamics has been extensively studied, but so far, the correlation of internal protein dynamics with the function of proteins was investigated only indirectly in steady-state experiments by variation of external parameters by variation of external parameters like temperature or hydration. the method comprises of an in-situ optical activation of a protein and a time-dependent sampling of the dymamic response using quasi-elastic neutron scattering. with the membrane protein bacteriorhodopsin, a light driven proton pump, we can demonstrate for the first time temporary alterations in the protein dynamics after triggering the working cycle. this observation is a direct proof for the functional significance of protein structural flexibility, in connection with the largescale conformational changes in the protein structure occurring during the operation of a "molecular machine". the slow vacuolar (sv) channels are ubiquitous in all tissues of higher plants. the sv channel is a non-selective cation channel permeable to both monovalent and divalent cations. sv currents recorded in a typical patch-clamp experiment require unphysiologically high cytosolic and low vacuolar calcium concentrations for full activation. we aim at looking for endogenous plant substances which might be able to modify or shift the voltage activation threshold of this channel towards more physiological conditions. flavonoid naringenin [nar] is present in all plant species where it plays a central role in the flavonoid biosynthetic pathway. nar is stored in the vacuoles in glycosylated form called naringin. when nar was added to cytosolic bath solution, we recorded a dose-dependent reversible decrease in sv channel activity. when we investigated the effect of nar on the voltage dependence of the channel, we observed that the activation threshold of the sv channel is shifted towards more positive voltages. our group has evidences that approximately % of the total sv current at high (e.g.> mv) positive voltages is mediated by calcium. therefore, in order to verify whether nar affects both potassium and calcium conductance, we performed experiments by combining the patch clamp technique with fluorescence measurements using the fluorophore fura- : both sv currents and calcium signals were abolished by mm [nar]. determination of calcium currents in cation channels using a novel fluorescence/patch-clamp approach p. v. k. gutla, a. gradogna, a. carpaneto istituto di biofisica, consiglio nazionale delle ricerche, via de marini , genova, italy the patch-clamp technique combined with fura- fluorescence detection is suitable to investigate calcium fluxes. we used the excised patch configuration and focused the photomultiplier to the tip of the recording pipette where the fluorescent dye was present (fluoresence combined with excised patch = flep). this configuration has several advantages, i.e. absence of delay in loading the fluorophore, of interference by endogenous calcium buffers and of photobleaching. here we present an application for the determination of fractional calcium currents (pf) in a plant non-selective cation channel, showing that pf can be modulated by cytosolic calcium and potassium. flep is very efficient for measuring small calcium currents (< pa) of sufficiently long duration; fluorescence signals are amplified by integration in time, as the calcium/fura- complex accumulates at the tip of the recording pipette and diffuses slowly. we propose this technique not only for the study of calcium transport pathways, but also for other transporters of divalent cations as nickel and manganese known to quench fluorescence thus reducing both and nm components. moreover, using the appropriate fluorophore the technique may be extended to further ion species, e.g. bcecf for investigating proton transport pathways. ref: we introduced an original method for the monitoring of the changes in the electrostatic surface potential, using the quenching of the intrinsic tryptophan fluorescence by acrylamide or iodide. this approach opens new way to understanding the dynamic processes within the proteins. our experiments revealed that the conformation of the na + /k + -atpase large cytoplasmic loop (c ) in the presence of the atp (without magnesium) substantially differed from the conformation in the presence of mg + or mgatp or in the absence of any ligand not only in the sense of geometry but also in the sense of the electrostatic surface potential. moreover, our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule. this property could be important for the communication between the cytoplasmic headpiece and the cation binding sites located within the transmembrane domain. influx of antibiotics into the periplasm of gram-negative bacteria is facilitated by porins that form channels in the outer membrane. we propose that certain natural antibiotics have been optimized by co-evolution to take advantage of the charge distribution in non-specific porins to achieve binding and thereby facilitating their uptake in bacteria. we investigate the permeation pathways of antibiotics into bacteria by reconstitution of a single porin into an artificial lipid bilayer and measuring the binding of antibiotic molecules through the time-resolved modulation of a small ion current. we have been able to characterize facilitated translocation of several antibiotics through escherichia coli and enterobacter aerogenes porins. noise analysis of ion currents through a porin in the presence of effective antibiotics revealed binding kinetics at a single molecule level. we report for the first time temperature dependent antibiotic translocation that revealed complete energy profile. combining these results with microbiological assays and molecular dynamics simulations, we conclude the molecular mechanism of antibiotic permeation. our approach may contribute to the rational design of new antibiotics against clinical bacterial strains for the most efficient delivery to target sites. mitochondria regulate ca + influx and determine patterns of er ca + refilling in acinar cel o. kopach, i. kruglikov, t. pivneva, n. voitenko, n. fedirko bogomoletz institute of physiology, kiev, ukraine store-operated ca + entry (soce) is mediated by activation of soc-channels of plasma membrane following the emptying of endoplasmic reticulum (er) ca + stores. the soce is required for calcium signaling, secretion of neurotransmitters and proteins, but the mechanisms of natural soce regulation are not well understood. we utilized several imaging methods to measure ca + signals in cytoplasm ( ] mit transients, observed both in egta-and bapta-buffering inside solutions. these data suggest that ca + sequestration by mit is associated with the formation of microdomains and prevents ca + -dependent socc inactivation. we also found that inhibition of mit under prolonged cell stimulation resulted in complete inhibition of soce as well as decrease and deceleration of er refilling. thus, mit regulate calcium recycling and maintain the soce controlling dynamic interplay between soce and sustained er refilling under prolonged stimulation. bioelectrochemical devices composed of au electrodes coated by self-assembled monolayers (sams) of different composition and thickness are ideal systems to probe et patterns and mechanisms for redox proteins. representative proteins, cytochrome c and azurin were studied by using the combinations of four different strategies including the variation of sam thickness ([−s−(ch ) n −ω], with n running over the range to , throughout), solution viscosity (varied by adding of the viscose additive -glucose), temperature ( to o c) and hydrostatic pressure (up to mpa), aiming the identification of different intrinsic et patterns and interplay between them in the framework of generalized charge-transfer theory. we demonstrated the full adiabatic (frictional) control for the case of thinner sams, the intermediate (mixed) regime, and the complete changeover to the nonadiabatic mechanism (long-range tunneling) for the case of thick sams owing to the variation of electronic coupling, in a nice agreement with theoretical predictions. h. nury , f. manon , b. arnou , m. le maire , e. pebay-peyroula , c. ebel institut de biologie structurale (ibs) cea cnrs ujf grenoble france, cea ibitec cnrs ura univ. paris-sud gif-sur-yvette france adp/atp carriers (aacs) are major and essential constituents of the inner mitochondrial membrane. they drive the import of adp and the export of newly synthesized atp. they were described as functional dimers from the s until the structures of the aac shed doubt on this consensus. we aimed to ascertain the published biophysical data claiming that aacs are dimers and to characterize the oligomeric state of the protein before crystallization. analytical ultracentrifugation sedimentation velocity experiments clearly show that the bovine aac is a monomer in -laurylamido-n,n dimethylpropylaminoxide (lapao), whereas in triton x- and reduced triton x- , higher molecular mass species can also be identified. neutron scattering data for monomeric bovine aac in lapao does not give definite conclusions on the association state, because the large amount of detergent and lipids is imperfectly matched by contrast methods. we discuss a possible way to integrate previously published biochemical evidence in favor of assemblies, the lack of well-defined multimers that we observe, and the information from the high-resolution structures, considering supramolecular organizations of aacs within the mitochondrial membrane. local anaesthetic binding to shaker channels: role of aromatic residues j. nilsson, h. ullman, k. sahlholm, p. arhem the nobel institute for neurophysiology, department of neuroscience, karolinska institutet, se- stockholm, sweden local anaesthetic, antiepileptic and antiarrhythmic drugs acting on nav and herg channels have been assumed to bind to aromatic residues in the internal vestibule; to f and y in nav (ragsdale et al., and to y and f in herg (mitcheson et al., ) . despite a lack of such residues in kv channels, local anaesthetics, antiepileptic and antiarrhythmics bind to kv channels with a considerable affinity. to explore the role of aromatic residues for the binding we investigated the effect of bupivacaine, benzocaine, phenytoin and quinidine on shaker channels mutated to residues corresponding to the most c-terminal of the two aromatic residues in the s segment of the nav and the herg channels, (v y and p f respectively). the channels were expressed in xenopus oocytes and the currents measured with the two-electrode voltage-clamp technique. the results suggest that aromatic residues do not increase the binding affinity of the studied compounds to kv channels. rather, the affinity decreases (as reflected in typical k d values for bupivacaine on v f, p f and wildtype channels, being , and µm, respectively). thus, aromatic residues seem not to be necessary for high-affinity binding of the studied compounds to kv channels. how this relates to their suggested roles in the nav and herg channels, remains to be evaluated. (molina et al, ) . the occurrence of a similar behavior in other channels points out to clustering and coupled gating as a potentially important drug target to modulate channel activity. we have identified molecular determinants involved in single and coupled channel gating in kcsa. first, we detected that clustering and coupled gating of kcsa is modulated by anionic lipid. also, a model for the interaction between two kcsa open channels was built. the docking predicts intermolecular sites which includes the non-annular lipid binding site. this explains how an excess of anionic lipid disrupts interactions between channels, destabilizing clustering and coupled gating in kcsa. in addition, the docking model reveals molecular determinants involved in single and coupled channel gating. this interaction involves w , which affects the neighbouring channel through specific interactions in the extracellular mouth stabilizing the selectivity filter in an open conformation. the coupled gating is also explained since this mechanism affects the opposite channel in a mutual manner. finally, mutants kcsa e a and kcsa w a disrupt the coupled gating of kcsa, thus, supporting the model. we think that this coupled gating phenomenon could correspond to the second gate previously detected by fluorescence methods (blunck, et al, ) . supported by grants from the spanish bfu - /bmc and csd - . a. kumar, e. hajjar, p. ruggerone, m. ceccarelli university of cagliari, monserrato, italy the striking presence of outer membrane (om) in gramnegative bacteria of e.coli represents a strong barrier for any molecule to penetrate inside bacteria. in particular for β-lactam antibiotics, which have their target located inside the bacteria; the first step towards reaching inner part of bacterium is the cellular uptake. ubiquitous presence of porins (such as for instance ompf, ompc) in the om, function as a channel facilitating the transport of molecules (such as, for instance antibiotics) across the om. bacteria can exhibit resistant towards antibiotics by: (i) decreasing their uptake by under-expressing the porins or/and (ii) production of inactivating enzymes such as ß-lactamases. to combat the latter mechanism ß-lactamase inhibitors (such as, sulbactam for instance) are prescribed together with the antibiotics. like the antibiotics, the inhibitors must penetrate the om, the main path being through porins. it is thus evident the biological relevance of investigating the mechanisms by which porins can regulate entry/exit across the om. to achieve this goal, molecular dynamics simulations were performed to explore the structure and dynamics of pores formed by ompf and ompc porin. from the analysis of data obtained from our simulations, we identified the key residues buried behind the l loop, which may play be crucial for porins to exert their biological role. as a case study, we report results about the diffusion of sulbactam through the two porins. the pentadecapeptide gramicidin forms a cation-specific ion channel in membrane environment. the two main conformations are the head-to-head helical dimer (hd) known as the channel conformation and the intertwined double helical form (dh) often referred to as non-channel conformation. in this comparative study [ ] , the energetics of single potassium ion permeation by means of the potential of mean force (pmf) for both gramicidin conformations embedded in a dmpc bilayer has been addressed by molecular dynamics simulations. a significantly decreased free energy barrier by ∼ kj/mol for potassium ion passage through dh as compared to hd is reported. favorable electrostatic side chain-cation interactions in hd are overcompensated by phospholipid-cation interactions in dh. the latter are coupled to an increased accessibility of the channel entrance in dh due to distributed tryptophans along the channel axis. this result underscores the importance of the lipid environment of this channel not only for the equilibrium between the different conformations but also for their function as cation channels. y. sawada , m. murase , m. sokabe dept. physiol. nagoya univ. grad. sch. med., nagoya, japan, icorp/sorst cell mechanosensing, jst, nagoya, japan the bacterial mechanosensitive channel of large conductance mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer α-helices, and its d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. although several models for the opening process have been proposed with molecular dynamics (md) simulations, as they do not include mscllipid interactions, it remains unclear which amino acids sense membrane tension and how the sensed force induces channel opening. we performed md simulations for the mechano-gating of mscl embedded in the lipid bilayer. upon tension in the bilayer, phe in the outer helix was dragged by lipids, leading to a tilting of the helices. among amino acids in the outer helix facing the bilayer, phe at the water-lipid interface showed the strongest interaction with lipids, thus may work as a major tension sensor. neighboring inner helices cross each other in the inner leaflet, forming the most constricted part of the pore. as tension increases, the crossings move toward the cytoplasm associated with an expansion of the constricted part. during the movement, a hydrophobic water block environment around the constricted part was broken followed by water penetration and permeation. the k + channel kcsa is an integral membrane protein from s.lividans, used as a model system for studies on ion channels and oligomeric membrane proteins. its atomic structure has been solved by x-ray diffraction, which shows an assembly of four identical subunits around a central aqueous pore, including the so-called selectivity filter. this channel is able to permeate k + at high flux rate and it is blocked by na + (physiological blocker). fluorescence, circular dichroism and fourier transform infrared experiments carried out in our laboratory demonstrated that k + and na + are able to bind to kcsa in a competitive manner. this binding is indeed associated with channel conformational changes, which seem to be related to the permeation and blockade processes. to further investigate this phenomenon we carried out a detailed study of chemical and thermal denaturation of wild-type and mutant kcsa channels. these two types of experiments were in agreement and indicate that both cations are able to stabilize the channel through conformational changes, being k + the more efficient one, even more when lipids are present. particularly, mutant channels with a structurally altered selectivity filter show that ion and lipid-induced global conformational changes are intimately associated to the conformation of this selectivity filter (conductive and non-conductive forms). modulation of the voltage-gated sodium channel nav . by rcssii, a toxin from the scorpion centruroides suffusus c. picco , g. corzo , l. d. possani , g. prestipino institute of biophysics, cnr, genova, italy, instituto de biotecnologia, unam, cuernavaca, mexico the main cardiac voltage-gating sodium channel, na v . , generates the fast depolarization of the cardiac action potential and plays a key role in cardiac conduction. its importance for normal cardiac function has been exemplified by the description of numerous naturally occurring genetic variants of the gene scn a, which encodes na v . , that are linked to various cardiac deseases. subsequently, studies of this channel localization have led to its identification in immature and denervated skeletal muscle and in the brain neurons. in our effort to identify high affinity ligands for this channel, we have investigated the effects of the recombinant cssii (rc-ssii), a four disulfide-bridged scorpion toxin isolated from the venom of the scorpion centruroides suffusus. human cardiac sodium channel α subunit scn a was expressed in cho cells and macroscopic na + currents were recorded with patchclamp technique in whole cell configuration. the electrophysiological experiments have highlighted a strong affinity for the channel at low nanomolar concentration. compared with control conditions, rcssii toxin affects in reversible way the kinetics of activation and inactivation and marked decrease the peak na + influx. the extrusion mechanism of substrates in rnd family efflux pumps: a molecular dynamics study a. v the rnd transporters of the acrab-tolc (e.coli) and mexab-oprm (p.aeruginosa) systems are able to export structurally and chemically different substrates outside bacteria through the membrane, being responsible of multidrug resistance. on the basis of crystallographic information, an extrusion process conceived as a three-cyclic peristaltic pumping has been proposed, but further microscopically well-funded investigations are needed to understand the mechanism. using different computational methods like adaptive bias force (abf) and targeted molecular dynamics (tmd), we have investigated the mechanism of substrate uptake and pumping at a molecular level. with the first method we have investigated the passage of antibiotics from the periplasm into the internal pore of the pump, while tmd has been used to assess the effect of conformational changes on the extrusion of drugs (which have been located into one of the proposed binding pockets). comparison between the active pumps acrb and mexb (which show different resistance patterns despite their homology) provide insights into the microscopic details of their functioning. in arabidopsis thaliana there are twenty genes, grouped into three subfamilies, encoding for homologues of animal ionotropic glutamate receptors (iglrs). each protein displays a pore-forming loop, flanked by two conserved helices (plus a third c-terminal helix), a glutamate-binding domain and an n-terminal region. through pharmacological and/or genetic approaches, many physiological functions have been attributed to plant glurs, such as the regulation of cytosolic calcium, photomorphogenesis, water balance and carbon/nitrogen sensing and assimilation. according to the endosymbiotic theory, the cyanobacteria are considered to represent the precursors of the present chloroplasts. the first prokaryotic glutamate receptor (glur ) was identified in the cyanobacterium synechocystis. the putative products of the atglr . and atglr . genes display a possible targeting sequence for chloroplast location and show a high degree of homology with glur . using specific antibodies and confocal microscopy, we have localized the two members of the atglr subgroup , glr . and glr . (splicing variant) , to the chloroplast in arabidopsis and to the inner envelope membrane in spinach. electrophysiological experiments indicate the presence of an activity which is compatible with that of glutamate receptors. furthermore, oxygen evolution measurements suggest that chloroplast-located glutamate receptors may play a role in the regulation of photosynthesis. under extreme conditions many cells control their volume responding to osmotic challenges by unloading or loading solutes to recover their original volume. a faster volume regulatory role triggered by membrane tension has been disclosed for aquaporins in kidney proximal tubule cells, where aqp is the main water channel, using isolated brush border membranes. in conventional osmotic studies in animal cells it is common to disregard internal hydrostatic pressures because they are insignificant compared to osmotic forces. however, by using low osmolarity buffers in small radii vesicles, we detected a rise on the internal pressure that creates surface tension and causes membrane stress, with a negative outcome on aquaporin water permeability. these findings suggested a mechanism for volume regulation in kidney proximal tubule epithelia where massive solute and fluid transport occurs. to further explore aquaporin regulation by membrane tension, yeast cells were used as a model that could bare surface tension some orders of magnitude higher than animal cells due to the existence of a cell wall. the effect of increasing levels of membrane tension on yeast water channel activity was evaluated. an impairment of aquaporin activity correlated with the increase of membrane tension corroborates the volume regulatory role of aquaporin in different cells. deuterium isotope effects on fast gating of the chloride channel clc- g. zifarelli, a. r. murgia, p. soliani, p. michael istituto di biofisica, cnr, via de marini, , i- genova, italy gating of the torpedo cl − channel clc- is modulated by intracellular and extracellular ph, but the mechanism responsible for this regulation has remained so far elusive. using inside-out patch clamp measurements we studied the dependence of the fast gate on ph int and [cl − ] int . only the closing rate, but not the opening rate showed a strong dependence on these intracellular factors. using mutagenesis we excluded several candidate residues as mediators of the ph int dependence. we propose a model in which a proton generated by the dissociation of an intrapore water molecule protonates e leading to channel opening. deuterium isotope effects confirm that proton transfer is rate limiting for gate opening and that channel closure depends mostly on [oh − ]. the model is in natural agreement with the finding that only the closing rate constant, but not the opening rate constant, depends on ph int and [cl − ] int . deletion of the c-terminus destabilizes phosphorylated na/k pump state containing na ions n. vedovato, d. c. gadsby the rockefeller university, new york, u.s.a. the na/k pump's extended c-terminus (compared to the serca ca pump's) links transmembrane helices, and its truncation lowers cytoplasmic na affinity for forming the occluded e p(na ) state. here we test the effects of c-terminal truncations on interactions with external na. we deleted the last (yy) or (kesyy) residues in xenopus α β pumps made ouabain resistant by mutations q r-n d (rd) or c y (c-y), and then used two-microelectrode voltageclamp recording in xenopus oocytes to measure pump currents as mm ouabain-sensitive currents while endogenous na/k pumps were silenced with µm ouabain. inhibition by external na of steady outward pump current ([k] o = mm) at large negative voltages was somewhat weaker in both rd and c-y pumps than in wt pumps, but was severely impaired in all c-terminal truncated pumps. consistent with this, the voltage dependence of transient charge movements under na/na exchange conditions ([k] o = mm) was strongly shifted to more negative potentials in the truncated pumps relative to the parent rd or c-y pumps, shifts comparable to those seen in wt pumps on decreasing [na] o several-fold. together, the results suggest that these c-terminal deletions lower the apparent affinity for external na ions to bind and become occluded in the na/k pump. the c-terminus therefore provides contacts important for stabilizing the occluded e p(na ) conformation, regardless of the route of na ion entry into the binding pocket. muscle contraction is driven by molecular motors that adapt their energy utilization according to the demands made on them. we test the hypothesis that rate constants controlling the biochemical steps involved in atp hydrolysis by myosin atpase are affected by the force of the muscle. here we use fluorescence lifetime imaging microscopy (flim) of a fluorescently labelled atp analogue to investigate changes in the environment of the myosin atpase, caused by different loads applied to skeletal muscle. single muscle fibres were subjected to cycles of stretches and releases in the presence of rigor solution and µm of coumarin-labelled atp. flim acquisition was synchronised with stretch/release cycles and force measurements, which allow us to investigate the effect of strain on the lifetime of the labelled atp bound to the actomyosin complex. characterization of the fluorescence decay by a bi-exponential function resolved the time constant of two populations, namely, free fluorophore (τ = . ± . ns; mean ± s.d.) and fluorescent nucleotide bound to the actomyosin complex (τ = . ± . ns at low strain). these experiments showed that while the time constant of the free fluorophore did not change with force, the time constant of the fluorescent nucleotide bound to actomyosin showed a linear dependence with the force applied to the muscle of . ± . ps/kpa. neck linker docking coordinates the kinetics of kinesin´s heads i. derenyi, a. czovek, g. j. szollosi department of biological physics, eotvos university, pazmany p. stny. a, h- budapest, hungary conventional kinesin is a two-headed motor protein, which is able to walk along microtubules processively by hydrolyzing atp. its neck linkers, which connect the two motor domains and can undergo a docking/undocking transition, are widely believed to play the key role in the coordination of the chemical cycles of the two motor domains and, consequently, in force production and directional stepping. although many experiments, often complemented with partial kinetic modeling of specific pathways, support this idea, the ultimate test of the viability of this hypothesis requires the construction of a complete kinetic model. considering the two neck linkers as entropic springs that are allowed to dock to their head domains and incorporating only the few most relevant kinetic and structural properties of the individual heads, we have developed the first detailed, thermodynamically consistent model of kinesin that can (i) explain the cooperation of the heads during walking and (ii) reproduce much of the available experimental data (speed, dwell time distribution, randomness, processivity, hydrolysis rate, etc.) under a wide range of conditions (nucleotide concentrations, loading force, neck linker length and composition, etc.). besides revealing the mechanism by which kinesin operates, our model also makes it possible to look into the experimentally inaccessible details of the mechanochemical cycle and predict how certain changes in the protein affect its motion. the positive role of noise on the transport efficiency of na, k atpase c.-h. chang , t. y. tsong institute of physics, national chiao tung university & physics division, national center for theoretical sciences, hsinchu, taiwan, institute of physics, academy of sciences, taipei , taiwan na, k atpase is a molecular motor which is able to transport ions through cell membranes, even against the transmembrane ion concentration gradient. while in vivo this nanoscale soft machine consumes atp, it may be driven by external fluctuating electric fields, no matter they are periodic or random. theoretically, the motor conformations can be described by a conformation vector v(t) governed by a multi-dimensional kinetic equation. given an oscillating electric field with a slight fluctuation, the boltzmann distributions of these conformations will change with time. the instantaneous transported ion flux is a functional of the quasi-cyclic trajectory v(t) of this non-autonomous dynamical system. various interesting dynamical properties of this ion pump, including stochastic resonance, can be studied theoretically, some of which have good agreement with recent experimental findings. in situ measurements of the molecular motor of muscle with nanometer-microsecond resolution in a contracting muscle, arrays of the dimeric motor protein myosin ii pull the actin filament towards the centre of the sarcomere during cyclical atp driven interactions. when the external load is smaller than the array force, the sarcomere works as a motor, converting metabolic energy into mechanical work; when the external load is larger than the array force, the sarcomere acts as a brake resisting the load with reduced metabolic cost. to investigate the molecular basis of the work production and the braking action of muscle, we use sarcomere-level mechanics and x-ray interferometry in intact single cells from frog skeletal muscle. during isometric contraction, each motor bears a force of about pn. during shortening against high and moderate loads, the number of myosin motors attached to actin reduces in proportion to the external load while the force per attached motor is maintained similar to the isometric value (piazzesi et al., cell , - , ) . rapid stretches of - nm between each overlapping set of myosin and actin filaments in a muscle sarcomere cause the stiffness of the array of myosin motors to increase up to twice the isometric value within ms (brunello et al., pnas usa , - , ) , indicating that the high resistance of active muscle to stretch is due to recruitment of the second motor domain of the myosin molecules with the first domain already attached to actin. supported by miur and ente crf (italy), nih (usa), mrc (uk), embl, esrf. kinesin- is a molecular motor that moves cellular cargo along microtubules. its functional mechanism is well understood for individual motors. however, the way that many kinesin- motor proteins bound to the same cargo move together is not. we addressed the structural basis for this phenomenon using video microscopy of single microtubulebound full-length motors and various spectroscopy methods were employed to study synthetic peptides derived from hinge- region. these peptides show an unexpected profile of secondary structure forming propensities. video microscopy of single microtubule-bound full-length motors reveal the sporadic occurrence of high compliance states alternating with longer-lived, low compliance states. the deletion of hinge- abolishes transitions to the high compliance state. from the results we hypothesize that strain accumulated during multiple kinesin motility populates the high compliance state by unfolding helical secondary structure in the central hinge domain flanked by unordered regions, thereby preventing the motors from interfering with each other in multiple motor situations. titin is a giant protein of vertebrate skeletal and cardiac muscles. cardiac titin is expressed in two main isoforms: short n b (∼ kda) and long n ba (∼ kda). we have studied changes of titin isoform composition in myocardium of hibernating ground squirrels and spontaneously hypertensive rats (shr). using electrophoresis we have revealed considerable decrease (by - times) in the content of titin relative to myosin heavy chains in shr heart as compared with that for normotensive rats. surprisingly that the data of qrt-pcr showed the increase in mrna content of n ba and n b-isoforms in hypertrophic heart more than times in comparison with norm. we suppose that such a result is an effect of depressed translation of mrnatitin in pathology. we have observed the decrease (by , times) of total titin amount in heart of hibernating animals in comparison with that for summer active animals. however n ba/n b ratio in the heart upon hibernation was increased by times. similar trend was not revealed for the mrna level of corresponding isoforms, although we have showed the decrease of mrna of both titin isoforms in heart of hibernating ground squirrels as compared to their content of summer animals. the decrease in total mrna level may be explained by repressed transcription or mrna degradation in the cell during hibernation. these discrepancies in protein and mrna levels may be considered as the posttranscriptional regulation of titin isoforms expression. actomyosin cross-bridges formed when the globular heads of myosins bind to actin filaments are the molecular engines that drive muscle contraction, fuelled by atp hydrolysis. critical to this process is the change in shape of the cross-bridge and the change in the interactions with actin, in response to force applied to the muscle, and to the status of the nucleotide in the binding pocket. although molecular detail is known from x-ray crystallography and biochemistry, understanding of the interplay between cross-bridge shape and chemical state requires studies in muscle fibres generating force. we use fluorescence life time imaging microscopy (flim) as a probe of the cross-bridge environment.with a fluorescent analogue of atp , fluorescence life-time (flt) changes when the crossbridge binds to actin. now, we show preliminary experiments on the effect of force on flt. the essential light chain of myosin (elc) is a ∼ kda peptide that wraps around a nmlong α-helix of the myosin cross-bridge known as the lever arm which tilts during force generation. using a recombinant elc, labelled with a fluorophore at a strategic cys, we replace the native elc and introduce the fluorescent elc in muscle fibres. preliminary experiments demonstrate that the elc fluorophore also is sensitive to force applied to the muscle fibre. in addition, förster resonance energy transfer occurs between the nucleotide and elc fluorophores, opening the way for studying structural changes in cross-bridges during force generation by fret. muscle contraction: pitfalls in the determination of the contractile response e. grazi dipartimento di biochimica e biologia molecolare, università di ferrara, ferrara, italy the contractile response of an active muscle depends on the load. the load is a force /cross-section. there are three fundamental dimensions: the mass, m; the space, l; and the time, t. from these three dimensions are built up all the physical dimensions. as an example the acceleration, a, is given by, a=l.t − . once the direction and versus are settled the modulus fully defines the physical effect of the acceleration. what about the force? the force, f, is given by, f=m.a. at variance with the acceleration, once the direction and versus are settled, the modulus does not define the physical and the biological effects of the force: the same force is generated by an infinite number of mass-acceleration couples that display different physical and biological effects. the same occurs with the load. thus defining the load that opposes the contractile force does not define the contractile system. in the studies on muscle contraction the acceleration of the load is not considered nor it is provided a way to extract this information. thus these systems are poorly defined from the physical as well as from the biological point of view. models of muscle contraction that consider explicitly both the mass and the acceleration of the load show that, at the same load, the decrease of the acceleration of the load significantly delays the pre-steady state of the contraction and decreases the stiffness of the active fibre. r. shahapure , f. difato , a. laio , d. cojoc , e. ferrari , j. laishram , g. bisson , v. torre int. school for advanced studies, trieste, italy, iit-sissa unit, trieste, italy, lab. nazionale tasc, trieste, italy polymerization of actin filaments is the main source of motility in lamellipodia and is controlled by many regulatory proteins. the underlying molecular mechanisms are only partially understood and now a determination of the dynamical properties of force generation is needed. using optical tweezers we measured with millisecond temporal resolution and pn sensitivity the force-velocity (fv) relationship and the power dissipated by lamellipodia of dorsal root ganglia neurons. when force and velocity are averaged over - s, fv relationships can be flat. on a finer time scale, random occurrence of fast growths and sub-second retractions become predominant. maximal power dissipated by lamellipodia over a silica bead with a diameter of µm is − w. due to the presence of adhesion forces, beads in close contact with a lamellipodium can seal on its membrane reducing the amplitude of brownian fluctuations often by more than times. under these conditions, when lamellipodia grow and push the beads, discrete jumps varying from about to nm are detected. when lamellipodia retract, pulling the beads, no discrete events are observed. our results on the dynamical properties of force generation are: a) force generation is a probabilistic process; b) underlying biological events have a bandwidth up to at least hz; c) fast growths of lamellipodia leading edge alternate with local retractions; d) force generation is produced in discrete steps with varying amplitude up to . pn. pushing on microtubules: dominant spindle centering mechanism in c. elegans embryo? j. pecreaux, s. redemann, a. a. hyman, j. howard mpi-cbg, pfotenhauerstr , dresden, germany asymmetric cell division, where the content of the two daughter cells -as well as their sizes -differ, is found in many organisms. strikingly, the spindle, first centered, starts to be displaced out of the center only in late metaphase. in c elegans embryo, the spindle rocks and is posteriorly displaced during anaphase by force generators asymmetrically localized on cell cortex. prior to anaphase onset, the spindle is usually assumed to be centered by the same pulling force. it thus requires the force generators to be carefully repressed to distribute forces symmetrically. on live embryos, we measured positional fluctuations of centrosomes during metaphase with nm accuracy. fourier analysis shows an extremely accurate centering respect to the number of force generators and microtubules. furthermore, spectrum is close to a lorentzian, modeled by a spring and a dashpot, suggesting a spindle centering more likely by pushing on microtubules than pulling. deviation at high frequencies indicates a subdominant pulling force. rnai of gpr- / , known to control force generation, increases slightly centering accuracy; this result supports the hypothesis of an independent centering mechanism. conversely, zyg- (rnai), a microtubule growing factor, decreases centering accuracy, modeled spring stiffness and damping modulus. conclusion: first, the spindle centering mechanism is independent of cortical pulling force generator. second, microtubules pushing is likely to center the spindle. mechanical forces are important in the regulation of cellular adhesion and migration. the focal adhesion kinase (fak) has been suggested to transduce cellular forces and govern cell migration. to obtain more insight in the functioning of fak, a fret-based optical biosensor for fak was designed to relate integrin-mediated conformational changes in its ferm domain to focal adhesion behavior during cell spreading and migration in living cells. imaging of the kinetics of ferm-based fak conformational changes in spreading cells revealed two consecutive stages of focal adhesion activation. heterogeneous ferm conformational responses were observed in individual focal adhesions of adherent motile cells, with the active ferm conformation being enriched in growing and sliding fas, but not in stable and shrinking focal adhesions. inhibition of the cellular actomyosin system revealed the involvement of rho-rock rather than mlckinduced tension signaling in the modulation of the ferm response. our results place the ferm conformational change of fak at the interface between integrin and force sensing. the time course of inorganic phosphate release in permeabilized cardiac trabeculae of the rat c. mansfield, t. west, m. a. ferenczi imperial college london, u.k. the rate of p i release was determined in permeabilized rat trabeculae. contraction was elicited at • c by laser-flash photolysis of npe-caged atp, and time-resolved p i release was monitored using mdcc-pbp, a coumarin-labelled phosphate binding protein, which increases its fluorescence intensity five-fold upon p i binding. the atpase rate during the first turnover of the total crossbridges (assuming µm myosin heads) was s − . the rate decreased to a steady state of s − after the eighth turnover ( . - . s after activation). this steady state rate is comparable to published values of - s − , made ∼ s after activation using an nadh-linked enzyme assay of adp release. the advantage of using mdcc-pbp is that the control of mechanochemical coupling can be examined from the onset of force production and as it progresses toward the steady state. force production and p i release were simulated using a seven step scheme. force was attributed to the states in the sequence a.m.adp.p i ↔ a.m.adp ↔ a.m.adp, with strain sensitivity incorporated into the isomerisation of a.m.adp. the a.m.adp.p i and a.m.adp states populated rapidly as force was increasing. in contrast, the a.m.adp state accumulated slowly after the force plateau was reached and became the dominant force bearing state at the time of the eighth crossbridge turnover. experiments are on-going to examine how the distribution of a.m states changes in response to rapid length-changes. pulling as a factor in forming the heterophasic structure of immunoglobulin proteins structure of proteins of immunoglobulin superfamily: human igg kuc and muscle protein titin, has been investigated by methods of electron microscopy and diffraction with the use synchrotron radiation. super elasticity of titin, the protein of immunoglobulin superfamily, is a key parameter that determines the mechanical properties of muscle. however, the structural-physical mechanism of titin elasticity under tension remains poorly understood. here both tension transduction and high elasticity of titin are explained in terms of crystalline polymer physics. x-ray data suggest a model of titin as a nanoscale, morphological, aperiodical array of rigid ig-and fn -type domains covalently-connected by conformationally variable short loops. the line group symmetry of the model can be defined as s m with axial translation τ ∞ . homologous domains would have similar stability, but the structure of different domains on stretching is subject to different forces because they have different orientations relative to the axis of the molecule. under the force influence the structure of any domain can become either rigid or flexible depending on its orientation in the titin strand. pulling geometry forms an active axial structure from latent isotropic random coil structure of titin strand. we are suddenly faced with nanophase-separated morphology of igg kuc. study was supported by rfbr grant - - . strain response of myosin essential light chain in permeabilized skeletal muscle fibres d. s. ushakov, d. ibanez-garcia, t. g. west, p. m. w. french, m. a. ferenczi imperial college london, uk we applied fluorescence lifetime imaging microscopy (flim) to investigate the relation between conformation of myosin head and mechanical force in skeletal muscle fibres. recombinant myosin essential light chain (elc) was expressed in e.coli and labelled at cys- with coumarin. the labelled elc was exchanged with native elc in single permeabilized rabbit m.psoas fibres. fluorescence lifetime was measured using leica sp upright confocal microscope equipped with becker & hickl time-correlated single photon counting module and x . na leica planapo dipping objective, with the two-photon fluorescence excitation at nm by sapphire pulsed laser. after acquiring flim images of muscle fibres in relaxed state, the solution was changed to ca-free rigor. further images were acquired in rigor with or without . - % stretch applied by a motor. both single and double exponential fluorescence decay analysis showed that the lifetime in rigor was lower compared to relaxed (about ps difference for single exponential fit) and to rigor fibres under strain (about ps). these data suggest a change in the microenvironment of coumarin induced by nucleotide binding and strain. this change is likely to be due to interaction between c-terminal domain of elc and the n-terminal domain of myosin heavy chain related to the lever arm re-orientation process. supported by bbsrc. alpha-synuclein and its a p mutant affects the actin cytoskeleton structure and dynamics v. sousa , s. bellani , g. ronzitti , f. valtorta , j. meldolesi , e. chieregatti department of neuroscience, hsr, milano, italy, department of neuroscience, iit, genova, italy alpha-synuclein (syn) is a soluble protein abundant in the brain, primarily enriched at pre-synapses. syn overexpression and the expression of its a p mutant participate in the pathogenesis of parkinson's disease. many roles have been proposed for syn, including the regulation of synaptic vesicle pools and of neurotransmitter release. the actin cytoskeleton regulates many aspects of synaptic function and its dysregulation may be a cause of neurodegeneration. working both in cell-free and in vivo conditions we demonstrate that syn and the a p mutant have different effects on the actin cytoskeleton dynamics. our results show that syn binds actin, and decreases actin polymerization rate probably by monomer sequestration. on the contrary, a p accelerates actin polymerization in vitro and disrupts the cytoskeleton of intact cells. in particular, during dynamic cytoskeleton remodeling, a p induces the assembly of discrete actin-rich foci. actin trapping and the impairment of filaments reassembly lead to inhibition of cell movement and of the re-establishment of cell-cell contacts. in a p expressing cells cytoskeleton-based processes, such as cell migration and the exo/endocytic traffic are inhibited. elucidating the dynamics of syn interaction with actin may contribute to the understanding of its role in neuronal physiology as well as in neurodegeneration. on the physics of muscle contraction m. l. shur ural state university, yekaterinburg, russian federation whichever energy source is chosen as an engine, its force will decrease with increasing velocity. this is connected with a limited power of any engine. thus, we state that hill's formula is a mere sequence of the law of energy conservation. to derive a mathematical dependence "force-velocity", all the means of consumption of fuel energy should be determined -in our case, the energy of the atp hydrolyze. moreover, the conformation energy of the crossbridges attached serves as the force source as well. we state that part of the energy release transforms into the energy of oscillations of myosin proteins; the other part goes into thermal energy of the sarcoplasmic solution. interaction of the oscillating myosin system with the sarcoplasmic solution controls the process of force generation by a muscle. it is just this interaction that leads to the temperature dependence of force. the presentation is devoted to constructing a theory based on these simple considerations. a discussion and constructive critic is especially wanted. -biological motility and molecular motors - meiotic nuclear oscillations in the fission yeast schizosaccharomyces pombe are crucial for proper chromosome pairing and recombination. we report a mechanism of these oscillations based on collective behavior of dynein motors linking the cell cortex and dynamic microtubules that extend from the spindle pole body in opposite directions. by combining quantitative live cell imaging and laser ablation with a theoretical description, we show that dynein dynamically redistributes in the cell in response to load forces, resulting in more dynein attached to the leading than to the trailing microtubules. the redistribution of motors introduces an asymmetry of motor forces pulling in opposite directions, leading to the generation of oscillations. our work provides the first direct in vivo observation of self-organized dynamic dynein distributions, which, due to the intrinsic motor properties, generate regular large-scale movements in the cell ( ) m. versaevel, s. gabriele, p. damman university mons-hainaut, mons, belgium the remodeling of blood vessels in response to changes in blood flow is mainly realized by endothelial cells (ecs) that convert mechanical stimuli from flowing blood into changes in cell signaling through a process called mechanotransduction. many of the biological responses to external forces originate at two types of microscale structures: focal adhesions linking cells to their extracellular matrix and adherens junctions that link adjacent cells. this study aims to elucidate the role of the cytoskeleton, cell-matrix and cell-cell junctions in transducing fluid shear stress into intracellular signals in ecs. by using microcontact printing of proteins, we design substrates with defined adhesive islands in order to control shapes of living cells. this confinement of ecs allows to study the organization and the contractile activity of the cytoskeleton in order to redistribute their intracellular forces in response to externally applied forces. we design microfluidic channels with sizes and geometries close to small blood vessels to apply a physiological range of shear stresses on ecs. our results indicate that cells deposited on a precisely defined adhesive area inside microchannels and subjected to shear stress reorganize their cytoskeleton, their focal adhesions and adherens junctions in response to blood flow. drugs interfering with the cytoskeleton are used to underline the role of its different components in the cellular adaptation to the mechanical environment. s. mahdavi , b. ranjbar , s. gharibzadeh , m. toosi , m. javan tarbiat modares university, tehran, iran, amirkabir university of technology, tehran, iran multiple sclerosis (ms) is the main known pathology of myelinating cells. an autoimmune reaction occurs against myelin sheets of neurons, so action potential (ap) propagation along the affected nerve fibers has been destroyed and it causes various disorders. here, we propose a novel strategy for ms symptoms treatment. we modeled neuron by orcad software and simulated the action potential propagation along the axon in normal condition. our model simulated normal neuronal behavior. then we destroyed the myelin sheet as it occurs in ms and observed destroyed ap propagation as it was reported in ms disease. we investigated the effect of changes in the voltage-gated sodium channel (vgsc) threshold on the efficiency of ap propagation. the results demonstrated that reduction of vgsc threshold improves the propagation of ap by increasing the amount of sodium flux during ap propagation. although, some researches have proposed vgsc blocker as ms symptom treatment, our result suggests that the increase of sodium current produced by reduction of vgsc threshold, improves ap propagation and probably cure some ms symptoms. so, we suggest that vgsc gating modifiers can be considered as novel strategy for ms treatment. surely, this results needs to be confirmed by experimental studies. a. gradogna, e. babini, a. picollo, m. pusch istituto di biofisica, consiglio nazionale delle ricerche, via de marini , genova, italy clc-ka and clc-kb are highly homologous cl − channels expressed in the kidney and the inner ear where they mediate transepithelial chloride transport. both channels heteromerize with the beta subunit barttin. mutations in clc-kb and barttin genes lead to bartter's syndrome. we analyzed the modulatory effect of extracellular ca + and h + on clc-k channels using the xenopus oocyte expression system. clc-ka currents increased with increasing [ca + ] ext without full saturation for [ca + ] ext up to mm. however, in the virtual absence of ca + , clc-ka currents are about % of currents measured in mm [ca + ] ext , demonstrating that ca + is not strictly essential for opening. vice versa, clc-ka was blocked by increasing the [h] + ext with an almost complete block at ph . among various reaction models tested, the model that best fitted all state-steady data predicts an allosteric regulation of channel opening by separate binding sites for ca + and h + . moreover, the best fit suggests that one ca + and two h + bind to the channel. kinetic analysis of current responses upon [ca + ] ext and ph jumps confirmed the allosteric character of modulation. in support of the presence of two separate binding sites we identified several mutations that selectively altered ca + or h + sensitivity. our data represent a first step towards a molecular picture of ca + and proton regulation of clc-k channels and suggest that it is of physiological relevance. the extracellular matrix molecule hyaluronic acid modulates l-type voltage-dependent ca + channels e. dvoretskova , g. kochlamazashvili , o. bukalo , c. henneberger , d. rusakov , m. schachner , a. dityatev italian institute of technology, genova, italy, centre for molecular neurobiology, hamburg, germany, institute of neurology, university college london, london, uk we studied the effects of hyaluronic acid (ha), a major extracellular matrix molecule, on activity of l-vdccs in a heterologous expression system and in hippocampal slices. we recorded currents mediated by a major neuronal subtype of l-vdccs (ca v . c, β b, and α δ ) expressed in cho cells. a five-minute application of . mg/ml ha potentiated l-vdcc currents at - , - , and + mv by approximately %. analysis of boltzmann curves showed that ha increased maximal conductance rather than other parameters (v . or k ). treatment with hyaluronidase removed endogenous ha in murine hippocampal slices and specifically impaired long-term potentiation (ltp) induced at ca -ca synapses by repetitive theta-burst stimulation. blockade of l-vdccs reduced ltp in control slices to the levels seen after hyaluronidase treatment. a potentiation of l-vdccs with bay k fully restored ltp after hyaluronidase treatment. removal of ha reduced ca + transients elicited by backpropagating action potentials in individual dendritic shafts and spines of ca pyramidal cells, whereas pretreatment with nifedipine fully occluded this effect. thus, ha potentiates postsynaptic l-vdccs and by this way influences use-dependent synaptic plasticity. whole-cell patch clamp recordings from a variety of human cancer cells showed that functional voltage-gated sodium channel (vgsc) expression occurred specifically with strongly metastatic cells. in addition, where studied, this was accompanied by down-regulation of outward (mainly potassium) currents. this has led to the celex ("cellular excitability") hypothesis of cancer according to which metastatic cell membranes are excitable and this promotes their hyperactivity. importantly, the vgsc genes expressed are embryonic splice variants, which are normally developmentally regulated, hence the phenomenon is 'oncofetal'. in breast cancer, where the predominant vgsc is nav . the neonatal and adult forms are significantly different and this is reflected in channel activity whereby the neonatal vgsc has much slower inactivation kinetics. the double-charge change at position is critical for this difference. the slow kinetics results in much greater influx of na + into cells and one consequence of this is activation of protein kinase a. this is a tonic effect and, under steady-state resting conditions, it results in a positive feedback effect promoting post-translational trafficking of vgsc protein to plasma membrane. the unique amino acid sequence of the spliced region has enabled the production of a polyclonal blocking antibody specific to neonatal nav . . it is concluded that vgscs represent novel biophysical targets for clinical management of metastatic disease. m. pusch cnr, istituto di biofisica, genoa, italy clc proteins form an evolutionary conserved gene-family that comprises members in mammals. four of the human clcs are passive plasma membrane cl − ion channels. the other clcs are expressed in intracellular organelles. clc- and clc- , mutations of which lead to dent's disease, are secondary active cl − /h + antiporters, similar to the bacterial clc-ec , and with identical cl − : h + stoichiometry. cl − and h + transport activity of the exchanger clcs depends on two glut residues. mutating a 'gating glutamate' (e in clc- ) converts the exchanger into anion conductances. neutralizing the 'proton glutamate' (e ), but not its replacement by some other titratable groups, abolishes cl − and h + transport. noise analysis indicated that clc- switches between silent and transporting states with an apparent unitary conductance of . ps, indicating a very large transport turnover. no − uncouples h + transport but mutating the highly conserved s to p, as found in the plant no − / h + antiporter atclca, led to coupled no − : h + exchange. clc proteins are a fascinating example of how a very similar protein architecture can be used to provide either a passive electrodiffusive permeation pathway or a strictly coupled secondary active ion transporter. (supported by telethon italy -grant ggp ). the role of ion dynamics in zebrafish fin regeneration the specific and directional ion transport across cell membranes or tissue layers results in differential accumulation of ions and endogenous electric currents. these phenomena have been shown to be important for vertebrate organs regeneration. however, the specific ion nature of such electric currents remains unknown, as well as the role of cellular ion dynamics during regeneration and the molecular signalling pathway that transduces electric cues into cellular responses. we use zebrafish caudal fin as an adult regeneration model to unveil the specific ion composition of the currents associated with wound healing and regeneration, using a non-invasive ion-specific scanning microprobe setup. our data suggests a role for potassium (k + ), calcium (ca + ) and protons (h + ) at different stages of the regeneration process. k + and ca + extracellular effluxes have both been detected during the wound healing stage. h + efflux is triggered during wound healing and is maintained throughout regeneration. we are validating these data with genetic and pharmacological approaches, as well as advanced ion imaging. overall, our results suggest ion-driven mechanisms underlie adult tissue regeneration and its comprehension may open way for new therapeutic strategies, both in regenerative and developmental medicine and in cancer therapy. cardiac effects of anabolic steroids: an electrophysiological approach anabolic androgenic steroids (aas) have been used by athletes and non athletes for almost five decades in order to improve performance. however, the illicit abuse of high-doses of aas has been attributed as a main cause of several cardiovascular disorders such as arterial hypertension, lipid profile abnormalities, heart failure, hypertrophic cardiomyopathy, arrhythmia and sudden death. the aim of this study was to investigate qt interval and transient outward potassium current (i to ) changes in rats treated with nandrolone decanoate (deca). male wistar rats received weekly mg/kg of deca (n= ) or vehicle (control, n= ). electrocardiogram was recorded weekly, and qt interval was measured. after weeks hearts were excised and single myocytes were isolated from the ventricles of animals. i to was recorded by means of the whole cell patch clamp technique. qt interval was larger in deca group from th to th week (p < . ). analysis of i to showed a decreased current density (p < . ) in ventricular cardiomyocytes of deca group, compared to control group. in conclusion, our results show that alterations on ventricular repolarizaton may constitute an early consequence of the chronic administration of high doses of anabolic steroids in rats, and demonstrated qt prolongation and i to density reduction, which may constitute an important marker of arrhythmia vulnerability and sudden death. -ion channels in channelopathies and cancer - denitrifying bacteria control no and no cytosolic levels by regulating the expression of denitrification gene clusters via redox signalling of specific transcriptional factors that may act as no sensors in vivo. a protein belonging to the subclass dnr (dissimilative nitrate respiration regulator) from pseudomonas aeruginosa has been recently suggested to be a heme containing protein. very recently the three dimensional structure of the apo-form of dnr (in the absence of heme) has been determined by x-ray crystallography, whereas the holo-form (in the presence of heme) has not yet been crystallized. we have investigated the heme local structure in solution of ferric, ferrous, co bound and no bound holo-dnr by x-ray aborption spectroscopy (xas) and we added a kinetic study of the co bound form by means of a flash photolysis setup using uv-visible absorption as a spectroscopic probe. the combination of fe k-edge xanes fingerprints and kinetic study reveal a heme pocket able to bind exogenous ligands like no and co with increased plasticity, thus supporting its role as the cofactor involved in no sensing activity. molecular examination of motifs that lead to the formation of s-nitrosylated proteins i. alicea, e. r. schreiter university of puerto rico rio piedras, san juan, puerto rico physiologically, a wide range of proteins experience structural and functional modifications after the addition of a nitric oxide (no) moiety to cysteine thiol. this posttranslational modification, known as s-nitrosylation, regulates a large number of cellular processes like vasodilatation, cell signaling and others, and the products of s-nitrosylation can be involved during the development of different human diseases. however, little is known about the mechanism by which different proteins specifically bind the no moiety to their cysteine. here we show a bio-statistical analysis of some properties of cysteine that are s-nitrosylated in different proteins, including the pk, electrostatic environment, solvent accessibility of the target cysteine and identity of surrounding amino acids. we also chose model proteins (human thioredoxin and s protein) to make specific targeted amino acid substitutions around selected cysteines to alter the properties described above. the reactivity and stability of these mutant proteins towards s-nitrosylation will be examined. sampling the flexibility of ppar-γ s. aci-sèche , n. garnier , d. genest , s. bourg , c. marot , l. morin-allory , m. genest upr cnrs , orléans, france, fr pcv cnrs , orléans, france, umr cnrs , université d´orléans, france a promising approach to consider the flexibility of proteins in docking studies consists in performing multiple rigid docking on a representative set of the receptor conformations. molecular dynamic (md) simulation is one of the best adapted methods for structural sampling, but exploring the conformational diversity of a protein is computationally expensive. we present a protocol for generating a wide range of conformational states of a receptor using restrained md and a partitioning protocol to select a few representative conformations of the binding site from this md. a way to speed up efficiently md calculation is using an implicit model to represent the solute-solvent interactions. we explore a protocol using a distance-dependant permittivity function to represent solvent effect and an ensemble of controlled restraints applied on a subset of specific atoms in order to prevent artefactual structural distortions, but preserving receptor's flexibility. ten ns simulations have been performed using different sets of parameters and compared to a reference ns simulation with explicit solvent. to select a representative set of conformations, partitioning (k -means algorithm) was applied on the ensemble of simulated conformations. this methodology was applied to the ligand binding domain of peroxysome proliferator-activated receptor-γ. department of biomedical sciences, university of antwerp, antwerp, belgium the complex system of cavities identified in human neuroglobin (ngb) has been postulated to be of functional significance to the putative no dioxygenase activity of the protein. the interconnected hydrophobic cavities may support this catalytic activity by acting as reservoir for reactants and providing preferential pathways assisting product removal from the active site. we thus decided to investigate co rebinding kinetics to ngb embedded in silica gels to expose ligand migration processes in the geminate phase. encapsulation of the co complexes of reduced neuroglobin, leads to a slight increase in geminate recombination after nanosecond laser photolysis. increasing the viscosity of the medium, by soaking the gels in glycerol, completely inhibits escape of the photodissociated ligand to the solvent, and highlights a complex, multiphasic kinetic pattern. this finding can be rationalized by assuming the existence of a discrete set of temporary docking sites, capable of trapping the photodissociated ligand for very long times, up to a few ms after photolysis. g. bartolommei, f. tadini-buoninsegni, m. r. moncelli bioelectrolab -department of chemistry, university of florence, italy ion pumps are integral membrane proteins devoted to ion transport through a lipid membrane phase. the ion pumps ca-atpase and na,k-atpase are prominent members of the p-type atpases family. due to fundamental physiological roles of these proteins, they are very promising drug targets. bioelectrolab has a wide expertise in the study of ion transport by these proteins [ ] . our attention has been recently focused on the interaction of these enzymes with molecules of potential pharmacological interest. frequently, drugs exert an inhibitory action on the transport activity of an ion pump, usually confining it in an inactive conformation. molecules like thapsigargin and cyclopiazonic acid belong to high (nanom) affinity inhibitors of the ca-atpase, whereas clotrimazole and curcumin are medium (microm) affinity inhibitors of both ca-atpase and na,k-atpase. for each of these compounds a mechanism of action is proposed. moreover, recent results concerning ca-atpase inhibition by clotrimazole analogues will be shown: the relevance of this type of molecules is due to their potential employment as an alternative to traditional drugs against malaria parasite. financial support of ente cassa di risparmio di firenze and of miur (prin project) is gratefully acknowledged. [ ] tadini-buoninsegni f., bartolommei g., moncelli m.r., fendler k. . arch. biochem. biophys. : - (review). s. asthana , s. shukla , g. giliberti , f. luliano , m. ceccarelli , r. loddu , p. ruggerone , p. la colla department of biomedical science and technology, università di cagliari, cagliari, italy, department of physics, università di cagliari, cagliari, italy studies of protein-inhibitors interactions are helpful to elucidate the mode of action of ligands and thereby providing clues for rational drug design. bvdv, is an important target of drug discovery activities largely because it is essential for viral replication. in bvdv rdrp no specific nni binding site has been reported till now. experimental results have shown that different class of inhibitors (benzimidazole, imidazoquinolines and pyridoxyquinolines), have resistant mutations located in the finger domain of the rdrp. all the reported mutations are spatially very close to each other. thereby, indicating that binding sites of nni's may lie in the finger domain for these different class of inhibitors.herein, we have utilized docking procedure to investigate binding sites, binding modes as well as binding affinity of different class of inhibitors.we then used all atom molecular dynamics (md) simulations to investigate the stabilizing interaction between inhibitor-receptor pairs. our md results are in good agreement with experimental data and provide deep insights into the dynamical features of the high affinity inhibitorreceptor binding. thus identifying the binding modes of our inhibitors and mechanism leading to inactivity of the enzyme can help us to build a microscopically well-funded picture of the functioning of these enzymes. we present an investigation of the molecular basis of ligand binding and reactivity of heme proteins using computer simulation. a combination of classical molecular dynamics and hybrid quantum-classical (qm-mm) calculations are applied to explore distal and proximal effects on diatomic ligand binding to the heme. trends in binding energies and in the kinetic constants are illustrated through a number of selected examples. an investigation of the interplay between ligand migration and protein dynamics obtained through classical molecular dynamics techniques in combination with advanced sampling tools is also presented to yield information about free energy profiles and possible secondary docking sites. results for truncated n hemoglobin of mycobacterium tuberculosis, presented as an illustrative example, suggest that the truncated hemoglobin n has evolved a dual-path mechanism for selective/distinct migration of o and no to the heme, to achieve efficient no detoxification. finally, we present also an analysis of the molecular basis of hexacoordination in human neuroglobin, which suggest that the flexibility of the cd plays a key role in determining the ligand binding properties. thermodynamic bases of nucleoplasmin-histone complexes recognition by the nuclear transport machinery i. arregi, j. falces, s. bañuelos, m. a. urbaneja, s. g. taneva unidad de biofísica (csic/upv-ehu), departamento de bioquímica y biología molecular, universidad del país vasco, spain the nuclear transport of the chromatin remodeling (nucleoplasmin) and chromatin building (histones) proteins is mediated by importins. nucleoplasmin (np) contains a classical bipartite nuclear localization signal (nls) that is recognized by importin α, while histones present multiple sequence elements (nls-like motifs) for nuclear targeting. besides, ternary importin/np/histone complexes might represent a putative coimport pathway for nuclear import of linker (h ), nucleosomal core (h ah b) histones and their chaperone protein np, enhancing the histone import efficiency. to better understand np and histone recognition by the transport machinery we studied the thermodynamics of complex formation of importin α (a truncated form) and importin β with histones and np, and with np/histone binary complexes by means of isothermal titration calorimetry. data show that importins interact with the two histone types and np, and that importin and histones can simultaneously bind to np. analysis of the binding energetics reveals an enthalpy driven formation of high affinity binary and ternary complexes. we demonstrate that different amount of importin molecules can be loaded on np/binary complexes dependent on the histone type, linker or core, and the amount of the bound histones. g. breuzard, i. di maïo, p. barbier, d. allegro, c. brault, v. peyrot cro inserm u , ufr de pharmacie, marseille, france since a decade, our laboratory has already studied the interaction of tau variant with tubulin (tub) or microtubules (mts) and phosphorylation process on this interaction. indeed, fret assay was a powerful tool to achieve binding parameters between tau and tub in vitro: / with mts stabilized by fluorescein-coupled taxol (flutax- ) as donor and rhodamine-labelled tau (rho-tau) as acceptor, and / in living cells by confocal laser scanning microscopy (clsm) with tau/α-tub fused to egfp/mcherry, respectively. results revealed ± % energy transfer efficiency from flutax- to rho-tau and a donor-to-acceptor distance of ± Å. by titration, the dissociation constant of tau was determined to . ± . µm. a cleavage procedure of αβ-tub was performed to determine the influence of the c-term tails of αβ-tub on the tau-mt interaction. no difference in distances and binding parameters was observed. clsm images displayed a heightened concentration of fluorescent tau in patches along mts. fret experiments revealed in particular higher efficiencies between gfp-tau to mcherry-α tub proteins in these locations. overall, our results suggested no involvement of the hypervariable and highly acidic c-term tails of tub in mt/tau binding. a molecular model is proposed in which flutax- is directly accessible to tau molecules. besides, the modified distribution of fluorescent tau could be implicated in local change of mechanical properties of mts. a. boreham , k. winkler , c. gebhard , k. rueck-braun , p. henklein , e. michalsky , r. preissner , r. misselwitz , a. ziegler , u. alexiev freie universität berlin, berlin, germany, technische universität berlin, berlin, germany, charité-universitätsmedizin berlin, berlin, germany peptide presentation by major histocompatibility complex (mhc) molecules is crucial for immune responses. photocontrol of peptide dynamics by means of photo-switchable peptide analogs will provide insights in peptide dynamics and its dependence on mhc polymorphism ( ) ( ) ( ) . we have designed a hemithioindigo (mhti) photo-switch bearing peptide using the viral epitope rrrwrrltv (plmp ) as a template. incorporation of mhti in the peptide backbone should result only in a minor change of the overall peptide structure given the relaxed conformation of the zisomer. indeed, the human mhc molecule hla-b* can tolerate nonpeptidic elements in plmp , as evidenced by normal peptide binding. using the autofluorescent properties of mhti we determined the stability of the hla-b* /mhti-plmp complex. photoswitching from z →e results in a decrease of hla-complex stability. based on computer modeling this decrease is due to a reduced interaction of the peptide c-terminus with hla-b* . truncated hemoglobins (trhbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. three phylogenetic groups (n, o, and p) have been identified in trhbs. the crystal structure of truncated hemoglobin o of b.subtilis, does not show an evident tunnel/cavity system connecting the protein active site with the solvent, a fact that cannot be easily rationalized considering the very high oxygen association rate. moreover, resonant raman results of the co bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. for these reasons we performed classical molecular dynamics simulations of the oxy, carboxy and deoxy protein, and computed the free energy profiles associated with ligand migration to the active site. our results suggest that there is a key residue, glne , that may present an alternate conformation in which a wide ligand migration tunnel is formed, consistently with the kinetic data. the results for the co and o bound protein show also that glne is directly involved in the stabilization of the coordinated ligand, playing a similar role as tyrb , and trpg in other trhbs. our results not only reconcile the structural data with the kinetic information, but also provide additional insight about the general behaviour of trhbs. patterned functionalization of surfaces for guided transport on molecular motors tracks m. bhagawati , s. ghosh , t. surrey , j. piehler department of biophysics, university of osnabrueck, osnabrueck, germany, european molecular biology laboratory, cell biology and biophysics unit, heidelberg, germany chelator head groups with multiple nitrilotriacetic acid (nta) moieties have been very well characterized and successfully utilized as high affinity adapters for functional immobilization of oligohistidine tagged proteins to surfaces. we have recently established a generic method for patterning nta functionalized surfaces by selective photodestruction via a light induced fenton reaction. efficiency of different transition metal ions for catalyzing this reaction was tested. functionality of the patterned protein was confirmed using the interaction between interferonα and its receptor. implementation of this technique in a confocal laser scanning microscope allowed us to control surface density of binding sites, providing the possibility to vary the surface concentration of immobilized proteins in a spatially resolved manner. we also applied this approach for exploring guided transport of microtubules by kinesin selectively immobilized onto trisnta patterns. rheumatoid arthritis (ra) is an autoimmune disorder, leading to pathological damage at the level of joints, associated with the hla class ii allele hla-dr . although etiology of ra is unknown, type ii collagen (cii) is a potential antigen candidate and it is believed that t cell responses in collagendependent ra are directed towards the immunodominant pathogenic epitope cii( - ). despite recent advances in characterization of class ii major histocompatibility complex (mhc) and t-cell receptor (tcr) contacts in this epitope, the atomic details of tcr-cii( - )-mhc complex are not known. here, homology modeling and molecular docking studies have been used to derive a three-dimensional model of tcr β chains, obtained from a dr + subject, in complex with cii( - )/hla-dr . the best complex from docking was further refined using molecular dynamics simulations for ns. the proposed model represents a reasonable structural basis for understanding cii( - )-mhcii complex recognition by tcr and for rational design of inhibitors targeting tcr-pmhc interface. probing bio-molecular bonds with magnetic force for biosensor applications a. m. de jong , a. jacob , x. j. janssen , j. m. van noorloos , l. j. van ijzendoorn , m. w. prins eindhoven university of technology, eindhoven, the netherlands, philips research laboratories, eindhoven, the netherlands we investigate new technologies to be applied in next generation biosensors, which not only measure biomarker concentrations but also probe bio-molecular interactions. the concept is based on the response of ligand-receptor pairs to an applied force or torque [ , ] . we use functionalized magnetic beads and magnetic fields to apply translational and rotational forces on the molecular bonds. in a model experiment, polystyrene surfaces were coated with anti-biotin and beads were coated with biotin. after incubation, a constant magnetic force was applied to the beads and the number of bound beads was measured as a function of time. this was repeated for a range of forces. the dissociation rate (k o f f ) is determined for each force and k o f f at zero force is extracted from these data. rotational forces were exerted on protein-g coated beads bound to igg antibodies immobilized on a surface. rotating fields revealed an oscillating behavior, which can be understood from the balance between the applied magnetic torque and the torque due to the deformation of the biological bond. studying interactions between cop regulatory protein and hy transcription factor d. s. dalafave the college of new jersey, ewing, nj , usa this work addresses important questions of protein-ligand interactions and selective protein recognition. proteinligand bindings are crucial for many cellular processes. dependable methods for predicting binding sites would lead to a better understanding of proteins' selective recognition and would, in turn, help research on fighting diseases. presented here is a study of interactions between the wd domain found in a regulatory protein cop and the motif v-p-e/d-Φ-g (Φ=hydrophobic residue) found in a transcription factor hy . cop and hy proteins have opposing roles in developmental regulation. cop can repress hy by directly binding with it. the repression involves specific interactions between the wd domain and the motif v-p-e/d-Φ-g. previous experimental research showed that mutations in the motif's v-p pair resulted in a large decrease in hy repression. to study effects of similar mutations, residues in the motif v-p-e/d-Φ-g were systematically substituted with other residues. interactions between the mutated motif and the wd domain were studied. distributions of binding sites, bond lengths, local shape complementarity, and interaction potentials were modeled for each residue substitution. the study identified binding sites critical for the cop -hy binding. some hy residues in the vicinity of the motif were also found to be important in the binding. the significance of the results for understanding selective protein recognition is discussed. determination of protein-ligand binding thermodynamics by thermal shift assay p. cimmperman, a. zubriene, l. baranauskiene, e. kazlauskas, j. matuliene, d. matulis laboratory of biothermodynamics and drug design, institute of biotechnology, vilnius, lithuania thermal shift assay determines the effect of ligand binding on the protein thermal denaturation equilibrium. several models have been derived to describe the general cases of protein-ligand binding. first, the model describing protein stabilization and destabilization by ligand binding to the native and unfolded states. second, the split of protein melting transitions by tightly binding ligands is presented when the transition of free protein and ligand-bound protein occur separately. mathematical equations describing the protein unfolding and ligand binding thermodynamic parameters at various temperatures are presented. the protein melting temperature shift can be determined by various techniques such as differential scanning calorimetry, circular dichroism, and intrinsic or extrinsic fluorescence. the advantages of fluorescent techniques are presented. the melting temperature shift caused by ligand binding is dependent on the thermodynamic parameters of protein unfolding and ligand binding, including enthalpy, entropy, and heat capacity, thus allowing determination of binding thermodynamics. application of the models in the design of hsp chaperone and carbonic anhydrase inhibitors is discussed. comparison with isothermal titration calorimetry data is presented. recent reports have shown that the bacterial redox protein azurin can enter into cancer cells and induce apoptosis by stabilizing p . the formation of a complex between the two proteins has been demonstrated, but little is known about binding features. for the first time, we show here that azurin binds to the n-terminal region of p with a dissociation constant in the - µm range. trp phosphorescence lifetime measurements revealed conformational changes of azurin induced by the interaction with p ( - ). acrylamide quenching of trp phosphorescence also indicated a significant increase of the overall flexibility of azurin upon binding to p . no change of the fluorescence emission of p ( - ) was detected in the presence of azurin. the latter finding suggests that w of p is not directly involved in domain binding to azurin, indicating that the binding site is distinct from that of mdm . the present results may assist the design of novel cancer treatments based on p stabilization by azurin. a. eleta , r. georgieva , h. bäumler , j. l. toca-herrera cic biomagune, donostia-san sebastián, spain, charité-universitätsmedizin berlin, berlin, germany human serum albumin (hsa) is the most abundant nonglycosilated plasma protein in the human body. this multifunctional protein has ligand-binding and transport properties, antioxidant functions and enzymatic activity [ ] . bilirubin interacts with albumin in order to be transported from the blood to the liver where it is secreted. the interaction occurs specifically in hsa i-domain of its three domains [ ] . in our work, we investigate the interaction between hsa and bilirubin by quartz crystal microbalance with dissipation (qcm-d) and atomic force microscopy (afm) [ , ] . albumin was adsorbed on negatively charged silicon oxide. however, hsa was removed after rinsing with pbs. hsa adsorbed on positively charged polyelectrolyte multilayers leads to a stable layer of surface mass density of ng/cm . cross linked albumin with glutaraldehyde after its adsorption on nh -terminated thiols is also stable reaching surface mass density of ng/cm . a preliminary bilirubin adsorption results on cross linked hsa substrate show that ng/cm is immobilized. taking into account that hsa-bilirubin stoichiometry is : , the outcome demonstrates that the % of hsa i-domains remain active. antimicrobial peptides (amps) are short positively charged polypeptides. they are important due to their potential to provide an alternative to conventional therapy against bacterial infections. rbpi is a kda peptide based on the nterminal region of the neutrophil bactericidal/permeabilityincreasing protein (bpi). it was shown that this amp possesses bactericidal effects on gram-negative bacteria and higher affinity for lipopolysaccharide (lps), neutralizing its effect. the peptide use against meningitis, is in phase iii clinical trials. here, we demonstrate that rbpi promotes aggregation of negatively charged large unilamellar vesicles (luv) and lps aggregates, by dynamic light scattering, while for zwitterionic phosphatidylcholine (popc) luv the size remains unchanged. the aggregation increases with peptide concentration until peptide promotes massive aggregation followed by sample flocculation/precipitation. with the rbpi -lipid interaction there is a progressive change in the zeta-potential of the luv systems and lps aggregates. luv systems composed of phosphatidylglycerol (popg) and popc:popg mixtures have higher zeta-potential variations than popc luv. for lps aggregates, rbpi neutralizes the surface charge and at higher peptide concentrations overcompensates it. results demonstrate that the interaction of the peptide rbpi with lps aggregates and luv systems has electrostatic and hydrophobic contributions. serratia marcescens heme acquisition system: heme transport and protein:protein interactions m. delepierre unité de rmn des biomolécules cnrs ura , institut pasteur, paris, france heme transport systems in bacteria are required and might be potential target for antibacterial drugs. the heme acquisition system, has, exists in pathogenic as well as in opportunistic bacteria but only for the latter one extensive studies have been conducted, constituting as such a model system. the outer membrane receptor hasr, the central component of this system, functions in synergy with a secreted high affinity heme binding protein, the hemophore hasa. hasa extracts heme from host hemoproteins and returns it to hasr. then, the energy given by a protein complex of the inner membrane is used to allow heme entrance across the bacterial membrane and to eject the empty hemophore from the receptor. reconstitution of this heme acquisition system in e coli, overexpression and purification of its various components have allowed us to obtain sufficient amount of protein to perform nmr and biophysical studies to analyse at the molecular level the different steps of heme acquisition by hasr. protein-protein interactions (ppi) are the central pillar supporting most of biological functional activity on the molecular level. a binding event between two proteins typically consists of two stages: ) diffusional search of the binding partners for each other, and ) specific recognition of the compatible binding surfaces followed by the formation of the complex. we focus here on the non-specific component of ppi, which refers to all physico-chemical properties of the binding partners (such as size, charge, isoelectric point, hydrophilicity etc.) that are independent of the exact details of their binding sites, but which could in turn affect their localization or diffusional search for one another. it is known that proteins co-localize due to segregation into different cellular compartments, sequestration via anchor and scaffold proteins or even chemical modifications. we suggest that the non-specific component of ppi determines in part the co-localization and clustering of the binding partners, which then directly in a non-specific fashion influences their interactions. we examine the possibility that such signature might be encoded within the experimental d structures of a large set of known mutually interacting proteins. we provide preliminary evidence that this indeed may be the case, and corroborate our findings by using different statistical tests to compare those features of the known interacting partners, and ascertain correlations and commonalities between them. a link between hinge-bending domain motions and the temperature dependence of catalysis in ipmdh i. hajdú, a. szilágyi, j. kardos, p. závodszky institute of enzymology, hung acad sci, budapest, hungary enzyme function depends on specific conformational motions. since conformational flexibility strongly depends on temperature, temperature dependent enzyme kinetic studies with measurements related to dynamics can give us some insight at atomic level into these functionally relevant motions. the catalytic efficiency (k cat /k m ) of -isopropylmalate dehydrogenase for its substrate (ipm) has unusual temperature dependence, showing a local minimum at • c. in search of an explanation, we measured the individual constants k cat and k m,ipm as a function of temperature, and found that the van't hoff plot of k m,ipm shows a sigmoid-like transition in the - • c temperature range. by means of various measurements including h-d exchange and fret, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperature above • c. the thermodynamic parameters of ligand binding determined by itc as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. because the binding of ipm is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with ipm binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency. a simulation approach to multiple sclerosis: study of a peptide with a pharmaceutical potential c. guardiani , s. marsili , p. procacci , r. livi centro dinamiche complesse, università di firenze, italy, dipartimento di chimica, università di firenze, italy, dipartimento di fisica, università di firenze, italy multiple sclerosis (ms) is an autoimmune disease of the central nervous system, leading to premature death. one of the potential targets of the autoimmune reaction is the myelin protein mog that has been crystallized in complex with the - c ms-autoantibody. the analysis of contacts and buried surface area combined with an alanine scanning computation reveals the key role of mog fragment - for the interaction with - c . a docking simulation shows that the - fragment, excised from mog, and kept in crystal-like conformation, is still capable of fitting into the binding pocket of the antibody. we then studied, through replica exchange molecular dynamics simulations, the structural equilibrium distribution of the free peptide and of a number of analogs stabilized by a disulfide bond. we found that the free peptide yields a significant fraction of crystal-like conformations and the proportion of native-like structures is further increased by the disulfide bridge. when we tried to dock the centroids of the most populated clusters to - c , we discovered the existence of a docking funnel whose bottom is populated by stable complexes where the peptide occupies the same spatial region as in the crystal. we therefore conclude that the mog - fragment may be used to develop a diagnostic assay or a drug for ms. the escherichia coli membrane insertase yidc reversibly binds its substrate pf coat protein u. gerken, s. winterfeld, a. kuhn institute of microbiology and molecular biology, university of hohenheim, germany the membrane insertase yidc of e. coli belongs to the oxa family of mitochondria and plays an essential role in facilitating the insertion and assembly of membrane proteins. we have previously shown with detergent-solubilized (c pc) yidc, labelled with ans, and pf coat that the initial step of the membrane insertion process, the binding of the substrate pf coat to yidc, is reversible [biochemistry , - ( ) ]. the dissociation constant k d for that particular system is about µm. in order to obtain data for the native system we used in this study membrane-reconstituted (dopc and dope/dopg) yidc. the effect of the initial binding was examined in vitro by fluorescence quenching of the tryptophan (trp) residues of yidc which are highly sensitive fluorescent probes for changes of the tertiary structure. quenching of the trp fluorescence after titration with a trp-free pf mutant indicates a change in the yidcs tertiary structure upon binding to its substrate. the binding data show a k d value in the range of . - . µm. the influence of different environments (lipid membranes, ddm micelles) on the secondary structure of yidc as well as on the yidc large periplasmic domain p was investigated by circular dichroism (cd). the cd data show that the secondary structure of yidc changes upon reconstitution into a membranes when compared to the detergent solubilized state. particularly, the p domain is considerably affected by the detergent c pc. b. karasulu, b. erman, o. keskin koc university, istanbul, turkey histone proteins are fundamental to the cells since they are involved in cell regulatory processes, such as chromatin regulation, gene silencing and transcription, cell cycle control, and epigenetics, which are controlled via post-transcriptional modifications of the histone protein tails. these modifications are categorized under four main groups: methylation, acetylation, ubiquitination, and phosphorylation. among them methylation has been very recently proven to be reversible with the discovery of histone demethylase proteins and this modification type has been extensively studied, because the abnormal methylation rates cause the excess proliferation of the cell, which, in turn, triggers the cancer. therefore, understanding of the details of reaction mechanisms of histone methylation/demethylation dynamics provide the required knowledge basis for preventing the abnormal methylation rates by designing proper inhibitor (drug) molecules. in this study, we display possible reaction mechanisms (such as amine oxidation via lsd ) for the demethylation of specific histone tail proteins. we try to explain the role/importance of residues that take place in or near to the reaction pocket for the demethylation reaction. we also carry out md simulations and reaction path (free energy profile) analysis using free energy perturbation (fep) method for qm/mm hybrid systems in order to compare different possible reaction pathways. the sonic hedgehog (shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors patched (ptc) and smoothened (smo) , which lead to birth defects, cancer or neurodegenerative diseases. however, little is known about ptc, the receptor of the shh protein, and the way ptc regulates smo, the receptor responsible for the transduction of the signal. to develop structure-function studies of these receptors, we expressed human ptc (hptc) in the yeast saccharomyces cerevisiae. we demonstrated that hptc expressed in a yeast membrane fraction is able to interact with its purified ligand shh, indicating that hptc is produced in yeast in its native conformational state. using surface plasmon resonance technology, we showed that fluorinated surfactants preserve the ability of hptc to interact with its ligand after purification. this is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor ptc. this work will allow the scale-up of hptc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by shh signalling dysfunction. dissecting the colicin translocon c. l. johnson , a. solovyova , p. callow , s. a. holt , l. a. clifton , k. weiss , j. h. lakey inst. for cell and molecular biosciences, newcastle univ., uk, inst. laue langevin, grenoble, france, isis, rutherford appleton lab., didcot, uk, oak ridge national lab., centre for structural molecular biology, oak ridge, usa pore-forming colicin n hijacks e. coli outer membrane protein ompf and exploits it as both a receptor and translocator to cross the outer membrane [ ] . it is currently a matter of debate if the translocation route is through the ompf lumen or the interface between ompf and the lipid bilayer. recent electron microscopy data from our laboratory suggests the latter route for translocation [ ] . the colicin n/ompf complex in detergent has been studied by sans to examine the translocation pathway undertaken by colicin n. by using a combination of deuterated ompf and hydrogenated colicin we have been able to derive a low resolution structure of individual proteins in the binary complex. low resolution structural studies supplemented by targeted mutagenesis and screening techniques including itc, potassium efflux assays and auc have allowed us further our understanding of colicin n translocation. dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined. here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. effect of osmolytes on the dhfr activity, structure and dynamics b. legrand , s. renaud , m. collen , c. tascon , s. bonnassie , e. gautier , j. mellet , c. blanco , e. le rumeur , j.-f. hubert , a. bondon rmn-ilp, duals, cbp, umr cnrs , univ. de rennes , france osmolytes are small molecules accumulated by a wide variety of organisms in response to hyperosmotic stress. they contribute to save the cellular integrity and to stabilize the macromolecules from environmental stress. dihydrofolate reductase activity is inhibited by several osmolytes. we studied the impact of osmolytes on the dhfr structure and dynamics by various techniques. we observe that substrate (dhf) and cofactor (nadph) diffusions are quite different in glycerol and betaine despite similar viscosities. we demonstrate that the overall structure is maintained at high osmolyte concentrations while no direct interactions can be detected with the enzyme. the k o f f of substrate analogues decreases with increasing the osmolyte concentrations. the enzyme dynamics, in various media, has been compared with the dhfr behaviour in water described in the literature. the osmolyte impact appears only partly conditioned by its viscogenic properties which reduce the molecules diffusion and the k o f f of the product controlled by the m loop of the dhfr. we suggest that the osmolytes decrease m loop mobility. comparing the results obtained with different osmolytes, we offer a better understanding of the osmolyte nature dependence of the dhfr inhibition. estrogen receptor (er) is a well characterized member of the nuclear receptor superfamily that modulates the expression of estrogen-responsive target genes in response to estradiol and other natural and synthetic chemicals mimicking the estradiol structure. in human, two ers, erα and erβ, lied on two distinct chromosomes, are known. er exhibits several functional domains: two conserved domains, a short domain c involved in dna-binding and a large domain e/f responsible for ligand-binding and hormone-dependent transcription activation, are linked by a hinge domain d; a poorly conserved a/b domain, at the n terminus, mediating interactions with the general transcription machinery, is involved in hormone-independent transcription activation. upon estrogen binding, ers can specifically bind to a dna fragment, called estrogen response element ere, and activate the transcription. the optimal ere sequence consists of two six base-pair half-sites, aggtca, organized as inverted repeats with a three base-pair spacing. in this study, we have investigated, by fluorescence methods, the effect of kcl concentrations, on the protein conformational flexibility and the thermodynamic stability of hers -eres complexes. we show, here, that electrostatic interactions, inside hers, contribute to its conformational flexibility and its thermal stability. moreover, the specific interaction between hers and eres is poorly sensitive to changes in ionic strength, in opposite to unspecific complexes. kinetic and structural explanation for the low enantioselectivity of human -phosphoglycerate kinase p. lallemand , j. rouhana , l. chaloin , b. roy , s. arold , t. barman , c. lionne cpbs umr , bd henri iv cs , montpellier cedex , france, ibmm umr , cbs umr l-nucleosides comprise a new class of antiviral and anticancer agents that are converted to pharmacologically active nucleoside triphosphates in vivo. the last step of the cascade may be catalyzed by -phosphoglycerate kinase (pgk), an enzyme that has low specificity for nucleoside diphosphate: ndp + , -bisphosphoglycerate ↔ ntp + -phosphoglycerate. here we compare the kinetics of formation of the complexes of human pgk with different d-and their mirror images, l-nucleoside diphosphates, and the effect of -phosphoglycerate thereon. two types of experiment were carried out: equilibrium experiments allow the estimation of dissociation constants, and stopped-flow experiments the transient kinetics of the interactions. in addition, by the rapid-quench-flow technique, we compare the kinetics of the phospho-transfer and product release steps with each d-or l-nucleotide. crystallographic and molecular modelling studies allow defining the structural reasons for the low enantioselectivity of pgk. the aim of this basic work on the mechanism of action of human pgk with non-natural nucleotides is to obtain information for the optimization of therapeutic nucleoside analogues that are poorly phosphorylated by pgk. anrs is gratefully acknowledged for financial support. a new itc assay for measuring high-and lowaffinity protein-ligand interactions g. krainer , j. fanghänel , s. keller leibniz institute of molecular pharmacology (fmp), berlin, germany, bayer schering pharma ag, berlin, germany isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions [ ] . however, reliable determination of the dissociation constant (kd) is typically limited to the range µm > kd > nm. nevertheless, interactions characterised by a higher or lower kd can be assessed indirectly, provided that a suitable competitive ligand is available whose kd falls within the directly accessible window [ ] . unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of high-and low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-affinity (kd < nm) and low-affinity (kd > µm) protein-ligand interactions. a molecular dynamics study of the cftr nucleotide binding domains interaction v. martorana , r. noto , o. moran cnr-istituto di biofisica, palermo, italy, cnr-istituto di biofisica, genova, italy the cystic fibrosis transmembrane conductance regulator (cftr), the dysfunctional protein in cystic fibrosis, contains two transmembrane domains, two nucleotide-binding domains (nbds), and a regulatory domain. opening of the pore have been linked to the atp-driven tight dimerisation of nbd. we have studied the nbd -nbd interactions on wild type and cystic fibrosis-related mutations by steered molecular dynamics simulations (smd). a fully solvated dimer, including the two bound atps, was separated by pulling one monomer with an external, increasing force. interestingly, the force needed to break the mutated (g d) dimer is significantly smaller than in the wild type case. the effect of a cftr potentiator, the genistein, has also been tested by repeating the smd simulations with the small molecule docked at the interface between the two nbd domains. to test the validity of our results we have repeated the separation process for different simulation lengths and force strengths. the amount of distortion on the pulled nbd domain has also been studied. this work is partially supported by the italian cystic fibrosis foundation (prog ffc # / ), with the contribution of "mille bambini a via margutta"onlus"blunotte", "lega italiana fc toscana" stability and aggregation studies of human septin (sept ) j. n. a. macêdo, r. c. garratt, a. p. u. araújo instituto de física de são carlos, usp, são carlos, brazil the septins are a conserved family of nucleotide binding proteins firstly identified in saccharomyces cerevisae as proteins required for the completion of the cell cycle. septins are implicated in several cellular functions such as cytokinesis, vesicle trafficking, exocytosis, cytoeskeletal dynamics, cell polarity and sperm motility. furthermore, they are associated with alzheimer's and parkinson's disease. sept was identified in rat brain being highly enriched in presynaptic nerve terminals. it colocalizes with synaptophysin and dynamin i and is associated with synaptic vesicle. in this work, human sept without its n-terminal domain (sept gc) was expressed in e. coli and purified by affinity and size exclusion cromatographies. structural stability studies were performed with recombinant sept gc using circular dichroism spectroscopy (cd), right-angle light scattering, and fluorescence spectroscopy. the sept gc cd spectrum showed a conformational transition from a predominantly α-helical starting structure to one dominated by β-sheet, just above physiological temperatures. the formation of irreversible aggregates, detected by light scattering, and their ability to bind thioflavin-t suggested that sept forms amyloidlike structures, as has been previously observed for human septins and . our data suggest that amyloid formation by isolated septins in vitro may be a general phenomenon whose physiological relevance needs to be further investigated. pln is an antimicrobial peptide produced from lactobacillus plantarum nric . analog pln (pln a) and a ser-derived (pln s, tyr to ser replacement) were synthesized on solid phase and their interactions with biomembrane model systems and inhibitory property on s.aureus and p.aeruginosa were investigated by cd, leakage assays, fluorescence spectroscopy, and differential scanning calorimetry. both peptides share the same unordered structure-like cd spectrum in aqueous solution, but a helicoidal induction in the presence of negative vesicles were observed, however pln s showed lower helical content than pln a. the ser-derived peptide presented % decrease of its leakage activity in different liposome compositions, a threefold increase in the dissociation constant from the liposomes than pln a, and close to % reduction for the inhibitory activity against p. aeruginosa growth. we can conclude that besides the electrostatic contacts between the amphipathic &alpha-helix, formed by the cationic residues from pln and negatively charged phospholipids, the pln -membrane binding is a process driven for the hydrophobic interactions from non-polar residues. in this case, there was a significant contribution of the tyr residue, which must be allocated in a lipid interface, described as the preferential position to this residue in membrane proteins. supported: fapesp label free detection using deep-uv laser-based fluorescence lifetime imaging microscopy q. li, s. seeger physikalisch-chemisches institut, universität zürich, winterthurerstrasse , ch- zürich, switzerland label free detection based on native fluorescence excited at uv region shows great potential for the life sciences. it offers simple, low-cost and fast method for sensitive detection of important biological analytes without modification. in this contribution we present a deep uv fluorescence lifetime imaging microscopy system (duv-flim) based on a picosecond deep uv laser using time-correlated single-photon counting method. the described setup is well-suited for biological applications for ultrasensitive detection. we have showed single uv dye (bmq) and single protein (ecβ gal ) molecules detection using duv-flim. further, the label free detection of protein interaction between ecβ gal and monoclonal anti-ecβ gal has been demonstrated by means of steady-state and time-resolved fluorescence spectroscopy. we achieved detection sensitivity for the ecβ gal/ anti-ecβ gal pair down to the nanomolar concentration range. we also extended this method to study the interaction of therapeutic drug porphyrin with bsa protein. fluorescence resonance energy transfer between protein and alexa fluor has been investigated using duv-flim. the intrinsic fluorescence and fluorescence lifetime changes of donor biotin β-galactosidase have been measured. energy transfer efficiency and donor acceptor distance have been obtained. fluorescence images of acceptor af due to fret have been observed when excited at nm. the monomeric heme-containing indoleamine , dioxygenase (ido) catalyses the oxidative cleavage of the indole moiety of l-tryptophan (l-trp). enhanced l-trp degradation contributes to various physiological disorders including depression or failure of the immuno-regulating system. the regulation of ido by inhibitors is extensively studied. however, the catalytic mechanism of ido on the molecular level is still unknown. in addition to l-trp, a wide range of substrates are degraded including d-tryptophan, melatonine or tryptamine. in contrast, indole or histidine do not function as substrates for ido. to understand the determinants for substrate specificity, we investigate the interaction of the heme iron, the heme-bound ligand and the substrate. we use (time-resolved) uv/visible and fourier transform infrared (ftir) spectroscopy over a wide temperature range ( - k) to monitor the binding of diatomic ligands to the heme iron and the binding of different substrates to coligated ido. changes in the spectra upon addition of l-trp are analyzed and compared to those induced by other substrates or inhibitors. archaeal protoglobin d-structure: novel ligand diffusion paths and heme-reactivity modulation protoglobin (pgb) from methanosarcina acetivorans c a, a strictly anaerobic methanogenic archaea, is the latest entry in the hemoglobin superfamily. our previous crystallographic studies on pgb have shown that protoglobinspecific loops and a n-terminal extension completely bury the heme within the protein matrix ( ) . access of o , co, and no to the heme is granted by protoglobin-specific apolar tunnels reaching the heme distal site from locations at the b/g and b/e helix interfaces. here we report structural and kinetic data on pgb mutants engineered to probe the protein structural and kinetic properties. six crystal structures (pgb mutants: ∆ (missing nter residues), y(b ) →a, y(b ) →w, f(b ) →w, f(g ) →w, i(g ) →f) show that the mutations engineered essentially restrict access to ligand tunnel . an accurate molecular level description of the signaling mechanism in ca + transducers necessitates the knowledge of the kinetics and energetics of conformational changes associated with ca + binding to various calcium binding proteins. with this in mind, we have developed an approach that combines the laser-induced photolysis of photolabile "caged" ca + compound, dm-nitrophen, with the photothermal beam deflection (pbd) technique to determine thermodynamic profiles associated with the ligand binding to calcium chelator, edta, and ca + sensor, calmodulin. this approach allows us to monitor time profiles of volume and enthalpy changes on the microsecond to millisecond timescale. the initial pbd study of ca + photo-relase from ca + loaded dm-nitrophen reveals the presence of two phases. the first step takes place within first µs upon and is associated with a volume decrease of - ml mol − and enthalpy change of kcal mol − . on the longer timescale (τ = µs), the second event with a positive volume change of ml mol − and enthalpy change of kcal mol − was detected. on the other hand, ca + photorelease in the presence of calmodulin is accompanied with an additional phase with a distinct lifetime and volume and enthalpy changes that reflects the metal binding to calmodulin and concomitant structural changes. antimicrobial peptides: linking partition, activity and high membrane-bound concentrations antimicrobial peptides (amps) have been intensively studied at micro and macroscopic levels for over twenty years. knowledge from these two domains has, however, contributed little to a comprehensive understanding of amp action; rather, in vivo amp performance has been only remotely correlated to biophysical properties. we focus on the assessment of peptide accumulation on bacterial membranes as an example of this separation: amp-bilayer interactions have been subject to extensive biophysical characterization, but conversion of that information into educated estimates of in vivo membrane-bound amp concentrations is lacking. this has led to the overlooking of important factors for activity. using simple partition models we were able to analyze available information on amp activity and interaction with membranes to show that unexpectedly high membranebound peptide concentrations are likely in vivo and may, in some cases, be required for triggering bacterial death. e. e. schäfer , s. schuy , a. janshoff georg august-university göttingen, germany, johannes-gutenberg-university mainz, germany retrovirus entry into cells occurs through fusion of the lipid bilayers that surround the virus and the lipid bilayer of the host cell. fusion proteins, present on the surface of the virus membrane play an essential role in the early stage of virus entry. understanding of the molecular mechanism is important for the design and function of modern fusion inhibitors. in this project we analyze the fusion of active conformation of the envelope gp from the human and simian immunodeficiency viruses (hiv and siv). during the infection process gp undergoes a sequence of conformational changes where the n -terminal nhr develop a trimer pre-hairpin intermediate. afterwards the three chr fold back towards the central nhr and a six helix bundle is formed. this rearrangement forces the viral and the host membrane into close contact and fusion pores may induce membrane fusion. this decisive molecular step in retroviral fusion has been modeled by rational design of lipopeptide assemblies that mimic a coiled-coil structure serving as a receptor for potential antagonists. for this purpose, sslbs were functionalized in an in situ coupling reaction with peptides originating from the nhr (n-peptides) of siv and hiv to monitor the interactions with the specific chr peptides (c and t ). binding of antagonists to surface confined coiled-coil structures has been quantified by ellipsometry, quartz crystal microbalance and was visualized by atomic force microscopy. a. rupprecht , e. sokolenko , e. e. pohl institute of cell biology and neurobiology, charité -universitätsmedizin, berlin, germany, frumkin institute of physical chemistry and electrochemistry russian academy of sciences, moscow, russia the production of reactive oxygen species (ros) in mitochondria is very sensitive to the proton motive force and can be decreased by mild uncoupling, mediated e.g. by uncoupling proteins (ucp) . in contrast, the activation of uncoupling proteins by ros as a negative feedback loop is a highly controversial hypothesis. ucp activation in mitochondria by -hydroxy- -nonenal (hne, aldehydic product of lipid peroxidation) was first demonstrated by echtay et al. . here we investigate the ability of hne to activate and/or to regulate the expression of ucp in two different systems: (i) in lipid membranes reconstituted with recombinant ucp and (ii) in primary neuronal cells. total bilayer conductance was enhanced in the presence of hne, but this effect was independent on ucp and ucp . the results concerning the hne-mediated ucp expression after induction of ros-production and/or after exogenous addition of hne are discussed for three brain-associated proteins (ucp , ucp , ucp ) in view of their possible functions. . beck, v et al. . faseb j. : - . . echtay, k. s. et al. . t. rudack, j. schlitter, k. gerwert ruhr university, department of biophysics, nd north, bochum, germany the gtpase ras p which is linked to the membrane via a lipid anchor, is a crucial switch in the cellular signal transduction processes that control cell growth and proliferation. docking simulations can be seriously hampered by the great difficulty to accurately estimate the ligand-protein binding affinity constant k, which is usually derived via the computation of the binding free energy ∆g. unfortunately, due to the involved logarithmic relationship, errors of less than . kcal/mol in the computation of ∆g result in about one order of magnitude inaccuracy on k. this can hinder computational methods from discriminating micromolar from nanomolar compounds. to improve the reliability of docking prediction, we make use of enhanced sampling methods, ranging from steered molecular dynamics to metadynamics . we also test several descriptors, such as the recently developed path collective variables , to identify the most suitable reaction coordinates accounting for binding and unbinding processes. using these approaches we investigate ligand docking and undocking and we attempt to describe at an atomistic level the kinetics of binding, which we intend to exploit for drug design purposes. here, an example of application in drug design is reported. . isralewitz, b. et al.; j. mol. graph. model. , , - . . laio, a. and parrinello, m.; pnas , , - . . branduardi, d.et al.; j. chem. phys. , , . prediction of protein-protein complex structures using wang-landau simulations a. solernou, barcelona supercomputing center, jordi girona , barcelona, spain protein-protein interactions are essential in the majority of life processes, so they have keen interest in several knowledge areas. however, experimental data on protein complexes is being produced at a quite low pace in comparison to that of the individual components. thus, in recent years attention has focused into computational approaches to the proteinprotein docking problem. a variety of docking protocols have been recently reported, sharing usually the following strategy: fast rigid-body search of the interacting subunits, followed by scoring and refinement of the interfaces. although this kind of strategy has proven some good results in the capri blind test it has two main limitations. on one hand one should include full flexibility on the protein structures. on the other, the evaluation should be made with free energy calculations instead of using a scoring function. in this work we propose to search the native complex structure in the minimum of the free energy landscape. we use the coarse grained potential unres to get the potential energy of the possible conformations. they are generated changing the dihedral angles, the side chain rotamers, and lastly the mutual orientation using a new sampling protocol we have developed (rotation-based uniform sampling; rotbus). finally, the free energy calculations are performed using an omp parallelization of the wang-landau algorithm. s. shukla , s. asthana , g. giliberti , f. luliano , m. ceccarelli , r. loddu , p. ruggerone , p. la colla department of biomedical science and technology, università di cagliari, cagliari, italy, department of physics, università di cagliari, cagliari, italy the virus encoded rdrp has emerged as a prime target in the search for specific hcv and other flaviviridae antivirals. recently, the determination of the hcv rdrp structure in complex with certain benzimidazoles has been reported, these nni's bind to the surface of the thumb domain, thereby disrupting its interaction with the finger domain, which is necessary for catalytic activity. on the other hand, we have found that, in bvdv, the mutations conferring resistance to same class of inhibitors lie in the finger domain of rdrp, indicating that the mode of inhibition of benzimidazole class of compounds is different in both hcv and bvdv rdrp. herein, we have applied docking approaches (binding orientation), molecular dynamics (md) simulations combined with metadynamics to elucidate the microscopic mechanism of the interactions between the ligand and the receptor in order to identify features barely seen in experiment. the recently designed algorithm overcomes the time scale problem by accelerating properly defined reaction coordinates. it mimics the real dynamics of a ligand staying or leaving the receptor and in doing so reconstructs the free energy surface, which in turn gives an idea of the residence time of the inhibitor in the cavity.finally we identified the binding modes and different mechanism of inhibition of benzimidazole class of compounds in rdrp of two closely related rna viruses. . c. seifert , w. stacklies , f. graeter protein mechanics and evolution, bioquant, inf bq , heidelberg, germany, ag graeter, picb, yueyang lu, shanghai , china we use a new method that detects force distribution in proteins. based on molecular dynamic simulations, changes in inter-atomic forces are calculated, here caused by different ligands. these changes will then be analyzed to detect a signal transfer through a protein initiated by the binding of a ligand to the protein. chaperones are ubiquitous proteins, which help other proteins to fold into a native conformation. they are able to refold misfolded proteins with a great variety of mechanisms. in this project, our group focuses on molecular dynamic simulations of htpg, an e. coli homolog of the human hsp (heat shock protein kda). the full structure of htpg was published by shiau et al. in , but the mechanism of how htpg performs its function is still not understood. the folding mechanism is an atp driven reaction cycle, in which the functional entity is a homodimer of htpg. we separate the cycle in three main states: apo, adp-and atpbound state. conventional molecular dynamics simulations are used to build a stable simulation system and provide structure trajectories and primary information about the behavior of htpg in its different states of the folding process. experiments indicate that the molecular movement is atp driven. we use force distribution analysis to elucidate how atp effects htpg conformation and dynamics. the small size of myoglobin makes it the preferred candidate to investigate the structure-function paradigm. in its interior five docking sites have been identified and for long time these xenon cavities have been classified as packing defects. recently, it was shown that they might be involved in ligands migration path. however, some questions regarding its role as oxygen carrier no scavenger remain yet open as well as the microscopic mechanism regulating these biological functions. in this work we made use of standard md simulations of solvated myoglobin to characterize internal cavities. our principal results is that we have found several secondary cavities showing volume and occurrence at least comparable to that of xenon cavities. in order to analyze and rationalize internal cavities we applied special cluster-analysis: we classified all cavities with respect to the position, size and occurrence ascribing them to different clusters. this analysis highlights possible intrinsic migration paths for small ligands within the protein matrix controlled by spontaneous fluctuations of the protein itself. moreover, we identified some key residues playing a fundamental role in controlling internal pathways. our suggestion that the secondary cavities constitute the preferred path for ligand escape is also supported by explicit metadynamics simulations of ligand escape. a. varga , p. lallemand , j. szabó , p. závodszky , m. vas , t. barman , l. chaloin , c. lionne institute of enzymology, brc, hungarian academy of sciences, budapest, hungary, cnrs-université montpellier -université montpellier , institut de biologie, umr , montpellier, france -phosphoglycerate kinase (pgk) is a promising candidate for the activation of nucleotide analogues used in antiviral and anticancer therapies. pgk is a key enzyme in glycolysis; it catalyses the reversible reaction , -bisphosphoglycerate + adp ↔ -phosphoglycerate + atp. here we explored the catalytic role in human pgk of the highly conserved lys that has been proposed to be essential for pgk function, by a transient and equilibrium kinetic study with the active site mutant k a. by the stopped-flow method we show that the kinetics of substrate binding and the associated protein isomerization steps are fast and identical for the wild-type pgk and mutant k a. by the use of a chemical sampling method (rapid-quench-flow) under multi and single turnover conditions and in both directions of the reaction, we show that the rate-limiting step with wild type pgk follows product formation, whereas with the mutant it is the phospho-transfer step itself that is rate limiting. these data are supported by saxs measurements which showed no direct role of lys in the domain closure of the enzyme i.e. the isomerisation step of the reaction. the results are explained by the structural data of the enzyme. characterization of different recombinant nrp proteins and interactions with heparin k. a. uniewicz, y. ahmed, d. g. fernig school of biological sciences and centre for glycobiology, biosciences building university of liverpool, l zb liverpool, u.k. neuropilin- (nrp ) is a mammalian membrane glycoprotein involved in tip sprouting processes like angiogenesis and neurogenesis. it has been shown that the interaction of nrp with heparin/heparan sulfate is implicated in its enhancement of growth factor signalling, however the mechanism is not yet known. commercially available extracellular domain of nrp is available either as a truncated his-tagged human protein (hnrp ) or as the rat protein fused to histagged human fc and expressed as a dimer (fcrnrp ). biochemical properties such as kinetics of heparin binding and structural requirements for sugar binding together with biochemical tests of protein properties were performed in order to characterise these commercial proteins. fc rnrp shown high affinity to heparin (kd= . nm) and required a minimum of dp to effectively compete with fc rnrp binding to immobilised heparin. competition experiments with various modified sugars show that interaction of fc rnrp with heparin is highly ionic and dependent on the position of sulfate groups along the heparin chain. the hnrp did not bind to heparin immobilised via nhs-biotin, though it did bind to some heparin affinity chromatography matrices. organic compounds of tin are among wide spread environmental pollutants. due to physical and/or chemical actions, poly-substituted alkyltins speciate into less substituted, more toxic species. tetra-or tri-alkyltins show a marked delay in their toxic action with respect to the corresponding di-and mono-alkylated analogs. it has been hypothesized that the delayed toxicity may results from the progressive dealkylation of alkyltins in more toxic di-and mono-derivatives, which bind and inhibit essential enzymes. it has been proposed that alkyltins preferentially target enzyme sulphydryl groups. previously, we showed that a nine amino acid linear peptide (i lgcwcylr ) containing a cxc motif is able to bind and dealkylate tri-substituted alkyltin compounds into the corresponding dialkyl derivatives. here, we investigated the time dependence of the degradation of the most common alkyltin derivatives by this peptide. we monitored the reaction kinetics using the intrinsic fluorescence of the tryptophan residue in position of the peptide. we found that all of the alkyltins analyzed are progressively degraded to dialkyl derivatives, following a pseudo-enzymatic reaction mechanism. the end-point of the reactions was the formation of a covalent complex between the disubstituted alkyltin and the peptide. these data agree with the speciation profiles proposed for polysubstituted alkyltins in the environment and reveal a possible biotic degradation pathway for these toxic compounds. parkinson's disease (pd) is a multifactorial neurodegenerative condition characterized by the progressive loss of dopaminergic neurons in the substantia nigra and by the presence of intracellular inclusions, composed predominantly of fibrillar alpha-synuclein (as). post-mortem studies indicate the presence of oxidative damage in the nigral neurons. dopamine oxidation, which leads to the formation of highly reactive quinones (daq), may account for the specificity neurodegeneration observed in pd. daq have many potential protein targets for chemical modifications. among them, we focused our attention on dj- , of which mutated forms have been found in familial cases of pd. a possible function of dj- is its redox-dependent chaperone activity that could prevent as aggregation and fibril formation. in the present work, we analyzed the structural and functional modification induced on dj- by daq. n-hsqc spectra of dj- were recorded in the presence of different amounts of daq and chemical shift perturbations were used to identify the dj- residues target of daq and their relative reactivity toward daq. aggregation assays were also performed to evaluate the functional effects of the daq modifications on the chaperone activity of dj- . rabies virus (rabv) infects neurons exclusively and causes lethal encephalitis. pathogenic rabv strains favor neuronal survival, whereas non-pathogen strains lead to neuronal apoptosis. the use of recombinant rabv showed: / the g protein determined the induction of the survival or death phenotypes; / the last cooh amino acids of the g protein cytoplasmic domain (cytog) are critical. these residues form a binding site for pdz domain (pdz-bs). one of the amino acid differences between survival and death gproteins are located in this pdz-bs. results of two-hybrid experiments showed that cytog survival interacted pdz domain of ser/thr kinases (mast), while cytog death interacted also with additional pdz containing host proteins. to understand the fine structural basis for the specificity of the pdz-cytog complexes, we determined the d structure and the dynamics of the mast-cytog complexes by nmr. the structures, as well as the affinities and constant kinetics, of the mast pdz complexes with both cytog are similar. we conclude that difference by one aa in the pdz-bs of the two strains cannot drastically modify the interaction with mast -pdz, in agreement with the two-hybrid data. preliminary results suggest that the interaction of cytog death with one additional cellular partner blurs the pro-survival signals engaging the infected cells through apoptotic trails. functional protein immobilization on glass-type surfaces s. waichman, m. bhagawati, y. podoplelova, j. piehler institute of biology, department of biophysics,university of osnabrück , barbara st. osnabrück, germany the immobilization of membrane proteins onto solid supports enables protein interactions and conformational changes to be probed by spectroscopic techniques under highly defined conditions. here, we present a novel method for covalent protein immobilization on glass-type surfaces using a bottom-up approach. in this approach the 'phosphopantetheinyl group of coenzyme a (coa) was transferred to the acyl carrier protein-derived ybbr tag of the target protein by means of the phosphopantetheinyl transferase sfp. the glass-support was rendered biocompatible by coating it with an ultrathin layer of peg (polyethylene glycol), followed by functionalization with coa through maleimide chemistry. immobilization of ybbr-tagged proteins in presence of sfp was followed in real time by label-free detection using reflectance interference spectroscopy. the immobilization procedure was thus systematically optimized by means of binding specificity, enzyme activity and functionality of the immobilized protein. this approach was employed for immobilizing the type i interferon ifnα in order to probe ligand recognition by ifnar and ifnar and ligand-induced ternary complex formation. the versatility of this technique was further enhanced by its combination with photopatterning methods. this immobilization technique can provide a beneficial tool for bioanalytical and biophysical applications at the single molecule level. r. vijayan, p. c. biggin department of biochemistry, university of oxford, south parks road, oxford, ox qu, uk ionotropic glutamate receptors mediate excitatory synaptic transmission in the brain and are heavily implicated in memory and learning as well as in numerous neuropathological conditions. one family of iglurs, the kainate receptors, show unusual sensitivity to changes in external ion species resulting in an apparent requirement for both sodium and chloride ions for activation. our recent work revealed the location and selectivity of the cation binding sites. despite this progress, it is still unclear how the cation binding sites confer sodium selectivity and how apparent affinity for chloride is influenced by the presence of cations. we have attempted to address these questions by performing extensive free energy calculations using all-atom molecular dynamics simulations. the rank order of cation binding obtained from relative binding free energy calculations is in agreement with experimental measurements of apparent affinity. these calculations also reveal that the pair of cation binding sites in the dimer interface act independently. binding free energy calculations performed using a reduced model of the binding site show that cation selectivity can been attributed to both the rigidity and high charge density of the binding sites. finally, a potential of mean force derived from umbrella sampling simulations indicate that the presence of cations stabilize the anion binding site considerably. the effect of toxins on the inorganic phosphate release during the actin filament formation a. vig, t. kupi, g. hild, m. nyitrai university of pécs, faculty of medicine, department of biophysics, szigeti str. , pécs, hungary actin can be found in monomeric (g-actin) and filamentous (f-actin) form in eukaryote cells under physiological circumstances. the first step of actin polymerisation is the formation of actin nuclei by atp-binding actin monomers. the next step in is the elongation when monomers are associated to the previously formed nuclei or to the ends of the growing filaments. after the association of monomers the bound atp is hydrolysed to adp.p i , and with first order kinetics the release of inorganic phosphate occures. the rate constant of the release is , s − , which is a slower process than the hydrolysis itself ( , s − ).phalloidin, a cyclic peptide from amanita phalloides can bind to the filaments and stabilizes their structure. jasplakinolide is another cyclic peptide from marine sponge (jaspis johnstoni) which binds actin filaments. the aim of this study was to investigate whether the binding of toxins to the newly created filaments has an effect on the kinetics of the inorganic phosphate release or not. we used absorption photometry measurements to measure the rate of phosphate release. phalloidin decreased the rate of the release substantially. although the effect of jasplakinolide was weaker, the results showed that the binding of these toxins to the actin can modify the rate of the release of inorganic phosphate from the filaments. these observations are in agreement with the molecular mechanisms by which these toxins stabilise the actin filamens. fluorescent proteins (fps) are invaluable fluorescent markers in cell biology. however, their use is often limited by photobleaching of the chromophore, notably in single-molecule, time-resolved or super-resolution imaging studies. we will present the crystallographic studies at near atomic resolution of a photo-activatable fluorescent protein irisfp that has been observed in a transient radical state en route to photobleaching. we took advantage of x-rays to populate the radical, which, under illumination with visible light, presumably forms with low probability from the triplet state. the combined x-ray diffraction and in crystallo spectroscopic data (from uv-vis and raman spectroscopies) reveal that radical formation in irisfp involves strong but reversible distortion of the chromophore, suggesting a transient loss of pi-conjugation. these results will help unravel the mechanisms of blinking and photobleaching in fps, which is of importance to rationally design variants of higher photostability. optimizing photoactivatable fluorescent proteins for live-cell imaging recently, novel fluorescent proteins (fps) have been reported which perform spectral changes in response to irradiation with light of a particular wavelength. reversibly photoactivatable proteins switch between a bright and a dim fluorescent state. this process is accompanied by photoisomerization of the protein chromophore. irreversible photoactivation results from photochemical processes within the protein, e.g., photolysis of an amino acid side chain, or an extension of the alternating π-electron system of the chromophore by a β-elimination reaction. fps have became valuable tools in live-cell imaging because they allow intracellular protein labeling by using them as fusion tag, and photoactivatable fps are powerful tools for application in novel subdiffraction imaging techniques. however, the available fps still offer potential for improvement in various ways. they frequently show a tendency to aggregate or oligomerize, incomplete chromophore maturation, fluctuating emission and low photostability. here, we will present our recent progress towards engineering the 'perfect' fp. simultaneous intracellular chloride and ph measurements using gfp-based sensor in ldcv d. arosio , f. ricci , l. marchetti , l. albertazzi , f. beltram italian institute of technology, udr pisa, italy, nest, scuola normale superiore, pisa, italy, nest, infm cnr, pisa, italy chloride ion participates in many physiological functions including control of neuronal resting potential, charge balance during endosome acidification, and regulation of cell volume. as a consequence dysfunctions in regulating membrane chloride permeability lead to severe diseases including motor disorders, cystic fibrosis and epilepsy. at present processes regulating intracellular chloride ion concentrations are still widely unexplored mainly as a consequence of limiting methods to quantify chloride fluxes in living cells. in the present work a highly specific, genetically encoded sensor is developed for detecting simultaneously intracellular ph and chloride concentration. the sensor is obtained by fusion of a red fluorescent protein (dsred-monomer), insensitive to chloride and ph, to a gfp variant containing a specific chloride-binding site (gfp-chl). dsred-gfp-chl binds the chloride ion following a fluorescence static quenching mechanism, which allows measurements of intracellular ph in a chlorideindependent manner. the sensor has been successfully tested in different living cells, in a ph range - and chloride concentration up to mm. for the first time, to the best of our knowledge, it allowed to measure the chloride concentration of dense core vesicles in the secretory pathway. applicability to high-throughput screening, range of validity and accuracy of time-lapse maps will be discussed. we aim to understand the basis of the photophysical changes in fluorescent proteins (fp) induced by reactive oxygen species (ros). indeed, ros might be involved in photochemistry of fp, leading to their photobleaching or photoconversion. in addition, fp may be used to investigate cellular events like phagocytosis or mitochondrial activity, where ros are naturally produced. in the latter cases, an accurate analysis of fp's fluorescence signals requires the full knowledge of reactions between ros and fp. in the future, this work may help in developing either photoresistant or photoswitchable fp and improving their use for imaging under oxidative stress conditions. using γ-radiolysis as a quantitative source of ros, we investigated the reactions of oh and o − radicals on the cyan fluorescent protein (cfp) and the modifications of the cfp's photophysical properties by oh radicals were explored in detail (submitted). in order to address the corresponding chemical changes in the protein, we devised a mild proteolysis protocol that for the first time offers a peptide mass fingerprint almost covering the cfp sequence (alvarez et al. biochemistry ). then, we achieved the meticulous characterization of the cfp oxidation products by mass spectrometry and proposed a mechanism to account for their formation by pulse radiolysis. since the cloning of the green fluorescent protein from aequoria victoria, numerous screens have been performed to improve the brightness of this protein, its spectral variants and fluorescent proteins from other species. the improvement is evaluated by comparing fluorescence intensity of individual bacteria or colonies. in this way also expression level, folding and maturation efficiency, and thickness of the bacterial colony contribute. here we report a screening method that, in addition to fluorescence intensity, quantifies the excited state lifetime of a fluorescent protein, which is independent of intensity or expression level, and provides a direct measure for the quantum efficiency of the fluorescent protein. the novel approach was used to screen a library of cyan fluorescent protein (cfp) variants randomly mutated at position , and , yielding an improved bright cyan fluorescent protein named, mposeidon, with a markedly increased fluorescence lifetime and quantum yield, increased photostability and improved fluorescence intensity in vivo. it is shown that mposeidon is the brightest cyan fluorescent protein in mammalian cells. in addition, several lifetime variants were identified that can be used for lifetime unmixing. it is demonstrated that three cfp variants can be separated and their distribution quantified in a single detection channel. a. r. faro , p. carpentier , d. bourgeois , e. rosny irtsv, cea, ufj, grenoble, france, ibs, cea, cnrs, ufj, grenoble, france, ibs, cea, cnrs, ufj, grenoble, france, ibs, cnrs, ufj, grenoble, france enhanced yellow fluorescent protein (eyfp) is extensively used as a fluorescent marker. like other photo activatable fluorescence proteins (pafp), it exhibits photo-switching properties. however, the mechanism by which fluorescence can be swiched on or off upon light irradiation is not fully understood at the molecular level. the bright to dark conversion involves a protonation step and structural rearrangements of the chromophore, but it is not clear which of these two steps is the triggering event. to answer this question, we carried out photo-switching experiments at cryotemperatures. our data suggest that a photo-induced protonation step (probably in the triplet state) is the primary event in the bright to dark conversion. our results may bear relevance to other pafps, such as iris, eos, dronpa, padron or kaede. how misfolding and aggregation of proteins constitutes a toxic insult to neurons remains largely unknown. in order to obtain insight into the molecular biology of neurodegenerative disease, we have developed a number of gfp-based biosensors for the detection and quantification of cellular clearing mechanisms for aggregated proteins. the high load on these protein quality control mechanisms, and their failure to meet normal physiological demand ultimately results in a "de-compensation" condition from which the nerve cell cannot recover. our fret/flim-based bioassays visualize protein ubiquitination and degradation; proteasomal activity; foldase activity using a folding-impaired gfp mutant which gains fluorescence conditional on the upregulation of chaperone activity; chaperone binding to unfolded proteins; and autophagosome formation/lysosomal integrity via the targeted and sensitive fret-based measurement of ph changes. these sensors are employed in cellular model systems for parkinson's and alzheimer's disease, and amyotrophic lateral sclerosis (als) to delineate the molecular pathway of cellular demise, and to gain a mechanistic understanding of the toxicity of protein aggregates and the basis for the vulnerability of neurons. millisecond photo-switching dynamics of e q gfp mutants for sensor and imaging applications m. collini , v. quercioli , l. d'alfonso , g. baldini , b. campanini , s. bettati , g. chirico dipartimento di fisica, università di milano bicocca, italy, dipartimento di biochimica e biologia molecolare, università di parma, parma, italy e q mutants of the green fluorescent protein are known to possess photo-chromic properties: the anionic emission, primed by a pump nm laser beam, can be switched between two levels of different brightness by irradiation with a blue, ∼ = nm, probe laser light. we have studied here the amplitude and the dynamics of the brightness enhancement of the e q mutant of gfpmut . the fluorescence emission increases almost threefold, under saturating probe laser excitation, for pump excitation intensity in the linear regime. two characteristic activation times, estimated by means of modulated two colour fluorescence correlation spectroscopy, are detected in the - ms range, independent of solution temperature and viscosity. the brightness enhancement factor and the characteristic activation times depend markedly on the solution ph. these results indicate that this mutant can be used as a high sensitive intracellular marker for local proton concentration and for modulated excitation imaging. the advent of fluorescent proteins (fp) gave researchers the opportunity to study proteins in situ. fluorescence resonance energy transfer (fret) benefited from this. cell fixation is a commonly used approach when working with microscopy. however, we have found that fret efficiency (e) in cells transfected with cerulean and venus chimeras could not be reproducibly measured after fixation. to evaluate this problem in detail, we measured e of cerulean-venus standard constructs by acceptor photobleaching fret, intensity-based ratiometric fret and flim-fret. the constructs were produced as standards (biophys j, , , ) with , and % e values, comprising donor-acceptor separations of , and amino acids, respectively. transient transfection of the fusion plasmids was performed into hela cells and e was measured in live and pfa or methanol fixed cells. literature e values were reproduced when measuring live cells. conversely, cell fixation caused a deviation of e values. methanol fixed cells showed e between - % for all the constructs. the effect of pfa fixation on both fluorescence intensity and fret varied vastly among independent experiments regardless of the measurement modality. thus, fixation should be avoided due to the effects it has on fp's fluorescence and consequently on fret efficiency. to explain the improvement in the fluorescence properties of cerulean when compared to ecfp, we have determined the x-ray crystallographic structures of these two proteins at physiological ph, and performed molecular dynamics simulations. both proteins exhibit a structural heterogeneity in the nterminal half of their seventh strand, which forms a specific set of van der waals interactions with the chromophore. the critical h d mutation present in cerulean induces a modification of these interactions, and allows the chromophore to be more planar and better packed, albeit only intermittently. as a consequence, the probability of non-radiative decay is significantly decreased. our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins, and provide insights which should allow the design of mutants with enhanced optical properties. the fluorescence lifetime of green fluorescent protein g. jung biophysical chemistry, saarland university, saarbruecken, germany biotechnological design of the green fluorescent protein (gfp) and the discovery of other proteins boosted the development of the life sciences in the past decade. tracking protein movements and high resolution microscopy are only a few recent applications which were realized by fluorescent protein technology. among these examples, the switching between two chromophore state is exploited. our aim is to establish fluorescent proteins for bioanalytical fluorescence lifetime measurements. despite the progress in other fields, quantification with gfp still imposes practical problems [ ] . in the past, we observed that the uv-light driven decarboxylation of the nearby aminoacids glu distorts the fluorescence lifetime of gfp [ ] . we found out that this chemical reaction also occurs under excitation of the anionic chromophore state with a high quantum yield [ ] . recently, we could show by time-resolved spectroscopy that, indeed, the more susceptible state for this kind of photoconversion is the anionic chromophore state [ ] . suppression of this reaction therefore enables the design of autofluorescent proteins which can be used e.g. for the quantification of ions and which are beneficial as donors in fret applications. the fluorescent properties of tryptophan residues (w) in proteins are highly dependent on their immediate protein environment. however, the multi exponential decay of single w proteins is not completely understood. the most cited hypothesis contributes a multi exponential decay to the existence of several micro conformations (rotamers) of the w residue within the protein matrix. to determine rotamers we apply a method based on dead-end elimination (dee) and molecular dynamics simulations (md). low energy rotamers are calculated by dee while dynamics and further refinement is accomplished using md. the method was applied on several test cases including the protein mutant bc-csp l e from bacillus caldolyticus, which contains a solvent exposed w residue. as resolved by x-ray crystallography, this w residue occupies two conformations. using dee and md we were able to retrieve the w conformations found in the x-ray structure. the results demonstrate the ability of the method to obtain valuable w rotamers, both for solvent shielded as exposed residues. the determined conformations were compatible with the findings based on a method using replica exchange simulations. k. nienhaus , h. nar , r. heilker , j. wiedenmann , g. u. nienhaus inst. of biophysics, universität ulm, ulm, germany, dept. of lead discovery, boehringer ingelheim, biberach/riss, germany, national oceanography center, university of southampton, southampton, uk, inst. of applied physics, universität karlsruhe (th), karlsruhe, germany eqfp is a red fluorescent protein (rfp) with the chromophore in a co-planar trans orientation, whereas the cis isomer is preferred by most other rfps. by using x-ray crystallography, we determined the structures of the dimeric variants d eqfp and d eqfp at high resolution (up to . Å). for d eqfp , we had previously seen a redshifted species upon irradiation with -nm light. concomitant changes in the raman spectrum were interpreted as evidence of a trans-cis isomerization of the chromophore. here we have combined x-ray crystallography and site-directed mutagenesis to assess whether we can create a stable fluorescent, red-shifted eqfp variant with a cis chromophore. in a first step, we introduced the n s substitution. this variant, d rfp , is highly fluorescent, with the absorption (emission) maximum red-shifted by ( ) nm. with an additional s c mutation, the chromophore is found completely in the cis form. the variant, rfp , is highly fluorescent, with excitation and emission maxima at and nm. still further red shifts appear to be in reach. k. nienhaus , v. adam , d. bourgeois , g. u. nienhaus of biophysics, universität ulm, ulm, germany, esrf, grenoble, france, institute of applied physics, universität karlsruhe (th), karlsruhe, germany dendra is an engineered, monomeric gfp-like protein that belongs to a sub-class of fluorescent proteins undergoing irreversible photoconversion from a green-to a red-emitting state upon exposure to purple-blue light. this process occurs in the neutral state of the chromophore and is known to result from backbone cleavage accompanied by an extension of the delocalized π-electron system. we have measured the x-ray structure of green dendra and performed a comprehensive characterization of the optical absorption and fluorescence properties of the protein in both its green and red forms. the structure, which is very similar to those reported for the closely related proteins eosfp and kaede, revealed a local structural change next to the chromophore, involving mainly arg and a water molecule. we propose that this structural change explains the blue shift of the absorption and emission bands, as well as the markedly higher pks of the hydroxyphenyl moiety of the chromophore. the -fold enhancement of the neutral species in dendra at physiological ph accounts for the observed higher photoconversion yield of this protein in comparison to eosfp. photochromic green fluorescent protein mutants: chromophore states unveiled by raman spectroscopy s. luin, v. voliani, g. lanza, r. bizzarri, r. nifosì, p. amat, v. tozzini, m. serresi, f. beltram nest, scuola normale superiore, cnr-infm and italian institute of technology, pisa, italy the most widespread genetically-encodable fluorescent markers used for studies in living cells and tissues belong to the green fluorescent protein (gfp) family. reversibly switchable fluorescent proteins (rsfps) were developed that can undergo repeated transitions between different states, e.g. bright and dark forms. this property makes rsfps particularly attractive as active labels in biological systems for selective photolabeling applications or subdiffraction imaging. we shall present pre-resonant raman results unveiling the photophysical mechanism underlying the observed photochromic behavior. the variation of spectral properties before and after photoconversion of chemically-synthesized isolated chromophores under different protonation and/or isomerization have been analyzed, and compared to results obtained for the case of complete folded proteins comprising the same chromophores. experimental results have been analyzed within a time-dependent density functional theory, allowing us to assign all relevant vibrational modes. these results make it possible to discriminate between the effect of cis-trans isomerization and of diverse protonation states of the chromophore in the photoproducts of these proteins. s. luin et al, j. am. chem. soc. , - ( ). r. bizzarri et al., anal. bioanal. chem. , - . a combined study of the interaction of outer membrane proteins with cephalosporin antibiotics m. lovelle, i. barroso, m. j. feio, p. gameiro requimte , fac. ciências, universidade do porto, portugal gram-negative bacteria characteristically are protected by an outer membrane that serves as a selective permeation barrier. most of the β-lactam antibiotics appear to penetrate the outer membrane through these non-specific channels, and it becomes important to understand the possible interactions between β-lactams and the porin. fluorescence techniques have been largely used to characterize both the conformation and the dynamic behavior of large biological structures such as membranes and proteins. the fluorescence of the tryptophan residues is quenched in the presence of the different cephalosporin antibiotics. this reaction between the excited state of the fluorophores and the drug can be described as a formation of a non-fluorescent complex. the dependence of the fluorescence intensity upon quencher concentration for static quenching is proportional to the binding constant for complex formation. since β-lactam susceptibility is closely related to the presence of these non-specific porins, minimum inhibitory concentration (mic) by micro-broth dilution in microplate were used to assess the bactericidal activity of cephalosporin antibiotics upon on escherichia coli strain bl (de ) and a series of bl (de ) mutated in different outer membrane proteins. this combined study of the interactions at single molecular level and at in vivo level provides new insights for a better understanding of the antibiotic translocation. when used in combination with e.g. a cyan fluorescent protein, keima offers the unique opportunity to perform dual color fluorescence cross-correlation spectroscopy using a single laser line to excite both fluorophores. the molecular determinants of the large stokes shift of mkeima have been characterized structurally by combining x-ray crystallography with in crystallo uv-visible absorption, fluorescence and raman spectroscopy. our results reveal a ph-dependant "reverse chromophore protonation" of mkeima, driven by the key residue asp . moreover, the chromophore protonation state is shown to be coupled with different chromophore conformations, cis conformation at ph . , and mostly trans conformation at ph . . these results will help unravel the mechanisms of fluorescence in fps, which is of importance to rationally design and develop new fluorescent markers. recently reversibly switchable fluorescent proteins (rsfps) have become a new branch of the green fluorescent protein (gfp) like family. the rsfps may be reversibly switched between a fluorescent and a non-fluorescent state by irradiation with light of distinct wavelengths. the key process of this switching behaviour is a light induced change of the chromophore between a cis-and a trans-isomeric state. the proteins are becoming increasingly important for diverse applications like protein tracking, sub-diffraction resolution microscopy and data storage. based on the switching mechanism, we created novel rsfps with unique and improved characteristics. padron and bs-dronpa are two of these new switchable proteins. padron features a reversed switching mechanism as compared to all other green rsfps known to date while bsdronpa exhibits a very broad absorption spectrum extending into the uv. utilizing the characteristics of both proteins, we performed multi label single detection colour microscopy as well as dual colour sub-diffraction microscopy, the latter with a resolution of nm. further, we recently introduced the first monomeric rsfp exhibiting red fluorescence: using a semi rational mutagenesis approach on the non-switchable mcherry, we generated the switchable monomeric protein rscherryrev. the use of this protein in single molecule switching microscopy allowed us to record time-lapse live-cell images of the endoplasmic reticulum with sub-diffraction resolution. a spectroscopic approach to the study of chromophoric dissolved organic matter (cdom) in the sea c. santinelli, r. lavezza, l. nannicini, a. seritti cnr-ibf, via moruzzi , pisa, italy dissolved organic matter (dom) in the ocean represents the largest reservoir of reactive carbon on the earth. it is produce at each level of the marine food web and it represents the food for heterotrophic bacteria, which can use the different pools of dom (labile, semi-labile, refractory) with a large range of turn-over times (from minutes to millennia). dom plays a central role in the global carbon cycle and it has a high ecological significance. chromophoric dom (cdom) is the fraction of dom capable of adsorbing light at uv and visible spectral regions. it determines the underwater light availability in open and coastal regions with important implication on primary production and biological activity. it is photodegraded to co , co, with a significant impact on the role of the ocean as source and/or sink of atmospheric co and to highly reactive compounds, dangerous for marine organisms. in its pool "humic-like" and "protein-like" fluorophores have been individuated. cdom optical properties (absorption and fluorescence) together with doc data collected in some key regions of the mediterranean sea will be presented and discussed, in order to (i) investigate the powerful of cdom optical properties to get information on cdom "quality" (i) asses the role of dom in carbon export at depth, (iii) underline the main unresolved question on dom and cdom dynamics in the ocean, with particular emphasis to their biological lability. mechanism and applications of photo-and redox-switchable fluorescent proteins s. j. remington, j. n. henderson, x. shu, j. lohman institute of molecular biology, university of oregon, u.s.a. photoswitchable fluorescent proteins have significant advantages over conventional fluorescent labels, and in a revolutionary application, now allow cell biologists to exceed the diffraction limit in light microscopy by a factor of ten. atomic resolution crystal structures and time resolved spectroscopy of both reversible (mtfp . ) and irreversible (pa-gfp) photoswitchable fluorescent proteins in the light and dark states explain the long term stability of either state, as well as how illumination at the appropriate wavelength causes the molecules to switch between states. the photoswitching mechanisms will be discussed in terms of photochemistry, light induced chromophore isomerization and excited state proton transfer (espt). mutagenesis of espt pathways provide insight into the nature of the ratedetermining steps in proton transfer and lead to practical applications, such as redox-sensitive gfp biosensors. k. i. willig, b. hein, s. w. hell max-planck-institute for biophysical chemistry, goettingen, germany stimulated emission depletion (sted) nanoscopy is a light microscopic technique offering a resolution far beyond the diffraction limit: the excitation beam is overlapped with a doughnut-shaped, red-shifted sted beam, which switches off the ability of the molecules to fluoresce in the outer region of the excitation spot. scanning the nanosized focal spot through the sample renders sub-diffraction images with a sub-second frame rate. we used the yellow fluorescent protein citrine to image individual structural elements of living mammalian cells: vimentin and the tubular network, structures of the cytoskeleton, were recorded with a lateral (x,y) resolution < nm inside the living cell, corresponding to a -fold improvement over that of a confocal microscope. also, time lapse sted imaging of dendritic spines of yfppositive hippocampal neurons in organotypic slices outperforms confocal microscopy in revealing important structural details. as an alternative to fluorescent proteins we used a genetically encoded protein tag which can be labelled with modified organic dyes within living samples. thus nanoscale imaging of structures in the interior of living cells greatly expands the scope of light microscopy in cell biology. pulling membrane tubes from solid-supported lipid bilayers with atomic force microscopy j. w. armond , j. v. macpherson , m. s. turner department of physics, university of warwick, u.k., department of chemistry, university of warwick, u.k. information on the elastic and dynamic properties of membranes is essential for a thorough understanding of biological processes such as exocytosis, endocytosis, cell division, and pore formation. we use an atomic force microscope to pull on solid-supported lipid bilayers and observe that an energy barrier must be overcome prior to the formation of membrane tubes. we have modified elastic models of lipid bilayer vesicles to describe the free energy of a planar lipid bilayer in adhesive contact with a surface. from this model we are able to extract force-distance curves for the formation of a membrane tether, including the associated energy barrier. the experimental data can thus be understood in a quantitative fashion. this work enables the atomic force microscope as a quantitative instrument for measuring membrane pulling mechanics, and in future work will allow two-dimensional mapping of elastic properties under tension. from pores to micelles -a peptide-membrane interaction study t. l. andresen, j. r. henriksen technical university of denmark, dtu nanotech, frederiksborgvej , b , roskilde, denmark. email: thomas.andresen@nanotech.dtu.dk. research in the partitioning of peptides into lipid membranes has been intense for several years. along with scattering techniques and nmr, isothermal titration calorimetry (itc) has proven to be a powerful tool for thermodynamic characterization of peptide-membrane interactions. we have studied two antimicrobial peptides, mastoparan-x and melittin, and found that these peptides induce a range of structural transitions of popc and popc/popg membrane systems at different peptide-lipid ratios. we have found that itc can be used to elucidate the threshold where transitions occur, including the threshold for pore formation and micellation. this has been achieved using a thermodynamic model based on gouy-chapman theory, which provides the partitioning constant of the peptide-membrane interaction and thereby the concentration of peptide on the membrane surface. the structural changes have furthermore been visualized by cryo-tem. we have further investigated the pore formation in detail and found that the thermodynamic parameters of pore formation can be fully characterized using a system specific temperature where the enthalpy of peptide partitioning becomes zero. this allows for an exclusive study of the pore formation process. lactoferrin is a glycoprotein with two globular lobes, with of two domains each. since its discovery, the research on antimicrobial properties has been extended to peptides derived from this protein. the largest family studied so far is known as lactoferricin b, obtained from the protein by digestion with pepsin. more recently, a new family of antimicrobial peptides derived from lactoferrin was discovered by bolcher et al and named lactoferrampin (lfampin). the original sequence of lfampin contained residues - from the n domain of lactoferrin. we studied peptides derived from lfampin obtained by extension and/or truncation at the c-or n-terminal sides, keeping the essential characteristics, in order to unravel the main structural features responsible for antimicrobial action. the peptides were tested against model membranes. the ability to adopt helical conformation was followed by cd, the perturbation of the membrane phase transition by dsc, the energetics of interaction by isothermal titration calorimetry (itc), the partition of the peptide to the membrane by trfs and the importance of charge effects assessed by zeta potential measurements. the results are discussed and compared to the antimicrobial and hemolytic activities obtained by microbiology techniques. defensins are small cysteine-rich cationic proteins or peptides found in both vertebrates and invertebrates. they can be active against bacteria, fungi and many enveloped and non-enveloped viruses; thus, being also classified as antimicrobial peptides (amps). in the present work a comparative study between psd (a plant defensin with antifungic properties obtained from the garden pea pisum sativum) and hnp (a human neutrophils defensin) was conducted, in order to shed some light on the mechanism of action at the molecular level of these defensins. large unillamelar vesicles with different lipid compositions were used for this purpose as biomembranes model systems; namely, popc/cholesterol (characteristic of the outer leaflet of vertebrates cell membranes) and popc/ergosterol (fungal) mixtures. changes on the intrinsic fluorescence of the tryptophan residues present in these peptides upon membrane binding/insertion were followed by fluorescence spectroscopy. experimental results show the affinity of both defensins for mammalian and fungal sterols. the partitioning behavior of psd showed a high selectivity for ergosterol rich membranes, while hnp has preference for cholesterol rich membranes. preliminary characterization of atomistic computer models of galactolipid bilayers k. baczynski, m. pasenkiewicz-gierula faculty of biochemistry, biophysics and biotechnology, jagiellonian university, krakow, poland the main lipid component of thylakoid membranes are galactolipids, which constitute more than % of its lipid composition. the most common types of galactolipids found in thylakoid are monogalactosyldiacylglycerol(mgdg), whose headgroup consists of a single molecule of beta-d-galactose and digalactosyldiacylglycerol(dgdg), whose headgroup consists of two galactose molecules: alpha-d-galactose and beta-d-galactose linked by o-glycosidic bond majorty of thylakoid galactolipid have gamma-linolenic acid both in the sn- and sn- position. atomistic computer models of mgdg and dgdg molecules have been constructed and parametrized in the opls-aa force field. the molecules were used to construct three bilayer systems for molecular dynamics (md) simulations:( ) composed entirely of dgdg molecules,( ) composed entirely of mgdg molecules,( ) composed of a mixture of dgdg and mgdg molecules the ratio : . the systems have been md simulated using the gromacs . . package. the generated trajectories were analysed to determine a number of structural parameters among them: membrane thickness, average area per lipid, electron density profile accross the bilayer, order parameters, organisation of the bilayer/water interface as well as several dynamic parametrers like diffusion coefficients, lifetimes of conformational states, lifetimes od lipid lipid interactions. single mixed-lipid guv method reveals interaction of viper venom with lipid membranes n. m. ayvazyan, n. a. zaqaryan, n. a. ghazaryan dpt. biophysics, yerevan state university, armenia studies on the interaction of snake venom and organized lipid interfaces have been conducted using a variety of systems, including bilayer lipid membranes (blms), small and large unilamellar vesicles. giant unilamellar vesicles (guvs) with a mean diameter of µm have a minimum curvature and mimic cell membranes in this respect. guvs are ideal for studying lipid/lipid and lipid/protein interactions using microscopy techniques with membrane fluorescence probes. guvs were formed from the total lipid fraction from bovine brain by the electroformation method, developed by angelova and dimitrov ( ) . vipera lebetina obtusa venom was added to the sample chamber before the vesicles were formed. the membrane fluorescence probes, ans and pyrene, were used to assess the state of the membrane and specifically mark the phospholipid domains. ans and pyrene allows us to quantify the fluidity changes in the membrane by measuring of the fluorescence intensity. the presence of viper venom in guvs media reveals a noticable decreasing of membrane fluidity compare the control, while the binding of fluorophores with guvs modified by venom lead to appearance of channel activity. it was recognized early that the vipers' venom components preferred an organized lipid substrate near the lipid's phase transition and were particularly active against micellar lipids. these studies also emphasize the importance of a membrane surface curvature for its interaction with enzymatic components of venom. the attenuated total reflection fourier transform infrared (atr-ftir) spectroscopy is ideal for highly absorbing samples such as water suspensions or even bulk water due to the small light penetration depth. it is also suited for experiments on lipid membranes in excess water. for example, we have studied the hydrogen bonding (h-bonding) between cholesterol (ch) and phosphatidylcholine (pc) or sphingomyelin (sm), which could be important for the stabilization of the cholesterol-rich lipid domains. the atr-ftir method enabled comparison of the carbonyl band shape for pc/ch samples in excess h o or d o, and has confirmed similar behavior for both [ ] . secondly, we were able to analyze the amide ii band for sm/ch samples in excess h o. the observations confirm the presence of h-bonds between ch and n-h group in sm [ ] . another example is the study of the interlamellar water structure, which could influence the water-mediated phenomena in membranes. h-bonds in interlamellar water in partially hydrated lipid multibilayers are stronger with respect to bulk water. in contrast, we show by atr-ftir spectroscopy that the h-bonds are weaker in multibilayers in excess water [ ] . the bactericidal activity of antimicrobial peptides is linked to their ability to perturb the permeability of bacterial cells. they often show α-helical conformation, and many peptides have a kink in the middle of this structure, caused by pro or gly. in order to understand the role of the kink we designed various analogues of p , in which the central pro residue was moved from its central position, or removed altogether (in analogue p f). displacement of the pro residue caused a decrease of the antimicrobial activity, and an increase in the toxicity against erythrocytes, with the less active and more toxic peptide being p f. fluorescence studies suggest that both p and p f bind on the membrane surface. fluorescence experiments show that the activities of the two analogues correlate with their affinity for different kinds of lipid bilayers: the kinked p peptide has a dramatically higher affinity for negatively charged vesicles (mimicking the composition of bacterial membranes) than for neutral liposomes (similar to mammalian cells), while analogue p f exhibits comparable affinities for both membranes. therefore, our data suggest that the central pro-induced kink is involved in selectivity, inhibiting peptide binding to the membranes of eukaryotic cells. d. behn , h. hoffmeister , r. witzgall , c. steinem institute for organic and biomolecular chemistry, university of göttingen, germany, institute for molecular and cellular anatomy, university of regensburg, germany polycystin- , encoded by pkd , is an integral membrane protein with a size of kda and amino acids. the protein, which is known to be a ca + permeable, non selective cation channel, interacts with several proteins such as polycystin- , pigea or pacs- /- etc. the interaction takes place through a proposed coiled-coil domain of polycystin- located at the c-terminus of the protein.if mutation of either pkd or pkd (gene product of polycystin- ) occurs, the interaction between the proteins is disturbed leading to increased formation of renal cysts. this autosomal polycystic kidney disease (adpkd) is one of the most common genetic diseases causing renal failure due to the enormous cyst formation. in this study, the interaction of the c-terminus of polycystin- with its postulated specific interaction partners has been investigated in terms of its biological relevance. binding affinities as well as kinetic parameters of the interaction were determined. in particular, the interaction of c-polycystin- and pigea immobilized on solid supported membranes with c-polycystin- has been quantified by means of the quartz crystal microbalance (qcm) method. a dissociation constant of about nm was obtained. the results are compared with those obtained by surface plasmon resonance (spr) technique using a different immobilization strategy. correlation of the lateral structure of lipid bilayers and monolayers using two photon excitation fluorescence microscopy l. a. bagatolli membrane biophysics and biophotonics group/memphys -center for biomembrane physics, bmb, university of southern denmark most of the reported fluorescence microscopy applications on langmuir lipid films are focused in obtaining fluorescence intensity images using particular fluorescence probes. in this type of experiments the probes are generally utilized to obtain "contrast" between membrane regions (lipid domains) displaying dissimilar physical properties. the obtained information largely depends on the partition of the fluorescent probes for the lipid domains and the obtained fluorescence intensity pictures are not providing any information about the local physical properties of the lipid film. local physical properties of these distinct regions can be determined by exploring fluorescent probe related parameters such lifetime, emission shift, polarization. however these fluorescent probe's properties are almost unexplored in this type of experiments. this presentation will focus in describing a new experimental setup that includes a specially designed film balance on top of a custom built multiphoton excitation fluorescence microscope. this setup allows obtaining laur-dan gp images ( ) many studies on phase separations of lipids in bilayers and their leaflet asymmetry make use of fluorescence techniques. we used a dithionite assay, steady-state fluorescence anisotropy and fluorescence resonance energy transfer (fret) for characterization. dithionite assay was performed to measure the fluorophore distribution in the inner and outer leaflets of symmetric membranes. for low concentrations, the fluorophore is distributed almost homogeneously, whereas for concentrations > . mol % it accumulates in the outer leaflet. we discuss these effects taking into account the presence of multilamellar liposomes and dynamic effects produced by higher local bending elasticities. the results point out possible artifacts in the use of fluorophores to characterize bilayers under the assumption of their homogeneous distribution. dithionite relative bilayer permeability is discussed. the results won by fret and steady-state fluorescence anisotropy regarding lipid phase transitions are in good coincidence to each other and to results reported in the literature. the photosensitizing properties of three chlorins are compared in solution and when incorporated in dioleoylphosphatidylcholine vesicules. in solution, they possess a similar efficacy to generate singlet oxygen and a similar ability to induce the peroxidation. however, the role of the photosensitizer localization within the lipidic bilayer is strongly highlighted, when chlorins are incorporated in liposomes, both by the changes in order of peroxidation efficacy but by the measurements of the photoinduced permeation of the liposomes. the results are discussed in relation with the technology of photochemical internalization, pci. then, using giant unilamellar vesicles, we asymmetrically oxidize the membranes. we observed different shape transitions, such as budding, typical of membrane curvature modifications. the asymmetry of the shape transitions are in accordance to a lowered effective spontaneous curvature of the leaflet targeted. this effect is interpreted as a decreased preferred area of the targeted leaflet compared to the other, due to the secondary products of oxidation. permeabilization of guv by photooxidation is interpret as the opening of a pore above a critical membrane tension due to the budding of vesicles. the effective spontaneous curvature of photosensitized vesicle at lysis was estimated. additionally photooxidation was shown to be fusogenic. influence of polyphenol extracts from fruits on biological and model membranes d. bonarska-kujawa, h. pruchnik, j. sarapuk, j. oszmiański, h. kleszczyńska univ. of environmental and life sciences, wroc law, poland biological activity of polyphenol extracts from apple, strawberry and chokeberry was studied. polyphenols were shown to be good antioxidants and to act as antyhemolytic agents. both the activities are the result of polyphenols incorporation into biological membranes. to check potential changes they induce in membranes some experiments were performed with the use of erythrocytes, and lipid membranes. it was found that the extracts studied induced shape transitions of erythrocytes. they were classified according to the bessis-brecher scale. strawberry extract induced mainly discocytes and discoechinocytes. populations of discocytes, echinocytes and some discoechinocytes were found on applying apple extract, while chokeberry induced mainly the formation of echinocytes and spheroechinocytes. the results evidence that the polyphenols incorporate into the external monolayer of lipid bilayer of the erythrocyte membrane. the results of the fluorimetric experiments showed that all the extracts changed fluidity in the hydrophilic part of rbc membrane. the changes observed were biggest for chokeberry extract and smallest for strawberry one. incorporation was also followed by changes in electrical resistance of black lipid membranes and in shifting the temperatures of main transition (t m ) and pretransition (t p ) in liposomes. this work was sponsored by ministry of science and education, scientific project no. n n . a. boll , n. czudnochowski , m. geyer , c. steinem institue of organic and biomolecular chemistry, university of göttingen, germany, mpi for molecular physiology, dortmund, germany hiv- tat belongs to the accessory proteins of hiv and has regulatory functions. tat is concentrated in the nucleus and nucleolus of infected cells. the protein is composed of amino acids with a molecular weight of kda. tat is a transcriptional activator protein, which stimulates rna polymerase ii-mediated transcription elongation. therefore, tat interacts with cyclin t and binds to the tar rna element. tat has different domains. with respect to the interaction with lipid membranes, the most important structural motif is its basic region, including arginine and lysine residues. the peptide derived from this basic region belongs to the cell-penetrating peptides and is able to translocate through lipid membranes. the major aim of this study is to investigate the influence of full length hiv- tat on artificial lipid membranes. as a starting point solid-supported membranes composed of -palmitoyl- -oleoyl-sn-glycero- phosphocholine (popc) immobilized on silicon dioxide were used. the influence of the lipid head groups on the interaction with tat was analysed by using membranes composed of the negatively charged lipid -palmitoyl- -oleoyl-sn-glycero- -[phospho-l-serine] (pops) in a mixture with popc. fluorescently labelled tat was used to localise the protein in the membrane. the impact of tat on lipid membranes was investigated by fluorescence and atomic force microscopy, showing that it is capable of perturbing lipid membranes. g. bocchinfuso , a. palleschi , b. orioni , g. grande , f. formaggio , c. toniolo , y. park , k. s. hahm , l. stella univ. of rome tor vergata, dept. of chemistry, italy, dept. of chemistry, univ. of padova and cnr, italy, chosun univ., rcpm, south korea. most antimicrobial peptides exert their activity by perturbing the permeability of bacterial membranes, but the molecular details of this process are still debated. here, we compare fluorescence experiments and molecular dynamics simulations regarding the interaction of two antimicrobial peptides (pmap- and trichogin ga iv) with lipid membranes, showing that their mechanisms of bilayer perturbation are significantly different. pmap- , a cationic peptide member of the cathelicidin family, associates to the membrane close to its surface and parallel to it, and in this arrangement it causes a severe perturbation to the bilayer, both regarding its surface tension and lipid order. on the other hand, trichogin ga iv, a neutral peptide member of the peptaibol family, undergoes a transition from a surface-bound state to an inserted orientation. in the first arrangement it does not cause any strong membrane perturbation, while in the second orientation it may span the bilayer from one side to the other, despite its relatively short length, by causing a significant thinning of the membrane. lipopolysaccharide (lps) is the main component of the outer membrane of gram negative bacteria. lps is also known as endotoxin because of its potency to induce sepsis, a serious source of mortality in many clinical cases. among lps-neutralizing agents, polymyxin b (pxb) is considered as the "gold standard" due to its high efficiency of binding and detoxification of endotoxin. in this work, we have further explored the interaction of pxb to lps from the rough mutant of salmonella minnesota (re-lps) both in the gel and in the liquid crystalline phase, using isothermal titration calorimetry (itc) and fluorescence based techniques. the effect of pxb binding on lps-membrane integrity was determined with a fluorescence quenching assay treating vesicles of lps labeled with nbd-pe with dithionite. thermodynamic parameters associated with the binding process as well as binding stoichiometry were obtained from itc experiments. finally, itc was conducted with enterococcus faecalis and salmonella typhimurium, as representative examples of a gram negative and a gram positive bacterium respectively. pressure jumps -an accessible trigger for biomolecular transformations high pressure can be used to induce many structural changes in biological systems: from triggering phase changes in model membranes to causing protein unfolding, in fact any change involving a volume reduction is promoted by pressure. as well as being broadly applicable, pressure changes can be applied very quickly both up and down in contrast to other structure change triggers such as temperature jumps. fast pressure jumps allow the trigger to be decoupled from structural changes, so with fast structure probe techniques such as time resolved x-ray diffraction, the out-of-equilibrium evolution of these systems can be monitored. despite great advantages, high pressure remains under-utilised primarily due to its technical difficulty. in response to this technology vacuum a high pressure user facility based around a pressure jump cell for small and wide angle x-ray diffraction has been commissioned at beamline i , diamond light source, uk and will be freely available to the user community. the cell is highly robust requiring virtually no user intervention during an experiment and the pressure system is computer controlled with a graphical user interface and is integrated with the beamline. pressures between and . gpa are accessible and jumps can be carried out in approximately ms at temperatures from - to • c. sample changing has been made simple and fast with a dedicated sample loading port and modular sample holders allow optimisation for a broad range of samples. the transformation of vesicle and lateral distribution of mobile membrane inclusions b. bozic institute of biophysics, faculty of medicine, university of ljubljana, lipičeva , si- ljubljana, slovenia membrane inclusions such as membrane embedded peptides or proteins exhibit curvature dependent interaction with the surrounding lipid matrix due to the mismatch between their intrinsic curvature and the local membrane curvature. this interaction causes an inhomogeneous lateral distribution of the inclusions and a corresponding adjustment of the vesicle shape. by taking into consideration that the membrane free energy includes elastic energy of the lipid bilayer and a contribution due to an inclusion-membrane interaction, the configurations of lipid vesicles with mobile inclusions have been studied theoretically. the variational problem to calculate equilibrium vesicle shapes is solved by applying a ritz method based on an expansion in spherical harmonics. in general, vesicle shapes adjust to the presence of inclusions by increasing regions with favorable curvature and decreasing regions of unfavorable curvature in such a way that the lateral distribution of inclusions becomes inhomogeneous. if the number of inclusions or the inclusion-membrane interaction exceeds a certain value, the prolate shapes become globally stable. investigating the structure of pores formed by antimicrobial peptides using epr spectroscopy m. bortolus , k.-s. hahm , a. l. maniero universita' di padova, padova, italy, chosun university, kwangju, south korea spin label electron paramagnetic resonance (epr) is a spectroscopical technique effective to study the molecular mobility of membrane components and the membranepeptide interactions, as the timescale of epr is optimally matched to the rotational motions of lipids in membranes. we applied epr to study the pore-forming mechanism of two antimicrobial peptides (amp) that create pores of different dimensions when interacting with liposomes. hp ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) is derived from the n-terminus of helicobacter pylori ribosomal protein l , and hpa is an hp ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) analogue where gln and asp at positions and were substituted by trp. we studied the interaction of the two amp with zwitterionic and negatively charged vesicles, doped with phospholipids spin labelled in the lipid head or at the c or c positions; the different phospholipids allow us to study the peptide-membrane interaction at different depths relative to the membrane surface. we studied the interaction of the amp with vesicles following the influence of amp on label mobility as a function of temperature and membrane depth. we also prepared spin-labelled dmpc/dhpc bicelles, doped with lanthanide ions (dy + /tm + ) that allow us to macroscopically orient the system using the magnetic field of the epr spectrometer. we studied the interaction of amp with the oriented bicellar system monitoring the effect of amp on the order parameter of the phase. a. chattopadhyay centre for cellular and molecular biology, hyderabad, india we addressed the organization and dynamics of the human serotonin a receptor fused to enhanced yellow fluorescent protein (serotonin a r-eyfp) expressed in cho cells. serotonin a receptors are prototypical members of the gprotein coupled receptor superfamily and represent a prime target for therapeutic actions of several anxiolytic and antidepressant drugs. interestingly, we observed significant retention in fluorescence of serotonin a receptors upon triton x- treatment of intact cells at low temperature demonstrating their detergent insolubility. we analyzed the role of cholesterol in the plasma membrane organization of the serotonin a receptor by fluorescence recovery after photobleaching (frap) measurements with varying bleach spot sizes. our results show that lateral diffusion parameters of serotonin a receptors are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin a receptors in the plasma membrane. interestingly, results from frap measurements performed under conditions of mild cytoskeletal destabilization suggest that receptor signaling is correlated with receptor mobility, in agreement with the 'mobile receptor hypothesis'. our current work is focused on exploring the oligomerization of the receptor using photobleaching anisotropy measurements and indicates the presence of constitutive oligomers of the serotonin a receptor in live cells. expression and reconstitution of connexin in pore-suspending membranes c. carnarius , s. kaufmann , m. tanaka , c. steinem institute of organic and biomolecular chemistry, university of göttingen, tammannstrasse , göttingen, germany, institute of physical chemistry, university of heidelberg, im neuenheimer feld , heidelberg, germany the intercellular communication and electronic coupling between adjacent cells of vertebrates are mediated by gap junctions. these proteins are composed of two connexon hemichannels, whereas each connexon consists of six connexin subunits. each subunit is characterized by two conserved motives: two extracellular loops and four transmembrane α-helices. in this study, we focused on cx . to obtain large amounts of this protein, we expressed cx and cx +gfp in a rather new expression system: dictyostelium discoideum. in contrast to human tissue cultures, the system allows for high cell densities up to million cells per ml and the cells can be cultivated by fermentation. cx +gfp was successfully visualized in d. discoideum by confocal laser scanning microscopy, where it was preferentially found in the plasma membrane.after the cells were harvested, plasma membranes were prepared and both proteins (cx and cx +gfp) were verified by western blot analysis. the proteins were solubilized by addition of % n-octyl-β-d-glucopyranoside and purified by ion metal chelate affinity chromatography. the activities of both proteins were confirmed by a cytochrome c assay. after the purification of both proteins, they were reconstituted in µm-sized pore-suspending membranes. in the near future, we plan to determine the mobility of cx and cx +gfp in these membranes by fluorescence recovery after photobleaching. o. cañadas, c. casals dpt. biochemistry and molecular biology i, and ciber enfermedades respiratorias, complutense university of madrid, madrid, spain inhaled bacterial lipopolysaccharide (lps) may incorporate into the lung surfactant monolayer. in this study, the effect of smooth lps (s-lps) on the surface activity of lung surfactant was evaluated. to that end we investigated the behavior of dppc films containing s-lps, with and without surfactant protein a (sp-a) in the subphase, using epifluorescence microscopy combined with a surface balance. our data show that s-lps injected into the subphase incorporated into dppc films forming mixed dppc/s-lps monolayers. cospread s-lps fluidized the dppc monolayer as demonstrated by epifluorescence images and changes in the compressibility modulus of the monolayer as a function of s-lps molar fraction (x s−lps ). the presence of low amounts of s-lps in the monolayer promoted early collapse, preventing high surface pressures to be reached. moreover, s-lps hampered the re-spreading of dppc molecules during dynamic compression at s-lps concentrations as small as x s−lps = . . such inhibitory effects could not be relieved by repeated compression-expansion cycles or by adding surfactant protein a. however, sp-a facilitated the squeeze-out of s-lps from dppc/s-lps mixed monolayers, suggesting that sp-a is an s-lps scavenger. cholesterol displaces ceramide from its tight packing with sphingomyelin in the absence of ld phase j. v. busto, j. sot, j. requejo-isidro, f. m. goñi, a. alonso unidad de biofísica (csic-upv/ehu) and departamento de bioquímica, u. país vasco (upv/ehu), spain a set of biophysical approaches have been applied to study the phase behaviour of palmitoylsphingomyelin (psm)/cholesterol (chol) model membranes upon palmitoylceramide (pcer) addition. fluorescence spectroscopy of di- -aneppdhq-stained psm/chol vesicles reveals no segregation of large liquid-ordered (l o ) microdomains. in contrast, the formation of disperse, compositionally homogeneous l o psm/chol ( : ) nanodomains over a psm gel (l β ) phase is proposed. dsc measurements show that pcer addition to vesicles with coexisting l o and l β phases (low [chol] ) induces the formation of large gel-like psm/pcer microdomains, coexisting with a l o psm/chol phase. the ∆h for the psm/pcer phase at high psm/(chol+pcer) ratio is close to that of the binary mixture in the absence of chol, supporting immiscibility, but no displacement, between chol and pcer-rich phases. on the contrary, both confocal microscopy of guvs and the dsc data coincide in showing that a rise in pcer increases the gel-like phase to a lower extent than in the pure psm/pcer mixture, revealing some cholinduced restriction. in the presence of a pure l o psm/chol phase (high [chol] ), pcer addition is unable to induce the formation of large psm/pcer microdomains. the present data support the role of chol as the key determinant in controlling its own displacement from l o domains by ceramide. hydroquinones modifying lipid membrane morphology c. di vitta , l. marzorati , v. rebbin , s. s. funari iqusp, university of são paulo, são paulo, brasil, hasylab, hamburg, germany quinones are important molecules in cells. flavina, ubiquinone and coenzyme q, coq, are associated with electron transfer. coq, having a hydrophobic tail, is soluble in the internal lipid membrane of mitochondria. their reduction leads to the equivalent hydroquinones. we synthesized novel alkylthioquinones aths, to investigate their interaction with phospholipids. one or more long hydrophobic chains attached to the quinone ring alter their hydrophobicity and electron availability. we aimed at their influence on the structure of membranes. different phs highlights the charge/polarity effect on the interaction and on the morphology of the bilayer. for that, pope and popc, lipids with different charges, but identical chains were selected. x-rays diffraction on pope/ , bath, / at different temperatures and ph, showed cubic phases coexisting with the usual phases formed by simply hydrated pope, at different phs. for investigation of the charge/polarity, we turned to the popc/ , bath system (smaller charge/polarity). despite decrease in temperature of phase transition and dimensions of the lattice, the structures were the same as in hydrated lipid, illustrating a less significant effect than on the similar lipid pope. another additive, , ath, mixed with pope showed the effect of multiple thioalky chains. no morphology change was seen, compared to pure lipid. interestingly, despite both additives differ by one thioalkyl chain only, their influence on the pope matrix is so different. cross-linking of phospholipid membranes by calcium-sensitive synaptotagmins synaptotagmins are vesicular proteins implicated in many membrane trafficking events. they are highly conserved in evolution and the mammalian family contains isoforms. we now show that the tandem c domains of several calcium-sensitive synaptotagmin isoforms tested, including drosophila synaptotagmin, rapidly cross-link phospholipid membranes. in contrast to the tandem structure, individual c domains failed to trigger membrane cross-linking in several novel assays. large-scale liposomal aggregation driven by tandem c domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic lightscattering and both confocal and negative stain electron microscopy. high-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem c domains results in a constant distance of approximately nm between the apposed membranes. our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem c domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion. membrane proteins can be challenging samples to work with and as such often require the use of multiple techniques. here we present an insight into the structure of the antimicrobial peptide melittin in lipid membranes using various techniques. we explore the conformational changes of membrane-bound melittin and its interaction with model membrane lipid systems with a series of spectroscopic methods that can be used in parallel. the techniques used include linear dichroism and ft-ir for orientation information and circular dichroism for conformation information. we use dynamic light scattering for molecular sizing, fluorescence emission for information on peptide environment, thin-layer chromatography for lipid identification, analytical ultracentrifugation to identify oligomerisation state and calorimetry to investigate the thermodynamics. we observe how the physical properties of both the peptide and the membranes affect the insertion kinetics of the peptide in the membrane. phospholipid membranes dynamics: molecular dynamics vs neutron scattering v. conti nibali , m. tarek , u. wanderlingh , g. d'angelo department of physics, faculty of science, university of messina, italy, unité mixte de recherche cnrs/uhp , université henri poincaré, nancy, france collective dynamics and single-particle dynamics of hydrated multilamellar phospholipid bilayers ( , -dimyristoylsn-glycero- -phosphatidylcholine, dmpc) have been studied by means of all-atom molecular dynamics simulations. here we report results of a gel phase bilayer at k and of a liquid crystal phase bilayer at k. coherent and incoherent dynamic structure factors and meansquare displacements have been calculated from the trajectories for both the inplane and out-of-plane lipid dynamics. moreover, the results have been compared to recent quasi-elastic and inelastic neutron scattering data. optical tweezers allow trapping of particles of different types in a wide range of sizes [ ] . among these, unilamellar vesicles are of interest as they are known to be effective vectors in drug delivery and they are also studied as models for cell membranes. this work focuses on the interactions between optically confined unilamellar vesicles and their cross-linking by proteins [ ] . synaptotagmins are vesicular proteins implicated in many membrane trafficking events having an accelerating effect on the membrane fusion. calcium-sensitive synaptotagmins are thought to confer calcium sensitivity to the fusion of secretory vesicles with target membranes [ ] . the novel optical assay reported here allowed us to visualize the cross-linking of nm liposomes mediated by synaptotagmin and calcium. the use of the optical tweezers approach to investigate the function of other fusogenic proteins related to exocytosis (i.e., snare proteins) is discussed as well. antimicrobial peptides (amps) have received increasing interest as the search for new potential antibiotics has become imperative due to increasing bacterial multiresistance. acylating the natural occurring polymyxin b (pmb) significantly enhances antibacterial activity towards two representative gram-negative bacteria e. coli and k. pneumonia compared to the nonacylated pmb. the aim of the presented work was to study various biophysical parameters, such as partitioning coefficients, zeta potentials and effective charges of a selected amp, mastoparan-x (mpx) and a propanoylated (pampx) and octanoylated (oampx) analogue, respectively. fluorescence spectroscopy, isothermal titration calorimetry and ζ-potential measurements were the main techniques used for the measurements. we employed different luv model systems; a partially charged (popg/popc : ) and a neutral system (popc). for the neutral luvs there was an increase in partitioning with increasing length of the acyl chain, whereas partioning into the partially charged luvs was governed by a balance of hydrophobic and electrostatic contributions. the selectivity for the partially charged luvs over the neutral luvs was in the order pampx, mpx and oampx. the modeled effective charge for the peptide followed the same trend as the partitioning coefficient for the three peptides for the respective luv systems. does the lysosomal membrane need triglycerides? a spectroscopic study of a simple model membrane l. duelund , k. pakkanen , m. vuento , j. h. ipsen memphys -center for biomembrane physics, university of southern denmark, odense, denmark, nanoscience center, university of jyväskylä, jyväskylä, finland lysosomes are intracellular organelles in which proteins and other macromolecules are degraded. morphological and functional changes in different compartments of the endocytic pathway are connected to several diseases. a crucial step in understanding biogenesis of lysosomes and their role in disease conditions, is to characterise the properties of the lysosomal membrane. by the use of tlc we have found that lysosomes contain non-neglible amounts of triglycerides (tg). to investigate how the presence of tgs could influence the lysosomal membrane, we have investigated the properties of a mixture of popc and triolein, as a simple model for the lysosomal membrane. we found the system to form two types of popc-rich membranes. these were determined as co-existing phases based on their spontaneous and stable separation and named heavy and light phase according to their sedimentation behaviour. by using epr and fluorescence spectroscopy the physical properties, including order, fluidity and water penetration, of these phases were found to differ markedly despite of their almost identical composition. the results suggest that presence of tgs on lysosomal membranes could have a crucial effect in the barrier functions and thus, the integrity of the organelle. model membranes with the capacity to align in magnetic fields such as bicelles and magnetically sensitive vesicles are of high interest, since their macroscopically alignment allows for the determination of structure, dynamics and topology of molecules within the membrane [ ] . we performed p solid state nmr on a magnetically sensitive lipid possessing a large positive magnetic anisotropy introduced in form of a biphenyl unit during lipid synthesis in one of its acyl chains ( -tetradecanoyl- -( -( biphenyl) butanoyl)-snglycero- -phosphocholine (tbbpc)). the phosphorus lineshapes of tbbpc mlvs studied at various magnetic fields ( . t, . t, . t, . t), revealed a drastic change in shape upon exposure to fields > . tesla: resonances resulting from phospholipid molecules oriented perpendicular to the magnetic field decrease whereas resonances resulting from parallel oriented molecules increase. this is a sign for magnetically induced vesicle deformation from vesicle to oblate ellipsoid shape. factors influencing this drastic deformation, such as magnetic anisotropy, membrane elasticity, lipid chain length and field dependency are discussed based on existing theories [ ] . hepatitis g virus (gbv-c/hgv) is an enveloped viruses belonging to the flaviviridae family. clinical findings have suggested that in people co-infected with gbv-c/hgv and human immunodeficiency virus (hiv), delayed progression of aids has been observed ( ). the mechanism by which this virus may inhibit the progression of aids remain to be elucidated. enveloped viruses acquire their lipid membranes by budding through host cellular membranes ( ) , in this process; fusion peptides play an important role. study of the interaction of hgv-c peptides with lipid membranes could lead us useful information about the mechanism that takes place. in this work we present a study on the effects of e peptides on the fluidity of and polarizability of model membranes (dmpc and dppc liposomes) by dsc and fluorescence polarization. both techniques showed that the presence of the studied sequences in phospholipid mixtures already affected the thermotropic properties of the gel to liquid-crystalline phase transition. systems were thermodynamically charac- small angle neutron scattering (sans) studies have been performed to study the structural changes induced in membranes of vesicles prepared from phospholipid and mixed phospholipid-sterol mixtures, in the presence of different concentrations of the anti-fungal drug, amphotericin b (amb).the vesicles, sonicated to a mean size∼ nm, were prepared using dimyristoylphosphatidylcholine(dmpc) or dmpc-cholesterol or dmpc-ergosterol mixtures -with both of the mixed systems involving mol% sterol. analysis of the sans data show that when the concentration of amb added is just above the drug's cmc (∼ µm) there is an increase in the membrane thickness of both the dmpc-chol and dmpc-erg vesicles (both cases + Å), but the thickness of the pure dmpc vesicle membranes remains the same as in the absence of amb. when amb is added at a concentration in excess of its cmc (∼ µm), the mixed-sterol vesicles show the same changes in membrane thickness as observed with the lower amb concentration, and the pure dmpc vesicles again remain unaffected. on the basis of these studies, therefore, there appears to be no difference in the structural changes induced by the insertion of amb into the model fungal cell membranes (mimicked by dmpc-erg vesicles and those resulting from its insertion into the model mammalian cell membranes (mimicked by dmpc-chol vesicles). the third transmembrane helix of bacteriorhodopsin, also known as phlip, is a unique model system for studying the interactions of a natural transmembrane domain with lipid membranes: depending on ph, the water-soluble peptide either adsorbs superficially or inserts as a transmembrane helix on addition of lipid vesicles [ ] . published values for the free energies of these processes were based on a stoichiometric model invoking two distinct sets of binding sites [ ] . however, discrepancies between data obtained from different experimental techniques and inconsistencies between experimental and expected temperature dependencies suggest that these values should be taken with caution. we therefore reassessed membrane interactions of phlip using titration calorimetry and fluorescence spectroscopy. if electrostatic effects at the membrane surface are taken into account, the data can be described quantitatively by a partition equilibrium, but not by a stoichiometric binding model. the thermodynamics of membrane partitioning differ substantially from those determined previously [ ] and draw a different picture of peptide-lipid interactions. beyond deepening our insights into the first step of the two-stage model of membrane protein folding, this also sheds light on the ability of phlip to drag cargo molecules across lipid membranes. adsorption of monolysine and polylysines at the surface of lipid membranes of varied composition was studied by two methods. both methods -the electrokinetic one, measured the surface potential of liposomes, and the intramembranouse field compensation sensitive to boundary potential of planar bilayer lipid membranes -detected the positive changes of the potentials for membranes with negatively charged component (cardiolipin, cl). neither monolysine, nor polylysines adsorbed at neutral membranes (phosphatidylcholine, pc). pentalysine show the difference between these potentials for the membranes composed from cl/pc mixtures. this difference is attributed to dipole part of boundary potential and indicates the changes in lipid packing. polylysines show high affinity to the membrane and saturation with plateau. these saturation levels correspond to surface charge densities . and . c/m for oligomers with and units, and . c/m for polymers with and units. these values do not depend on the ionic strength of background electrolyte but proportional to the content of negatively charged components in the lipid bilayer. the polylysine layer at the mica surface was studied by atomic-force microscope (afm) technique. it was shown that pentalysine molecules cover the surface by the layer of . nm thickness and polylysines of high molecular weight by the layer up to nm. effects of lysolipids on the mechanical stability of lipid membranes j. r. henriksen , l. feldborg , j. h. ipsen , t. l. andresen technical university of denmark, dtu nanotech, frederiksborgvej , b , roskilde, denmark., university of southern denmark, department of physics and chemistry, memphys center, campusvej , odense m, denmark lysolipids (lpcs) and fatty acids (fas) are the natural products formed by the hydrolysis of phospholipids. lpcs and fas form micelles in solution and thus act as detergents in the presence of lipid membranes. in the present study we investigate the detergent strength of a homologous series of lysolipids (lpcs) on popc lipid membranes by use of isothermal titration calorimetry (itc) and vesicle fluctuation analysis (vfa). the membrane partition coefficient (k) and critical micelle concentration (cmc) are determined and found to obey an inverse proportionality relation (cmc x k ∼ constant). the partition coefficient and critical micelle concentration are used for the analysis of lpc's effect on the membrane bending rigidity. the dependence of the bending rigidity on the lpc membrane coverage has been analyzed in terms a phenomenological model based on continuum elastic theory which yield information about the curvature inducing properties of the lpc molecule. the results reveal: (i) an increase in the partition coefficient with lpc acyl-chain length and (ii) the degree in acyl chain mismatch between lpc and popc determines the magnitude of the membrane mechanical perturbation per lpc molecule on the membrane. patch clamp electrophysiology remains the gold-standard for ion channel research because of the information richness of the data produced. automated planar patch clamp devices, with their higher throughput and high data information content, have made the technique accessible to a wider audience. nanion technologies offers two planar patch clamp workstations combining higher throughput with high data quality. the port-a-patch records from a single cell at a time and the patchliner from up to cells simultaneously with high success rates (typically - %). both, the port-a-patch and the patchliner are bench top patch clamp rigs which uses a planar borosilicate glass chip for obtaining a giga-seal for electrophysiological recordings. suction applied from the underside of the chip is used to attract a single cell to the recording site without the need for optical visualization. both workstations have been successfully used for whole cell, perforated patch, and cell attached recordings as well as for guv-bilayer recordings. due to the versatility of nanion`s products it is possible to study a wide variety of ion channels including nav, kv, cav, ligandgated, herg or reconstituted proteins such as ksca, cx , ompc. special features unique to nanion products, including but not limited to internal solution exchange and temperature control, expand the experimental possibilities. waves on lipid monolayers j. griesbauer, s. boessinger, a. wixforth, s. f. matthias univiversity of augsburg, experimental physics i, biological physics group, augsburg, germany " for the sake of illustration we shall try to provide a physical basis for the equations, but must emphasize that the interpretation given is unlikely to provide a correct picture of the membrane." [ hodgkin&huxley, ] to explain the occurrence of reversible heat production during nerve pulse propagation it has been suggested by multiple authors [wilke,kaufmann,heimburg] that travelling sound waves and not ion channels may offer a better explanation than the hodgkin&huxley theory. therefore, if sound waves are an essential feature of the nerve membrane they should also appear on lipid monolayers where ion conductivity is evidently absent. here we demonstrate that sound waves can be excited on a lipid monolayer by using a set of planar electrodes incorporated into the monolayer and driven by an alternating voltage. not only do our results indicate propagating waves on lipid monolayers in accordance with their thermodynamic predictions, but importantly, no significant attenuation is detected proposing an adiabatic phenomenon. in order to provide evidence that our physical explanation provides a correct picture of the membrane, direct detection of the waves was done, whereas a clear transduction of signals was shown. finally, the impact of toxins, neuropharmaca and anaesthetics needs to be integrated in our physical picture of the nerve membranes, what can easily be done now and could deliver a new approach for understanding their physical mechanism. our earlier studies showed that organometallic compounds (otc) in the presence of uvc radiation enhanced the degree of phosphatidylcholine (pc) liposome oxidation, whereas quercetin effectively protected the membrane. the present investigation is concerned with the effect of uvb and otc (chlorides of diphenyl-and dibutyltin, triphenyl-and tributyltin), both in combined action and separately, on oxidation of erythrocyte proteins, pc liposome and albumin. the degree of oxidation of proteins and liposomes induced by otc and radiation, also in the presence of selected antioxidants (trolox, quercetin) was determined on the basis of changes in the number of sulfhydryl groups, carbonyl groups and malone dialdehyde, respectively. the studies indicate that uvb induces pc liposome and erythrocyte oxidation, whereas in the case of albumin it causes both an increase in the number of c=o groups and free sh groups (most probably, braking sulphonic bridges). otc compounds interact with membrane biomolecules both as weak pro-and antioxidants, also in combined action (uvb plus otc). the prooxidative effects are markedly diminished by application of antioxidants. the action of quercetin results from its ability to incorporate into membranes and formation of complexes with otc (liposome>erythrocyte>albumine). this work was supported by grant n n . a phospholipase c/ sphingomyelinase from pseudomonas aeruginosa has been assayed on giant unilamellar vesicles (guv) consisting of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and cholesterol, at equimolar ratios. the enzyme activity modifies the chemical composition, thus the physical properties of the bilayer, and conversely the latter influence the enzyme activity. biochemical assays of enzyme activity, together with confocal fluorescence microscopy examination of guv provide novel information about the system. the original lipid composition in the absence of enzyme gives rise to lateral phase separation of liquid-ordered and liquid-disordered domains in the guv. the two enzyme end-products, diacylglycerol and ceramide, have opposite effects on the bilayer physical properties, the former abolishes lateral phase separation, while the latter generates a new gel phase. morphological examination of individual guv shows that the enzyme binds preferentially the more fluid (or more disordered) domains, and that, in most cases, it causes the fluidification (disordering) of the other domains.. cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. to this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures ( - mol% cholesterol) have been measured and compared with the original mixture before lipid hydration. there is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the bilayers is much lower than expected. the maximum solubility of chol in bilayers containing saturated pc or pc with less than four unsaturations is - mol%, while in polyunsaturated pc, e. g. dic : , and in sphingomyelin the maximum chol contents is mol%. a quantitative analysis of the vesicles is thus required before any experimental study is undertaken. membrane fusion assay based on poresuspending lipid bilayers i. höfer, c. steinem institute of organic and biomolecular chemistry, university of göttingen, germany membrane fusion has attracted significant interest due to its biological relevance. thus in recent years a great variety of fusion assays has been developed. since the process of membrane fusion is not yet fully understood, our aim is to develop a new fusion assay based on pore-spanning membranes, which have been proven mechanically stable, highly insulating and rather tension free. the application of these so-called micro-blms should allow simultaneous monitoring of lipid mixing, content release as well as electrical readouts. furthermore both membrane sides can be addressed individually to apply transmembrane potential or fusion modulating compounds. first results show that the micro-blms provide the opportunity to investigate lipid bilayer fusion by means of fluorescence microscopy. the formation of porespanning membranes is achieved by the painting technique of dphpc doped with oregon green dhpe dissolved in ndecane. the addition of large unilamellar vesicles doped with texas red dhpe allows direct observation of single fusion events. lipid mixing during fusion leads to a decrease of the donor fluorescence (oregon green) and an increase in acceptor fluorescence intensity (texas red) in the plane of the planar bilayer due to fret. in future work simultaneous monitoring of lipid-mixing and content release combined with electrical measurements are planned to gain further insight into different intermediate steps of membrane fusion. the kinetics of membrane-peptide folding and orientation m. r. hicks department of chemistry, university of warwick, uk. understanding interactions of peptides with lipid membranes is essential if we are to be able to design new and better antibiotic peptides. there are different steps in these interactions and following the kinetics of these processes requires that we combine the information from different biophysical techniques. here we present data on the kinetics of peptidemembrane interactions using circular dichroism to report on conformation and linear dichroism to report on orientation of the peptides in/on membranes. these are combined with other techniques such as dynamic light scattering and fluorescence spectroscopy to elucidate the mechanisms of action of the peptides. one of the most important aspects of membrane-active peptide design is that of specificity. we investigate specificity for different types of membranes by using libraries of lipids with different properties of charge, chain saturation and curvature stress. in this way one can test for effects of e.g. negatively charged head groups found in bacterial membranes or cholesterol found in animal and human membranes. using these approaches it is proposed that we will be able to modify peptide sequences, test what part of the kinetic processes are affected and subsequently use this information to design new and better antibiotics. k. kubica, h. misiak wroclaw university of technology, wroclaw, poland reversible membrane electroporation, that can be observed as a effect of electric field influence on biological membranes, finds application in cell delivery of biologically active compounds. the membrane susceptibility for creation of stable pores, depends on electric field parameters applied to the membrane as well as on the membrane environment (ionic strength, ph, temperature) and membrane molecular composition. we have already shown that the changes of van der waals lipid chain interaction effect the process of pore formation. there are known factors which increase or decrease the membrane respond to electric field. because pores appear in lipid membrane matrix it make sense to study this phenomenon using model planar lipid membranes. as lipids are very receptive on oxidizing factors we decided to study the relation between lipid oxidation and membrane reaction on electric field. with the use of four electrode galvanostat we are examining the influence of factors initiating lipid oxidation (uv, fe + ) and the efficiency of some antioxidant compounds of lipid protection on electric properties of lipid membrane. the product of lipid oxidation is determined by characteristic reaction with tiobarbituric acid. we want to determine the role of polyunsaturated fatty acids on membrane properties also. results of our work will be useful in optimization of medicine distribution, aided by electric field, into cells. budding of giant phospholipid vesicles induced by β -glycoprotein i j. kovacic, b. bozic, s. svetina institute of biophysics, faculty of medicine, university of ljubljana, ljubljana, slovenia β -glycoprotein i (β gpi) is classified among amphitropic proteins: in its inactive form it is dissolved in plasma and in its active form it is bound to membrane. in comparison to other amphitropic proteins it exhibits a distinct membrane interaction behavior. a patch of positively charged amino acid residues contacts anionic phospholipids via electrostatic interactions, and a hydrophobic loop anchores the protein into the outer leaflet of the membrane via hydrophobic interactions. the binding constant depends on physical properties of the membrane such as membrane lipid composition, surface potential, lipid packing density and curvature. the binding of an amphitropic protein alters the membrane's spontaneous curvature as well as the difference between the equilibrium areas of membrane leaflets. theoretical model has been developed to predict vesicles shape transformations realized by the formation of buds. it was shown that the effects are stronger for flaccid vesicles which were therefore chosen for our experimental investigations. vesicles were composed of % pops and % popc, and flaccidity was achieved by an osmotic adjustment. the solution containing β gpi was subsequently injected to a chamber with flaccid vesicles by a syringe pump. observation took place under a phase-contrast microscope. experiments showed a concentration dependent occurrence of buds. their number and size were related to the degree of vesicle flaccidness. the interplay between detergent cohesion and peptide adsorption on the structure and dynamics of a glycophorin a tm-detergent complex j. khao, j.-p. duneau, j. n. sturgis lism, cnrs -aix marseille university, marseille, france in spite of the major interest of membrane proteins at functional, genomic and therapeutic scales, their biochemical and structural study remain challenging as they require, solubilization in detergent micelles. the complexity of this task arises from the structural dependence of membrane proteins on their anisotropic environment and in particular by a delicate balance between different physico-chemical properties. in order to study these properties in a small protein detergent complex, we have used molecular dynamics simulations on the transmembrane part of glycophorin a (gpatm) solubilized in micelles of the detergent di-hexanoyl-phosphatidylcholine(d pc). we show that the molecular aggregates organizes to give distinct populations of detergent molecules. those molecules which loosely interact with the peptide are preferentially involved in highly cohesive inter-detergent interactions that impose a global bilayer structure to the micelle. interaction profiles of other detergents with gpatm depend upon the nature of residues along the surface of the peptide. this topology dependence leads to different modes and strength of interactions that ultimately constrain the orientation of the micelles around the peptide. this simple model illustrates how differential detergent selectivity for faces and strong constraints coming from purely environmental features could influence transmembrane helix packing, membrane protein structure and assembly. in this paper we present a study of a new family of bolaamphiphiles. these amphiphiles are unsymmetrical bolalipids having one sugar polar head and the second glycine betaine polar head. they are potentially useful for pharmaceutical or cosmetics applications as vectors for drugs. therefore it is important to investigate their self-assembled properties. the chemical variations that we introduced in this new family concern the length of the main chain that connects two polar heads as well as the length of the side chain placed on the position of the sugar moiety. another variation concerns the introduction of a diacetylenic unit into the main chain in order to rigidify it. we have performed the saxs (small angle x-ray scattering) measurements on the dehydrated compounds as a function of temperature and observed the lamellar liquid crystalline structures. we also measured the saxs spectra of aqueous solutions of these compounds that have shown lamellar l α structure in all cases. these measurements are compared with polarised optical microscopy measurements that confirmed our interpretation. formation of membrane tubular structures induced by phase separation in giant vesicles y. li, r. lipowsky, r. dimova max planck institute of colloids and interfaces, science park golm, potsdam, germany tubular membrane structures (also known as tethers) exist widely in eukaryotes. abundant work has been done on tube extrusion from cells and model membranes under the application of external forces. we present a novel system allowing tube formation in the absence of external forces. aqueous solutions of two chemically dissimilar polymers, polyethylene glycol (peg) and dextran are encapsulated in giant vesicles, a cell-size model system. the exposure of vesicles to hypertonic solutions induces phase separation of the internal aqueous polymer solution. the excess membrane area created by this vesicle deflation, engages in the formation of tubular structures. membrane tube formation and phase separation are coupled processes. hydrodynamic flows and changes in the membrane spontaneous curvature during phase separation might be the driving forces for tube formation. the tubes are rather stable: without external perturbation, they can exist for several days. they prefer to be located in the peg-rich phase at low polymer concentration. at high concentration, they are absorbed at the interface of the liquid phases to lower the surface energy of the system by decreasing the contact area between the liquid phases. the membrane tubes can be retracted back to the vesicle surface by increasing the membrane tension via vesicle aspiration. membrane tubes, which can form and be retracted easily, might be relevant to lipid storage in cells. insight into the antimicrobial mechanism of a de novo auto-assembling peptide b. legrand , m. laurencin , e. duval , c. zatylny , j. henry , m. baudy-floc´h , a. bondon rmn-ilp, umr cnrs -univ. de rennes , france, icmv, umr cnrs -univ. de rennes , france, pemm, umr ifremer -univ. de caen, france a short ( residues) de novo antimicrobial peptide (k ) composed of a cationic polar head and a hydrophobic tail was studied. it exhibits a broad spectrum of antimicrobial activity on bacteria. no haemolytic activity or cytotoxicity on eukaryotic cells are observed at mic. when bacteria are lysed by the k , spherical objects are observed on the sem micrograph. k was structurally studied using various membrane mimetic media such as different micelles and small unilamellar vesicles (suv) of different composition. cd revealed that k adopt various structures (random, β-turn, α-helix) in the presence or the absence of detergents or phospholipids. nmr structures confirmed the α-helical structure of k hydrophobic tail in presence of sds. k self-assembles at high concentration as observed by sem and dls. we suggest that k may act as a surfactant building mixed microsomes composed of peptide and lipids. this destabilization mechanism of the bacterial membrane support the "detergent like model" previously described in the literature. oxidative stress and the membrane dipole potential; modulation with tocopherol s. le-nen-davey , b. m. davis , j. l. richens , k. a. vere , m. w. tilley , p. g. petrov , c. p. winlove , p. o'shea biomedical physics group, university of exeter, uk, cell biophysics group, university of nottingham, uk tocopherol, a component of vitamin e well know for its antioxidant properties, has recently been thought also to influence the structure of cellular membranes. tocopherol treatment reduces hyperglycaemia induced oxidative stress and the associated endothelial dysfunction which is a precursor to the vascular complications of diabetes. tocopherol and insulin interactions are also modified by hyperglycaemia. it is unclear whether these clinically important effects arise from the anti-oxidant and/or structural properties of tocopherol. we have therefore measured the dipole and surface potentials of phosphatidylcholine vesicles containing different amounts of cholesterol, ketocholesterol and tocopherol. we have also investigated the effects of hyperglycaemia, ketocholesterol and tocopherol, alone and in combination, on microdomain formation and interactions of insulin within the membranes of cultured endothelial cells. both sets of experiments indicate that tocopherol causes significant modifications of the membrane dipole and surface potentials. the physiological significance of these changes will be discussed. t. d. lazzara , a. janshoff , c. steinem georg-august university göttingen, institute for organic and biomolecular chemistry, tammannstr. , , germany, georg-august university göttingen, institute for physical chemistry, tammannstr. , , germany cell penetrating peptides (cpp) have been shown to penetrate cellular membranes. they have been of interest for their ability to translocate not only themselves through cellular membranes, but also carry along with them, cargo as large as iron nanoparticles. the exact entry mechanism remains unclear, but has been shown to vary with peptide sequence. their translocation properties have been demonstrated through different experiments involving vesicles, cells and living animals. we plan on using nano-black lipid membranes (nano-blm), which span the pores of nanoporous materials as a model system to study the interaction between cpp and lipid membranes. the nanopores can be used as cellular containers whose interior surface can be functionalized with receptors for biotin-streptavidin recognition. we hope that this system will provide greater control over experimental variables, such as the type of cpp and lipid used, as well as provide kinetic data that can be used to evaluate cpp activity and the kinetics of cargo transport. ethanol induces phospholipid acyl chain interdigitation. while much is known, important issues remain unclear, such as the role of lipid domains. the main purpose of this study was to follow, in real time, the changes induced by ethanol on supported lipid bilayers with nano to microdomains by in situ atomic force microscopy. to this goal, a pure lipid in the fluid phase (dopc), a pure lipid in the gel phase (dppc), binary phospholipid mixtures with gel/fluid phase coexistence, and cholesterol-containing, raft -forming mixtures (dopc/dppc/cholesterol and dopc/sphingomyelin/cholesterol) were investigated. from the height differences observed upon ethanol addition to pure lipids (dppc and dopc), and to dopc/dppc mixtures, it is shown that in the binary system the interdigitation of the fluid phase occurs prior to the gel phase. however, for the lipid rafts mixtures the simultaneous interdigitation of both raft and non raft portions of the membrane is observed both in mica and silicon substrates. for all compositions studied, domain formation or rearrangement accompanied by lipid bilayer expansion occurs as a consequence of interdigitation. these results show the ability of ethanol to influence the bilayer properties in different ways according to membrane composition. ethanol may exert its biological effects by reducing bilayer thickness, and also by changing membrane proteins conformation and lateral distribution, as a consequence of the altered properties of the lipid bilayer. interactions between non-steroidal anti-inflammatory drugs and a pc/cholesterol bilayer m. markiewicz , t. librowski , p. serda , m. pasenkiewicz-gierula faculty of biochemistry, biophysics and biotechnology, jagiellonian university " department of pharmacodynamics, medical college, jagiellonian university " regional laboratory, jagiellonian university, krakow, poland. the non-steroidal anti-inflammatory drugs (nsaids) are among the most frequently prescribed and used drugs [ ] . there are several side effects connected with frequent use of nsaid, mainly gastrointestinal ulcers and bleedings. a plausible molecular mechanism of these effects are direct interactions of nsaids with gastric phospholipids [ , ] . the influence of three well-known non-steroidal antiinflammatory drugs with a diverse gastric toxicity (aspirin, ketoprofen and piroxicam) and three newly-synthesized xanthone derivatives (belonging to the nsaids) on the structure and dynamics of lipid bilayers was studied using small angle x-ray scattering and molecular dynamics simulations. the results showed some correlation between nsaid toxicity and its binding to the lipid polar groups. this binding increases membrane fluidity by reducing its density due to an increased membrane surface area. a reduced lipid packing in the membrane most likely increases gastric mucosa permeability, which can result in a decreased resistance of the gastric mucosa to luminal acid. we studied the partition of the anionic amphiphile pyrenesulfonate (psa) into mlv and luv (produced by extrusion), composed by zwitterionic popc and zwitterionic/anionic mixtures with pops (until mol %). psa is an anionic amphiphile that mimics several xenobiotics (e.g. pharmaceuticals, pesticides) and endogenous substrates that interact with biological membranes. we found increasing b. lorenz, s. schuy, a. janshoff georg august university, göttingen, germany cell-cell and cell-virus interactions are ubiquitous in living organisms. the analysis of the forces acting between two cells or a cell and a virus gives insight into details of these interaction processes such as their stochastics, cooperativity, reversibility, and energy landscape. using colloidal probe microscopy in conjunction with solid-supported lipid bilayer techniques, we mimic the contact between two membranous compounds displaying sponge glycolipids or viral peptides on their surfaces. after spreading functionalized lipid bilayers on both -a colloidal probe and a silicon wafer surface -the molecular interactions are quantified by means of force-distance curves. by probing the dynamic interaction strength between the viral peptides n and c we aim for a deeper understanding of the role of these peptides in the complex process of the formation of the prehairpin intermediate as the key step in retroviral fusion. as far as the cellular interaction of marine sponge cells is concerned, we intend to investigate the strength, specificity, and the ca + dependency of the self-recognition between sponge glycans. the systems advantages are the flexibility of the membrane composition and the control over the distribution of receptor molecules in the membranes. since adhesion in biological systems relies heavily on cluster formation within the biomembrane, we plan to mimic the clustering by "printing" interaction domains and comparing the results to homogeneous samples. development of video-rate imaging microscope using laurdan and its applications to lipid raft t. ohba, k.-i. muto, t. kiuchi, k. ohki department of physics, graduate school of science, aobaku, sendai, japan - , ohki@bio.phys.tohoku.ac.jp various biological functions are located on cell membranes, and the biomembranes maintain heterogeneity as microdomain even in its dynamic structure. existence of such domain was reported as phase-separated 'rafts' in a cell. and the bio-functions of membrane protein are affected by physical property of its surrounding lipid bilayers. laurdan is a useful fluorescence dye to monitor membrane fluidity, and we have developed an instrument to image spatial and temporal change in membrane fluidity by use of laurdan. in order to examine role of 'micro-domain' in biomembrane, we applied this imaging instrument to a giant liposome, cho cells and pc d cells. fluorescence microscope was equipped with a home-build dual-view optical unit. microscopic image of membranes stained with laurdan is separated into an image at nm and an image at nm by the combination of monochromatic filters and dichroic mirrors. the each image is focused on an image plane of a ccd camera side by side. and generalized polarization (g.p.) image is calculated from those images by personal computer according to the definition of g.p. the g.p. imaging at video rate was applied to a giant liposome of composed of dmpc and dmpe in order to observe phase separation. it was also examined that specific interaction of sphingomyelin and cholesterol in living cho cells and neuritis protrusion from raft region of pc d cells stimulated by neuron growth factor. current fluctuations in biological lipid membranes from human cell lines s. nuschele, a. wixforth, m. f. schneider university of augsburg, experimental physics , univer-sitätsstrasse , d - augsburg, germany lipid membranes can undergo phase transitions at physiological temperatures. not only do single lipid component membranes show melting behaviour but also biological lipid membranes from eukaryotes as well as from prokaryotes. performing calorimetric and monolayer studies we are able to detect phase transitions in extracted membranes from different human cell lines (e.g. keratinocytes and melanoma cells). close to and in the melting transition regime we measured distinct channel like current fluctuations. the opening times of these current fluctuations can be predicted based on the lateral compressibility measured from the monolayer isotherm and agree well with the experimental data [*]. the applied method of extraction excludes the presence of functional proteins in the membrane rendering the lipid bilayer as the source of the observed current fluctuations. the molecules are composed of single hydrophobic tail and two hydrophilic aldonamide-type groupings (gluconyl c -dga or lactobionyl c -dla) linked by the propylene chain at the nitrogen atom. the micellization processes of c -dga and c -dla were studied by means of itc. the critical micelle concentrations, the enthalpies (∆h m ) and the entropies (∆s m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (∆g m (hy)) were calculated. qspr analysis was also used to predict cmc of studied compounds. the interactions of c -dga and c -dla with model membranes (dppc and dppc/chol. bilayers) were studied by means of dsc. using quantum computations some basic molecular properties were calculated. the conformational space was explored using molecular mechanics. obtained results were compared with those for analogical compounds with single head groups, e.g. with also synthesised by us n-alkanoyl-nmethyllactitolamines (c n mela) and common sugar-based surfactants c gluc and mega- . enfuvirtide (t- ) was the first hiv- fusion inhibitor peptide approved for clinical use. t- is an inhibitor still under evaluation. previous studies, based on tryptophan intrinsic fluorescence, showed that the peptides interact differentially with membrane model systems (luv) with different lipid compositions. studies with human blood cells were necessary to further establish the role of membranes on these peptides mode of action. an experimental strategy was applied based on the membrane dipole potential, as measured by the fluorescent probe di- -anepps. human erythrocytes and peripheral blood mononuclear cells (pbmc) were successfully labeled. for both systems, a fusion inhibitor concentration-dependent decrease on di- -anepps fluorescence excitation ratio (a measure of the spectral shift and dipole potential) was observed. the quantitative analysis of these variations indicated that t- has an approximately ten-fold higher affinity towards erythrocyte and pbmc, when compared with enfuvirtide. this is in agreement with the previously known adsorption of t- on cholesterol-rich membrane domains and with its higher partition constants. hiv associates with erythrocytes in vivo, which can constitute a route to deliver peptide to the viral membranes (also rich in cholesterol). lymphocyte membranes can concentrate and accelerate the drug interactions with its molecular target, gp in its exposed conformation. oxidation of low density lipoprotein (ldl) is known to be a key step in atherogenesis, leading to inflammation, proliferation and apoptosis of cells of the arterial wall. these effects are largely exerted by oxidatively fragmented phospholipids, which are highly exchangeable between cells, tissues and lipoproteins. in particular, pgpc has been identified in minimally modified ldl and has been reported to elicit a wide range of pathophysiological responses in vascular cells, e.g. the activation of apoptotic signaling pathways. we investigated here the behavior of the fluorescent oxidized pgpe-alexa compared to dhpe-bodipy in artificial supported lipid bilayers with different cholesterol contents. the two labeled lipids differ in the type of membrane insertion: while dhpe-bodipy is anchored to the membrane via two fatty acids, pgpe is incorporated with only one fatty acid. the second chain is an acyl fragment in sn- position, which represents the oxidation product of an unsaturated acyl chain. with increasing cholesterol content we observed a decrease in the diffusion coefficient for both lipids. interestingly, the diffusion of the oxidized lipid was reduced in a higher degree compared to that of the non-oxidized lipid. the calculated ratios of the diffusion constants of pgpe-alexa and dhpe-bodipy suggest a different type of interaction with cholesterol. membrane proteins account for a third of all proteins encoded for by the human genome, and play a vital role in a number of cellular processes. few membrane protein structures have been determined to date in comparison to soluble proteins. this discrepancy is due to experimental difficulties in preparing membrane protein samples for structural analysis. traditional techniques for studying membrane proteins by x-ray crystallography or solution nmr use detergent solubilised proteins which can differ from their native confirmations. solid-state nmr allows the study of transmembrane proteins in lipid bilayers, representing a more native like environment in which to obtain biologically relevant structural information. we have been working on the development of reliable methods for reconstitution of transmembrane peptide into liposomes using glycophorin a as a model tm protein. using reconstitution methods based on the removal of detergent by bio-beads, we have used electron microscopy to screen the ideal conditions for insertion of gpa into liposomes in preparation for mas nmr experiments. em has allowed us to identify conditions favourable for insertion of peptide into lipid vesicles and those that result in aggregation. in order to confirm the secondary structure and insertion of gpa into lipid vesicles, techniques such as cd, ocd, ftir and dls have been used to provide quantitative information in addition to the visual results from em. nonesterified fatty acids regulate a broad spectrum of metabolic activities and are involved in many physiological, pathological and/or pharmacological processes in living cells. they spontaneously transfer between donors and acceptors such as fatty acid binding proteins and lipid membranes. we focus on protein-lipid dispersions of human serum albumin (hsa) and sterically stabilized liposomes (ssl) composed of dppc and appropriate amount of peg: -dppe in which stearic acids (sa) are inserted either in the protein or in the ssl. exploiting the fact that hsa has a single tryptophan residue and that the intrinsic trp-fluorescence emission signal is quenced by the presence of sa, the kinetics of sa transport between hsa and peg: -grafted dppc membranes is studied by means of fluorescence. it is found that the transfer of sa between hsa and ssl is a first-order process and the kinetics of transfer depends on the type of donor and acceptor matrix, on the temperature (i.e., on the physical state of the lipid bilayers), and on the grafting density of the peg-lipids at the protein/lipid interface. indeed, in the absence of polymer-lipids, the rate of transfer increases with temperature in both directions of transfer and it is faster for the passage from dppc bilayers to hsa. the presence of polymer-lipids reduces the rate of transfer both in the mushroom and in the brush regime of the polymer-chains, especially for lipid membranes in the fluid phase. a. orth , w. römer , l. johannes , c. steinem institute for organic and biomolecular chemistry, university of goettingen, germany, laboratoire trafic et signalisation, institut curie, paris, france shiga toxin (stx) from shigella dysenteriae is an ab -class bacterial toxin. infections with stx lead to the haemolyticuraemic syndrome, which is known to be a major cause for renal failure at an early age. the interaction of the homopentameric b-subunit (stxb), which is responsible for binding and intracellular transport of the holotoxin, with its cellular receptor, the glycosphingolipid gb , is the first step for endocytosis of the toxin. one b-subunit can bind up to gb -molecules to form stxb-gb -clusters causing negative curvature of a membrane. the binding of stxb to giant unilamellar vesicles, composed of dopc, cholesterol and gb induces tubular membrane invaginations, which were also found in experiments with energy-depleted hela-cells. in recent studies, protein and lipid reorganization processes after attaching stxb to solid supported membranes, composed of dopc/sphingomyelin/cholesterol/gb have been investigated. the compaction of gb -molecules led to an additional stxb-gb -enriched phase, which was also observed in lipid monolayers at the air/water interface. in this study, the influence of stxb on model membranes will be investigated combining the advantages of free-standing lipid membranes with those of ssms. the impact of stxb on pore-suspending membranes will be followed by confocal laser scanning mi croscopy and atomic force microscopy. a. robaszkiewicz , c. spickett , p. sicinska , g. bartosz , m. soszynski department of molecular biophysics, university of lodz, lodz, poland, strathclyde institute of pharmacy and biomedical sciences, glasgow, u.k. hypochlorite generated in vivo under pathological conditions is a known oxidant, able to initiate lipid peroxidation process, which affects the stability of biological membranes. the aim of this study was the analysis of the products formed during the reaction of hypochlorite with phosphatidylcholines containing unsuturated fatty acid residues ( -stearoyl- -oleoyl-, -stearoyl- -linoleoyl-, stearoyl- -arachidonylphosphatidylcholine) and their effects on the human erythrocytes. using electrospray mass spectrometry we observed complete conversion of the lipids into chlorohydrins, which resulted in the decrease of the rotational correlation time and rotational motion freedom of liposomes estimated by epr using spin probes ( -and doxylstearic acid). unilamellar chlorohydrin liposomes had lower diffusion coefficient for calcein than liposomes made of parent lipids. flow cytometry demonstrated fast incorporation of uni-and multilamellar chlorohydrin liposomes labeled with nbd-pe into erythrocytes. this effect was accompanied by the formation of the erythrocyte subpopulations of higher volume, decrease of the rate of fluorescein diacetate hydrolysis, estimated by flow cytometry, and increase of affinity and maximal velocity of the membrane enzyme acetylcholinesterase. extensive bilayer perforation coupled with the phase transition region of an anionic phospholipid k. a. riske , l. q. amaral , m. t. lamy depto. biofisica, universidade federal de sao paulo, sao paulo, brazil, instituto de fisica, universidade de sao paulo, sao paulo, brazil at low ionic strength dimyristoylphosphatidylglycerol (dmpg) exhibits a broad phase transition region characterized by several superimposed calorimetric peaks. peculiar properties, such as sample transparency, are observed only in the transition region. we use differential scanning calorimetry, turbidity and optical microscopy to study the narrowing of the transition region with the increase of ionic strength. upon addition of salt, the temperature extension of the transition region is reduced and the number of calorimetric peaks decreases until a single cooperative event is observed in the presence of mm nacl. the transition region is always coupled with a decrease in turbidity, but a transparent region is detected within the melting process only in the presence of up to mm nacl. optical microscopy of giant vesicles shows that bilayers first rupture when the transition region is reached and subsequently lose optical contrast. fluorescence microscopy reveals a blurry image in the transparent region, suggesting a different lipid self-assembly. overall sample turbidity can be related to the bilayer optical contrast. our observations are discussed in terms of the bilayer being perforated along the transition region. in the transparent region the perforation is extensive and the bilayer completely loses the optical contrast. financial support: fapesp. label-free imaging of biological membranes using surface imaging techniques j. l. richens, k.-a. vere, p. o'shea cell biophysics group, university of nottingham, nottingham, uk surface plasmon resonance (spr) is a detection technique traditionally used for specific protein detection, which is now exploited routinely as a generic label-free sensor. spr responds to changes on a metal surface conditioned to sense the binding of analytes. thus, the composition of the external medium and metal surface will be fundamental to the signal output. here we use a model phospholipid membrane system to investigate the effect of altered buffer and surface compositions on spr signals. comparisons are undertaken between continuous gold surfaces and gold nanoparticles. we demonstrate that surface electrostatics and the salt composition and molarity of a buffer all have significant impacts on spr output. the association of respiratory syncytial virus matrix protein with membrane microdomains the association of the matrix protein from respiratory syncytical virus with membranes has been characterised by tensiometry, brewster angle microscopy, and atomic force microscopy following deposition of langmuir monolayers onto modified silica substrates. association of the protein with monolayers containing phosphocholines and cholesterol leads to the formation of materials with new properties that differ from those of either of the pure components. the behaviour of the protein in monolayers rich in cholesterol and sphingomyelin exhibits a significant concentration dependence. at low concentrations, the protein exhibits a simple partioning behaviour. at higher concentrations, lipids are extruded from the monolayer below a critical surface area. these findings are discussed in relation to the recently published structure of the protein (pnas, , , - ) , the documented formation of viral filaments during key stages of the infection cycle and the isolation of the protein from detergent-resistant membrane fractions. structure and dynamics of closed melting membranes v. m. rondelli , s. santorsola , p. brocca , e. del favero , l. cantù , m. zimbone dep. of chemistry, biochemistry and biotechnologies for medicine, university of milan, segrate, italy., dep. of physics, university of catania, catania, italy. we studied the dynamical and structural properties of large unilamellar vesicles (≈ nm luvs) of phospholipids (dmpc, dc pc and dmpc : dc pc = : molar mixture) in the temperature range around the chain-melting transition, ≈ • c wide, with . • c resolution and . • c accuracy. small-angle (saxs) and wide-angle x-ray scattering (waxs) measurements show that across the transition the vesicle behaves as an 'evolving membrane', passing through several different states, each of them being characterized by different proportions of coexisting fluid-and gelchains molecules. noteworthy, no kinetics has been detected. on the same samples, a unique and very sensitive laser light scattering technique allows to determine the characteristic times of thermally induced shape fluctuations, connected to the elastic properties of the membranes. results indicate a clear softening of the membranes in correspondence to the chain-melting transition, as indicated by a manifold increase of the corresponding fluctuation characteristic time. meanwhile the overall size of the vesicle is not sensibly changed. this softening is likely to be due to the presence of structural defects, eventually driving to local morphological modifications. thermodynamics characterization of isolated lung surfactant assembled as lamellar bodies e. x. rodriguez , r. alvarez , r. barrio , c. irles , a. ortega biochemistry depto., school of medicine, inst. of physics unam, nat. inst. of perinatology, mexico city, mexico lungs are a large extension of ∼ m of one layer cells, which is structured as compacted sacs called alveoli, where lung function takes place: the gas exchange. alveolar endotelium is formed by two kinds of cells: neumocyte type i (nti), which covers ∼ % of the alveolar surface where gas exchange occurs and, ntii where lung surfactant (ls) is produced. ls are a mixture that lay over a water film in the luminal surface of the alveoli and it is thought to decrease the surface tension to avoid alveolar collapse during expiration. ls are made of phospholipids and proteins, which are determinant for the structure of ls under particular conditions of pressure and temperature. ls inside the cell is packed in organelles called lamellar bodies (lb), and is released to the water/air interphase in other conformation. in the present study we isolate lb from pig's lung and study lb thermodynamic characteristics by microcalorimetry in the presence and in the absence of structural proteins. phase transition profile of lb with and without proteins is basically the same; while ls in the alveolar lumen have a higher transition temperature (tm). mayor changes in tm are observed between lb and ls from alveolar lumen. although ls lipid composition in and outside the cell is assumed to be the same, the ground for the differences in tm under these two conditions is unknown. m. rodrigues , g. rádis-baptista , b. g. de la torre , m. castanho , d. andreu , n. c. santos instituto de medicina molecular, portugal, pompeu fabra university, spain, federal university of cearà, brasil nucleolar-targeting peptides (nrtps) have been recently designed by structural dissection of crotamine, a toxin from the venom of a south america rattlesnake (radis-baptista et al. . j med chem : ) . at µm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work is to decipher the molecular mechanism for the translocation of nrtps into cells. quantification of partition into membranes was carried out, based on intrinsic tyrosine fluorescence. the role of the bilayer phase, anionic lipids, reducing agents and peptide concentration on the extent and kinetics of partition were studied. both nrtp and nrtp exhibited high partition to popc (neutral) lipid vesicles (k p ≈ × ), which was enhanced by the anionic lipid popg for nrtp , but not nrtp . the peptides showed a decrease in partition for popc:cholesterol (liquid ordered state) or dppc (gel) membranes. depending on the lipid composition, the peptides either increased or decreased their quantum yield upon membrane insertion. quenching experiments with acrylamide showed no peptide aggregation in solution. once the translocation mechanism is fully understood we will test nrtps as carriers of relevant cellular cargos, evaluating their potential clinical application in drug delivery or gene therapy among other applications. the ingestion of trans fatty acids (tfa) formed during the partial hydrogenation of vegetable oils has been linked to a detrimental impact on health by an, as yet, unknown mechanism. we synthesized deuterated analogs of -elaidoyl- -stearoylphosphatidylcholine (t : - : pc) that contains a single "unnatural" trans double bond and -oleoyl- -stearoylphosphatidylcholine (c : - : pc) that contains a single "natural" cis double bond. solid state h nmr, complemented by molecular dynamics (md) simulations, was then employed to compare molecular organization in model membranes prepared from these isomeric molecules. analysis of spectra recorded as a function of temperature reveals a higher chain melting temperature for the trans isomer, indicating tighter molecular packing in the gel state. in the liquid crystalline, however, the difference between the trans and cis isomers is subtle. order as probed by the perdeuterated [ h ] : sn- chain, and corroborated by computer simulation, coincides within < %. only in the conformation of the double bond is an appreciable difference implied. thus, our results contradict the conventional view that tfa resemble saturated fatty acids, which is > % more ordered. (supported by acs, prf -ac .) photooxidation and lateral membrane diffusion of dipole molecules v. s. sokolov , e. a. sokolenko , a. a. lents , p. pohl a.n frumkin institute of physical chemistry and electrochemistry, russian academy of science, moscow, russia, institut fuer biophysik, johannes kepler universitaet linz, austria the photodynamic oxidation of phloridzin, of the styryl dye di- -anepps and of unsaturated lipids was monitored "online" by measuring the collapse of the dipole potential which has been introduced to the membrane by these molecules. their photodamage occurred at different rates when the target molecules and the singlet oxygen generating photosensitizer (phthalocyanin) were adsorbed to the same or to opposite sides of the planar lipid bilayer. the difference in the oxidation rates were attribute to singlet oxygen transport through lipid bilayer and therefore we were able to estimate the permeability of lipid bilayers to singlet oxygen. however, the apparent permeabilities derived from experiments with different targets were differ from each other. therefore, we tested the hypothesis that the lateral membrane diffusion of target molecules and oxidation product may have biased our analysis. in line with this anticipation we found that the apparent permeability is dependent on the size of the planar bilayer. the development of a new mathematical model, which takes the mobility of all reacting species in the aqueous and lipid environments into account, allowed estimation of the real membrane permeability to singlet oxygen. it appeared to be very close to that of oxygen in the ground state. a number of complex three-dimensional lyotropic liquid crystal phases are already known, such as the bicontinuous cubic phases, but so far only a single example has been found -a cubic phase of spacegroup fd m -of a structure based upon a complex close packing of inverse micelles ( ) . we now report the discovery ( ) of a novel lyotropic liquid crystal phase, of space group, p /mmc, whose structure is based upon a hexagonal close packing of identical quasi-spherical inverse micelles. the model membrane system consists of a hydrated mixture of dioleoylphosphatidylcholine, dioleoylglycerol, and cholesterol. this novel phase has a number of unique features which may render it useful for a range of applications. firstly, it is the only known self-assembled lyotropic phase whose structure consists of a periodic close packing of identical inverse micelles. secondly, it is stable in excess aqueous solution, which is very important for potential biological or biomedical applications. references ( ) v. luzzati, v., r. vargas, a. gulik, p. mariani, j.m. seddon, and e. rivas, biochemistry , - ( ) . dystrophin is a rod-shaped muscular subsarcolemal protein. its deficiency is one of the root causes of duchenne muscular dystrophy. dystrophin rod domain contains homologous repeats, where the sub-domain constituted by the repeats to (r - ) was reported to bind actin and membrane lipids. we analyzed the interaction of r - with lipid monolayers. to better understand the assembly mechanism of this protein with lipids, we studied its adsorption behavior at the air-liquid and lipid/liquid interface using ellipsometry, surface pressure, and atomic force microscopy (afm). using two different mixtures of phospholipids, ellipsometry and pressure surface data show that r - interacts with the lipid monolayers, but is inserted into the monolayer formed by dopc-dops while it lies below the monolayer of dopc-dope. this indicates that r - interacts more strongly with dopc-dops than with dopc-dope. afm images show that the pressure of lipid monolayer influences the organization of r - . when the lipid surface pressure is mn/m, r - forms a striking network, indicating a protein-protein interaction in addition to the protein-lipid interaction. this unique behavior of one part of the central domain of dystrophin may explain its key role in muscle cell. studies on polyphenol extracts from helichrysum l., fagopyrum mill., crataegus l. and hypericum l. were performed in order to find if they may be applied as a natural free radical scavengers protecting biological membranes against peroxidation. ghosts erythrocytes were used in the experiments. they were suspended in tris-edta solution, ph , , then.irradiated with a bactericidal lamp without (control) or with a proper amount of the extracts studied. the product of lipid peroxidation was malonic dialdehyde (ma). the colour reaction of ma with thiobarbituric acid (tba) enables to determine the concentration of ma spectrophotometrically. it was found that peroxidation increased with the irradiation time. however, it significantly decreased when concentrations of poliphenols increased. the best antioxidtive property was found for hypericum l. the antioxidative efficiency sequence of the plant extracts studied was the following: hypericum l. > crataegus l. > fagopyrum mill. > helichrysum l. the results obtained indicate that polyphenol extracts exhibit excelent antioxidative properties that make them good free radical scavengers for efficient protection of biological membranes. this work was sponsored by ministry of science and education, scientific project no. n n . interaction of cationic porphyrins with neutral and negatively charged liposomes i. voszka , g. corradi , p. maillard , h.-j. steinhoff , g. csík institute of biophysics and radiation biology, semmelweis university budapest, hungary, research institute for solid state physics and optics, hungarian academy of sciences, budapest, hungary, institute curie, section de biologie, orsay, france, fachbereich physik, universitat osnabrück, germany porphyrin derivatives are used in photodynamic therapy of tumors. the knowledge of the photosensitizer location within the cell is important. the effect of porphyrin derivatives containing cationic side groups was examined on neutral and negatively charged liposomes by esr. the esr signal was first examined as a function of temperature and porphyrin concentration in the dark. a significant change related to the appearance of quasi free spin labels was obtained for spin probes at the th carbon atom and was more expressed for the asymmetrical derivative. illuminating the samples the esr amplitude decreased for all positions of the spin probe but to different extent. the effect was more expressed in case of the symmetrical derivative, especially for label positions and . for the asymmetrical derivative the effect changed from moderate to weak from the th to the th position. this indicates that the asymmetrical derivative is incorporated nearer to the lipid head groups while the symmetrical one may be located deeper in the membrane. under oxygen-free conditions both derivatives showed weaker but still pronounced effects.. single channel recording of α-hemolysin in nanopore-spanning tethered bilayer lipid membranes n. t. thet , i. pfeiffer , w. knoll , i. köper max planck institute for polymer research (mpi-p), ackermannweg , mainz, austrian research centers gmbh -arc, tech gate vienna, vienna, austria artificial lipid bilayer membranes mimic biological cell membranes in many aspects and can be used to study functional processes such as ion channeling, signal transducing, transport of nutrients etc. as most of the functions of a cell are accomplished by membrane proteins, research has been ongoing in studying and characterization of membrane proteins embedded in model lipid bilayer membranes. black lipid membranes (blms) were studied for decades but their potential for practical applications is hindered, mainly due to their lack of stability and limited lifespan. recently, tethered bilayer lipid membranes (tblms) proved to have long life span of up to several months without significant changes in membrane resistance and capacitance. in order to combine advantages of blms and tblms, we have designed a membrane system which is freely spanned across a single nanopore in a silicon nitride membrane. this system not only mimics cell membranes, but also it allows control over the chemical composition of buffer with unlimited ionic reservoirs on both sides of the membrane. this freestanding tblm maintains structural stability and lifetime of up to hours without significant decrease in its structural integrity and electrical sealing. for the validation of the tblm formation, we have inserted well known α-hl pores. we are able to control the amount of α-hl pores insertion and measure single channel ion transport across the tblm by α-hl pores using a capacitor feedback amplifier. the language of shape: biological reactions are dramatically affected by the shape of lipid membrane d. stamou university of copenhagen, copenhagen, denmark a plethora of biological process are taking place on the surface of lipid membranes. as a rule membranes in vivo are curved in a variety of complex geometries. here i will present a quantitave study on the influence of membrane curvature on protein-membrane and membrane-membrane interactions. to gain systematic access to a continuum of membrane curvatures we immobilized liposomes on a surface at dilute densities. using confocal fluorescence microscopy we imaged single liposomes of different size, and therefore different curvature, and monitored their interaction with a binding partner (proteins or other liposomes). i will discuss unpublished data on two important classes of biomolecular interactions that exhibited dramatic curvature dependence: a) snare-mediated docking and fusion b) anchoring of peripheral proteins. the following references provide partial information on the single-liposome assay: a. zettergren, c. gudmundsson, t. nylander, e. sparr physical chemistry , lund university, lund, sweden there is accumulating evidence of substantial amounts of phospholipids in the cell nuclei , although the function of these lipids is still not fully understood. it has been shown that the chromatin complex composed of dna, rna and proteins also includes phospholipids, and that rna colocalize with these . although the rna-phospholipid interactions may have important implications to biological function, in gene therapy and in medicine, very little work has been dedicated to the characterization of rna interaction with phospholipids. the objective of this work is to investigate the adsorption behavior of short single stranded bases long rna (ssrna ) molecules (similar to mirna) to lipid monolayers at the air-water interface as well as to study how the presence of rna affect the domain formation in the monolayers using fluorescence microscopy. monolayer studies have shown adsorption of ssrna to monolayers consisting of zwitterionic dppc as well as to monolayers consisting of cationic dodab. the adsorption behavior of these very short nucleic acids differ significantly from the adsorption process for longer nucleic acids as for example a base pairs long ds dna (dsdna ) which has been used as a reference system . the presence of ssrna significantly changes the compression isotherm of both dppc and dodab monolayers. plant defensins are cysteine-rich antimicrobial peptides found in various plant species. they share a common threedimensional structure, stabilized by eight disulphide-linked cysteines consisting of three antiparallel β-strands and one α-helix. most plant defensins show antifungal activity with no effect on mammalian and plant cells. expression of these peptides in plant tissue is induced by pathogen infection. the mechanism of defensin action is based on membrane permeabilization. this occurs through an interaction with high affinity binding sites on fungal membranes, resulting in alteration of membrane potential. the genes encoding for a peach ppdfn and a grape vvamp defensins were expressed in e. coli and purified to homogeneity. they were tested for antimicrobial activity against some fungi and showed to have an inhibitory effect on the spore germination. biophysical analysis showed that defensins were able to interact with artificial membranes. binding of defensins to membranes was dependent on lipid composition, increasing with the sphingolipids content. interaction between peptides and sphingolipids could lead to insertion of the defensins into the membrane resulting in its destabilization. extracts of the plant perilla frutescens have many uses in the asian kitchen, e.g. as a popular garnish used in sushi. the plant is also employed in eastern traditional medicine to treat a variety of ailments including colds, food allergy and depression. two of the main constituents of the plant are limonene and perilla aldehyde. by bio-oxidation, these molecules can be converted to perillic alcohol and perillic acid. these cyclic terpenes possess antibacterial and anticarcinogenic properties. the modes of action for these compounds are at present not understood, but their remarkably diverse pharmacological properties suggest that they might target the phospholipid matrix of the cellular lipid membrane. indeed, the cyclic terpenes can bind to, and alter the physiochemical properties of the lipid bilayer of the membrane, the effects of which can cascade down to several essential cellular processes. here, we use molecular dynamics (md) simulations to investigate the effect of limonene and its derivatives on the properties of lipid bilayers including the changes in acyl chain order parameters, bilayer thickness and the area per lipid. md can afford molecular-scale dynamic information, often not easily accessible from experimental measurements. this information can be used to interpret existing experimental data obtained by e.g. isothermal titration calorimetry, electron paramagnetic spectroscopy and differential scanning calorimetry. catalysis in the membrane: interfacial mechanism of phospholipase a h. p. wacklin , r. k. thomas institut laue langevin, grenoble, france, physical and theoretical chemistry laboratory, oxford university, u.k. phospholipase a cleaves the sn- acyl chains of membrane phospholipids and performs a range of physiological functions. one of the least well understood aspects of the its mechanism is how its activity is regulated by the interaction with the substrate membrane. we have used neutron reflection to monitor changes in membrane structure during lipid hydrolysis [ , ] . the penetration depth of pla depends on lipid packing and increases during the lag phase of porcine pancreatic pla . by using a selectively deuterated lipid substrate, d -popc, we determined the relative membrane-water partitioning of the lipid products in-situ. the lyso-lipid product partitions into the solution phase, while fatty acid accumulates in the membrane and increases the affinity of pla [ ] . pla is inhibited at ph , which is consistent with protonation of the catalytic histidine. however, irrespective of ph, pla is fully activated by me-betacyclodextrin, which facilitates the release of the lyso-lipid from the enzyme-substrate complex. me-beta-cyclodextrin does not interact directly with the membrane surface or the substrate lipids, indicating that product release occurs outside the immediate membrane-water interface. diffraction limits resolution in optical microscopy, but many interesting biological problems occur on shorter (molecular) length scales. recently, methods to circumvent the diffraction limit have been presented. fluorescence photoactivation localization microscopy (fpalm) uses activation of many small subsets of photoactivatable or photoswitchable fluorescent probes (pafps) to generate images with effective resolution in the tens of nanometers. pafp molecules are photoactivated, imaged, localized, and photobleached in small numbers. the process is repeated for many subsets to build up data on thousands to many hundreds of thousands of molecules. the positions of all localized molecules are used to construct an image of the sample with resolution limited not by diffraction, but by the localization precision and molecular density. results will be shown from a variety of biological systems, including live and fixed cells expressing a variety of pafp-tagged proteins. bi-plane fpalm can image in three dimensions with demonstrated resolution of nm x nm x nm. polarization fpalm can image both molecular positions and anisotropies simultaneously with lateral resolution of ∼ - nm. using these powerful capabilities, many potentially interesting biological problems can be addressed. binding of the hiv- ncp on oligonucleotides at the single-molecule level j. godet, p. didier, a. jouonang, y. arntz, y. mély laboratory of biophotonics and pharmacology, umr cnrs , university of strasbourg, france the nucleocapsid protein (ncp ) of the hiv- is a small basic protein which plays key functions in the viral life cycle. the activity of ncp mainly rely on its potent rna-and dna-chaperone activities that direct the rearrangement of numerous nucleic acid molecules into their most stable conformation. two main features of nc's chaperone activity are its abilities to aggregate and destabilize nucleic acids. in addition, the rapid kinetics of ncp interaction with nucleic acids was recently proposed as another major component of nc's chaperone function based on the apparent correlation between an indirect measurement of the nucleic acid dissociation kinetics of ncp and its overall chaperone activity. but so far, no direct measurement of the on/off rates of ncp binding to oligonucleotides was performed. in the present work, we realized single molecule fluorescence resonance energy transfer (smfret) measurements to probe the transient interactions of a tmr-labelled ncp with a short cy -labelled dna oligonucleotide confined into nanovesicles. after confirming the efficiency of the ncp /odn complex encapsulation into the nanovesicles by fcs, the vesicles were tethered to the surface for immobilization. integrity of the entrapping vesicles attached on the surface was confirmed by afm. finally, the association and dissociation constants retrieved from these smfret measurements were discussed in the context of the dna-chaperoning activity of the protein. high-resolution spatiotemporal organization of the integrin lfa- m. f. garcia-parajo , t. s. van zanten , g.-j. bakker , r. diez-ahedo , a. cambi , c. g. figdor bionanophotonics group; ibec, barcelona, spain, tumour immunology dept; ncmls, nijmegen, the netherlands lfa- is a leukocyte-specific integrin involved in different steps of the immune response. on monocytes, lfa- plays a key role in the regulation of monocyte-endothelial interaction during rolling, arrest and extravasation into the underlying tissue. i n-vivo experiments showed that blood borne lymphocytes can 'switch' within seconds from rolling to arrest. furthermore, tem observations of pro-active, ligandindependent nanoclusters confirmed that affinity and clustering are complementary processes required in adhesion. yet, the mechanisms leading to fast-switching remain obscure. in our group, we used a combination of single molecule fluorescence techniques to study the spatiotemporal organization of lfa- on monocytes. we performed optical nanoimaging of lfa- nanoclusters in relation to membrane rafts with a resolution of nm and accuracy of ∼ nm. in quiescent cells, lfa- nanoclusters do not associate with membrane rafts and diffuse freely on the membrane. binding of the integrin to its ligand icam- induces the formation of microclusters that further associate with rafts and exhibit reduced mobility, consistent with cytoskeleton interactions. our work highlights the markedly different spatiotemporal organization of lfa- that might explain its concerted action to form larger and stable platforms on the cell surface required for rapid and effective cell adhesion. c. ciobanasu, u. kubitscheck rheinische friedrich-wilhelms-universität bonn, germany cell penetrating peptides like the hiv tat peptide have the property to rapidly translocate the cell membranes and the capability to deliver a wide range of cargoes. the mechanism of the membrane translocation is still under investigation and object of considerable controversy. we applied and single-molecule and confocal laser scanning microscopy (lsm) to study peptide-membrane interactions. electron-multiplying ccd cameras yield images of single fluorescent molecules with a time resolution in the range of a few milliseconds only, which allows the tracking of fluorescently labelled peptides and lipids at bio-interfaces in realtime with a localization precision of a few nanometers. we formed giant unilamellar vesicles (guvs) from different lipid mixtures and examined their interaction with fluorescently labeled tat peptides. we found that the passive peptide internalization process depends on lipid composition, charge of the lipid bilayer, and the ionic properties of the medium. a translocation of cationic tat peptides was observed in membranes containing at least mol% of lipids with a phosphatidyl ethanolamine or a high mol fraction of the phosphatidyl serine head group. in salt-free solution tat efficiently bound to guvs, however, in a physiological nacl solution tat binding was completely abrogated, but the peptides efficiently equilibrated across the guv membrane. new approaches to measure interactions in the live cell plasma membrane g. j. schütz biophysics institute, johannes kepler university linz, austria in my lecture, i will show examples how to obtain insights into the organization of the cellular nanocosm by single molecule experiments. our primary goal is an understanding of the role of such structures for immune recognition. brightness and single molecule colocalization analysis allows us to study stable or transient molecular associations in vivo. in particular, i will present results on the interaction between antigen-loaded mhc and the t cell receptor directly in the interface region of a t cell with a surrogate antigenpresenting cell. in addition, we developed a method for in vivo micropatterning of plasma membrane proteins to measure molecular interactions. the method allows identifying and characterizing interactions between an arbitrary fluorescence labeled protein ("prey") and a membrane protein ("bait") directly in living cells. cells transfected with a fluorescent fusion protein of the prey are plated on micropatterned surfaces functionalized with specific antibodies to the extracellular domain of the bait; the fluorescence copatterning is used as readout for the interaction. we applied this tool for the study of the interaction between cd -the major coreceptor for t cell activation -and lck, an important tyrosine kinase in early t cell signaling. in addition to the well-known zinc-clasp structure, we found strong contributions of lck membrane anchorage for the binding of the two proteins. developing a fluorescent redox sensor for monitoring metal-ion mediated catalysis in biosystems a. rybina , a. kiel , b. thaler , a. sprödefeld , r. krämer , d. p. herten bioquant and, department of inorganic chemistry, heidelberg university, germany a fluorescent redox sensor is an electron photo-switching device that can be used for the characterization of the redox state of a given environment. it combines a fluorescent fragment with a redox-active unit that senses the media by a redox reaction and controls the light emitting properties of the fluorophore. such reversible sensor can help to examine the electrochemical state and changes in biological systems during biochemical processes in real time. recently a new fluorescent molecular sensor with a redox-active hydroquinoneunit covalently linked to fluorophore rhodamine b was developed. (kierat r.m. et al., bioorg. med. chem. lett., -accepted) . the reduced hydroquinone-form of the sensor is fluorescent while its oxidation to benzoquinone-derivative leads to a significant decrease of the fluorescence. although the above method shows great promise for applications in biological systems, the exact mechanism of this process is not fully understood yet. we use fluorescence spectroscopy to investigate oxidation reactions on the ensemble and single-molecule level and study kinetic rates. the proposed strategy is to use cu (ii) complex as oxidation mediator immobilized on surface via dna linker to examine the oxidation mechanism. fluorescently labeled atp as a probe of the outer mitochondrial membrane barrier: role of vdac fluorescence correlation spectroscopy (fcs) was applied for studying the distribution of fluorescently labeled atp (bodipy-atp) in isolated mitochondria. the setup and peak intensity analysis (pia) was described in our recent paper (perevoshchikova et al. biochim.biophys.acta : . the binding of bodipy-atp to mitochondria was maximal in the non-energized state, whereas the addition of succinate (respiratoty substrate) or atractyloside (adenine nucleotide translocase inhibitor) led to a decrease in the binding. nadh reduced the fcs signal from bodipy-atp added to non-energized mitochondria more than nad+ did under the same conditions suggesting the control of nucleotide transport through voltage-dependent anion channel (vdac) residing in the outer membrane. konig's polyanion also decreased the bodipy-atp binding to mitochondria with the effect being reduced by alamethicin or digitonin. control experiments showed that bodipy-atp did not bind to liposomes showing minor role of unspecific binding. it was suggested that bodipy-atp in combination with fcs can be used to monitor the functional state of mitochondrial vdac which is considered to be a principal regulator of mitochondrial function. fluorescence correlation spectroscopy studies of lysozyme partition to phospholipid vesicles a. m. melo , a. coutinho , m. prieto cqfm and in, ist, - lisboa, portugal, dqb, fcul, - , lisboa, portugal binding to membrane lipids has been increasingly recognized as an important step in the aggregation and cytotoxicity of several amyloidogenic proteins [ ] . in addition, it has been recently reported that membranes containing negatively-charged phospholipids can also trigger rapid amyloid-like fiber formation by a variety of several nonamyloidogenic proteins, such as cytochrome c and lysozyme [ ] . our study aims to elucidate the factors that govern the formation of these lipid-protein complexes. given the importance of electrostatic interactions between the proteins and the acidic phospholipids in the putative membrane-induced protein misfolding step, it is essential to first characterize quantitatively the protein partition behavior towards liposomes prepared with variable anionic lipid content. in this study, lysozyme was chosen as a model protein and fluorescence correlation spectroscopy (fcs) was used to monitor its binding to liposomes after its conjugation to alexa fluor . most organic dyes labelling techniques produce a mixture of populations of molecules labelled with a different number of fluorophores. the influence of this poli-dispersity of labelled molecules on the protein partition behaviour will be explored, namely the ability of the fcs technique to detect the production of non-competent membrane-binding species. t. wohland , p. liu , x. shi , y. h. foo , t. sudhaharan , s.-w. chong , v. korzh , s. ahmed chemistry dept., singapore nat. univ., medical biology inst., singapore, molecular & cell biology inst., singapore biomolecular interactions have been measured mostly under in vitro conditions because of higher accuracy and ease of measurement. however, it has become clear that the cellular environment has an important influence on these interactions. it was therefore necessary to develop new tools to allow the measurement of interactions in cells and organisms. recently, we have developed a modality of fluorescence cross-correlation spectroscopy (fccs) called single wavelength fccs (sw-fccs), which uses one-photon excitation to excite two fluorophores with overlapping absorption but separable emission spectra. sw-fccs has been used to determine e.g. dimer fractions of proteins in live cells. here we aim to extend the use of sw-fccs to cells and organisms. in the first part we determine the dissociation constants of a small rho-gtpase (cdc ) with an effector domain (crib) and two effectors (n-wasp or irsp ). in the second part we measure the binding between cdc and a scaffolding protein (iqgap ) in dependence of their expression levels in cho cells and in live zebrafish embryos. by using gfp/mrfp fusion proteins we can excite both fluorophores at nm and detect them separately in two different wavelength channels. we quantitatively determine the dissociation constants and compare their differences in vitro, in cells, and in embryos. these experiments demonstrate that sw-fccs is a powerful biophysical tool for the quantitation of biomolecular interactions in cells and organisms. addressing plasma membrane structure at the nanoscopic length-scale s. wieser, m. axmann, g. j. schütz biophysics institute, johannes kepler university linz, altenbergerstr. , a- linz, austria there is increasing interest in a detailed understanding of the structure and dynamics of the cellular plasma membrane, primarily based on recognizing its essential role for controlling cellular signaling processes. we employed single molecule fluorescence microscopy to study diffusion of cd , a gpi-anchored protein, in the plasma membrane of living t cells at sub-wavelength resolution, both on the cell body and on tunneling nanotubules connecting cells. the lateral motion of this single fluorescence labeled molecule was imaged on a millisecond time scale. within the experimental errors, no indications for confined diffusion for cd on the cell body in t cells have been found. furthermore by separating longitudinal and transversal mobility, we found isotropic diffusion behavior on the surface of tunneling nanotubules. in both studies we analyzed the mean square displacement as a function of the time-lag and the distribution of displacement steps. however, a closed analytical theory for these analysis is only available for the simplest models. to address a suspected diffusion process we reasoned that a full analytical description may not be required; it may well be sufficient to compare the experimental data with monte carlo simulations of the process. we demonstrated the working principle for this simulation based analysis for free diffusion, hop diffusion and transient binding of the tracer molecule to slowly moving receptors. n. chakroun , f. fraternali , m. malfois , h. rezaei , c. a. dreiss king's college london, u.k., diamond light source, didcot, oxfordshire, u.k., inra, jouy-en-josas, france prion(prp) diseases are fatal neurodegenerative diseases affecting mammals including human and sheep.they are characterized by the accumulation of extracellular βrich fibrillar deposits of a structurally modified form (prp sc ) of the cellular prp c .despite the increasing interest for prp diseases,the mechanism of prp c /prp sc conversion is still unknown.studies on prp diseases suggest that neurotoxicity arises from small pre-fibrillar oligomers.we have used a range of biophysical techniques combined with molecular dynamics simulations (md) to resolve the oligomerization pathways of sheep prp (sprp).we have shown that under well established conditions, sprp oligomerizes into three oligomers, which form in parallel.in addition, we have now identified the minimal region of sprp leading to the same oligomerization profile of the entire sprp,namely h h .low resolution shapes of sprp, h h and resulting oligomers have been determined by small-angle x-ray scattering.time-resolved studies have been used to follow the oligomerization of sprp and h h monomers into the oligomers.the conversion of sprp sc at the molecular scale was studied by md.simulations of the h h region recreating experimental conditions revealed a complete unfolding of h helix followed by h helix.these crucial steps are followed by the formation of β-sheet structures leading to a stable βrich double hairpin structure. single-molecule force spectroscopy investigation of the conformational equilibria of alphasynuclein m. brucale , a. rampioni , m. sandal , i. tessari , l. tosatto , l. bubacco , b. samorì istituto di biochimica g.moruzzi, università di bologna (italy), dipartimento di biologia, università di padova (italy) alpha-synuclein (asyn) is an abundant intrinsically disordered protein (idp) primarily located at presynaptic terminals. mutations in the gene encoding asyn have been linked to early-onset parkinson's disease (pd). by means of single molecule force spectroscopy (smfs) experiment, we show how the conformational equilibrium of monomeric wild type (wt) asyn shifts toward more compact structures in several unrelated conditions linked to pd pathogenicity [ ] . the conformational heterogeneity of pathological alpha-syn mutants a p, a t and e k has also been characterized, revealing marked differences in the conformational behaviors of the mutants with respect to wt asyn [ ] . all the mutants show a distinctively higher propensity, with respect to wt, to acquire a monomeric compact conformation that is compatible with the acquiring of beta structure. the same smfs experimental methodology is then used to characterize the conformational behavior of wt and mutant asyn in a variety of conditions, in an attempt to gain insight about the multiple and contrasting parameters controlling the equilibrium. in vitro protein folding studies using chemical denaturants have contributed tremendously to our understanding of the folding thermodynamics and kinetics of water-soluble proteins. this is not the case for integral membrane proteins, which constitute about one third of all eukaryotic proteins and more than half of all validated and potential drug targets. fully reversible denaturant-induced unfolding remains limited to a few β-barrel porins, whereas the much larger and more relevant class of α-helical membrane proteins has thus far evaded this approach. we report here the first example of an α-helical membrane protein that can be unfolded completely and reversibly by a chemical denaturant: mistic, a -residue protein from bacillus subtilis [ ] , dissociates from detergent micelles or lipid vesicles and assumes an unfolded monomeric state on titration with urea. using spectroscopic and microcalorimetric techniques, we exploited this unique property to provide (i) a quantitative comparison of membrane protein stability in different membrane-mimetic systems; (ii) an experimental test of controversial predictions [ ] regarding the folding core of mistic; and (iii) a convenient setup to study the spontaneous, translocon-independent membrane insertion of this unusual membrane protein. the mechanical functioning of biological tissues is important from many viewpoints such as diseases, clothing and even food. the protein collagen makes up the greater part of these tissues, and is remarkable for its many uses in the body, however there are at least two other major components. one of the most interesting properties of these tissues is their non-linear behavior under stress. this behavior is essential to prevent a catastrophic failure such as an aneurysm. at least three major theories have been proposed within the past few years to explain this behavior, but have been impossible to verify. in order to determine a correct description of the mechanical structure of the tissue we have been using cutting edge technological solutions to address the single molecules within the extra cellular matrix. this technique combines optical methods with single molecule force spectroscopy, allowing stiffness measurements over the nanoscale as well as determining the individual protein tensions within the extra cellular matrix. the results show that this method can be used to determine the network properties even in the complicated aortic wall enabling better understanding of disease states, which in this case include marfan's syndrome and ascending aortic aneurysms. beta amyloid peptide abetapy - shows a faster aggregation kinetics than abeta - c. d'arrigo , m. tabaton , a. perico institute for macromolecular studies, national research council, genova, italy, department of neurosciences, university of genova, genova, italy we test directly the differences in the aggregation kinetics of three important beta amyloid peptides, the full-length abeta - and the two n-terminal truncated and pyroglutamil modified abetapy - and abetapy - , found in different relative concentrations in the brains in normal aging and in alzheimer disease. we find by following the cd signal and the tht fluorescence of the solution in phosphate buffer, a substantial faster aggregation kinetics for abetapy - . this behavior is due to the particular sequence of this peptide which is also responsible of the specific oligomeric aggregation states, found by tem, during the fibrillization process which are very different from those of abeta - , more prone to fibril formation. in addition abetapy - is found here to have an inhibitory effect on abeta - fibrillogenesis, coherently with its known greater infective power. this is an indication of the important role of this peptide in the aggregation process of beta-peptides in alzheimer disease. the puzzle of the anomalously long denaturation kinetics of green fluorescent protein (gfp) mutants still is largely unveiled. in this study we have followed the effect of mutation h g on the stability of gfpmut (mut g) in the presence of guanidinium hydrochloride (gdnhcl). different techniques of fluorescence spectroscopy have been employed in order to obtain information concerning the unfolding event: time resolved fluorescence, fluorescence correlation spectroscopy (fcs), and fluorescence anisotropy. the substitution of the histidine with glycine affects protein stability versus ph: in particular mut g kinetics is not ph dependent and at basic ph values the protein is less stable. the fluorescence properties (quantum yield, lifetime) and the rotational correlation time are unchanged during the unfolding dynamics, while the number of fluorescent proteins decreases exponentially. an extrinsic probe, bound to cysteine , has been employed in order to gain more insights on the unfolding process, monitoring the stability of a different region of the protein. in particular, it has been found that a softer region is present around cysteine in both gfp variants, showing that the unfolding process does not follow a simple two step mechanism. recently, negatively-charged membranes were reported to catalyze amyloid fiber formation by amyloidogenic peptides/proteins and also to induce formation of "amyloidlike" fibrils by nonamyloidogenic proteins. here, we used an approach which combines steady-state and time-resolved fluorescence measurements to obtain structural information about these supramolecular assemblies and to gain insights about the factors that control their formation. by exploring a wide range of lipid concentrations, the interaction of alexa -lysozyme with phosphatidylserine-containing membranes was found to be a complex multi-step process, critically dependent upon the protein-to-lipid molar ratio (p/l) used. upon increasing the total lipid concentration in solution, there was a balance between an increased overall protein binding to the lipid vesicles and a progressive protein dilution on the membrane surface. as the surface potential of the vesicles decreased upon increasing the protein interfacial coverage of the liposomes, the protein binding mode was found to switch from a peripheral binding of lysozyme to the anionic headgroups (at low to intermediate p/l) to a partial insertion of the basic protein into the hydrophobic core of the membrane (at a high p/l). it is hypothesized that disruption of the protein tertiary structure might be a stepwise process beginning with loosening of the structure caused by its deeper insertion in the membrane bilayer. unexpected scaling laws in the mechanical unfolding of single protein molecules m. clusel, e. i. corwin, h. lannon, j. brujic center for soft matter research, physics department, new york university, new york, ny, usa it is a question of fundamental importance to understand the response of proteins to a stretching force, particularly in the case of mechanically active proteins, such as those in muscle fibers. we aim to understand how the structure and topology of a protein affect its resilience to external forces and presumably its function. owing to recent advances in single molecule force-clamp spectroscopy using the atomic force microscope (afm), we are now able to probe the structure and dynamics of single proteins under a constant stretching force by measuring their end-to-end length over time. the probability distribution of unfolding times at a given force allows us to estimate the strength of the protein in terms of a characteristic energy barrier, while the shape of the distribution provides a window into the microscopic mechanism by which the protein breaks apart. here we show a novel scaling of the unfolding kinetics with the stretching force, which deviates significantly from the currently accepted bell model. instead, we propose a physical picture for forced unfolding that is analogous to the mechanics of fracture. v. garcía-gonzález, j. mas-oliva instituto de fisiología celular. universidad nacional autónoma de méxico. méxico, d.f. méxico. studies focused on the thermodynamic and kinetic analysis have demonstrated that transfer of neutral lipids such as cholesterol esters through an aqueous phase is a highly costly biophysical event. therefore, nature has developed a series of lipid transfer proteins such as the cholesterylester transfer protein (cetp) designed to efficiently lower the energy barrier for transfer of cholesterol-esters between lipoproteins through an aqueous environment. employing circular dichroism we evaluated the secondary structure stability of a small peptide derived from the carboxy-end of cetp (helix y ) in a wide range of ph's. the percentage of α-helix is diminished only at extreme temperatures and acidic ph's in a reversible way. we report that while a mixture of phosphatidylcholine/cholesteryl-ester forms large aggregated particles independently of ph, inclusion of helix y to the mixtures close to neutral ph's allows the formation of small micellar-like structures confirmed by dynamic light scattering and electron microscopy. these results suggest that helix y when close to physiological ph values presents the ability to organize a micellar structure around itself. this type of organization allows the process to dramatically lower the energetic barrier for lipid transfer through aqueous media, phenomenon directly related with the facilitation of lipid transfer between lipoproteins. mimicking metastasis by a novel microfluidic approach there is increasing evidence that cancer metastasis shares commonalities with thrombosis. the von-willebrand-factor (vwf), a mechanical active blood clotting protein appears to be a particular potent candidate to bridge the gap between clotting and cancer extravasation. modeling the crucial physiological conditions of the blood circulatory system, for an in situ study of blood clotting-metastasis connections is not only, absolutely necessary, but also a challenging task. here, we present acoustically driven flow as a novel microfluidics method for mimicking the blood flow. this method enjoys very beneficial advantages of possibility of handling very little volumes of fluids, together with freedom to model most of the geometries present in our microcirculatory system. one technologically challenging, yet physiologically important factor, is the hydrodynamic condition in a bifurcated vessel, where complex shear profiles arise. we present a model to mimic these conditions and discuss the impact of hydrodynamics on vwf mediated cancer cell adhesion in bifurcated vessels of our microcirculatory system. protein structural changes occurring in flows stresses inherent to viscous fluid flow have previously been associated with protein unfolding, although structural changes have not been well documented as a function of relevant hydrodynamic parameters. we have used raman spectroscopy to monitor the structure of various protein solutions in situ for multiple flow scenarios within a concentric cylinder fluidic device ( ) . the flows, which ranged from circular couette to wavy taylor-couette flow, were characterised experimentally using particle image velocimetry. several proteins were observed to change conformation when exposed to these flows, although the nature of these changes was protein specific. shearing hen egg white lysozyme in water altered the protein backbone structure, while similar shear rates in a % glycerol, % water solution affected the solvent exposure of the side chain residues near the exterior of the α-domain. the solventdependent response may be due to the flow topology, viscous stress, or the surface hydration properties. comparison of spectra acquired at different time points, including before and after flow, confirmed that the observed changes are reversible and independent of fluid stress exposure time. the scripps research institute, la jolla, ca, usa. intrinsically disordered proteins are increasingly found to play major roles in cell biology and disease. we are utilizing single-molecule fluorescence methods to probe these complex and highly dynamic molecules, allowing more direct measurements of structural distributions and dynamics, while avoiding loss of information due to ensemble averaging. in one example, we investigated the structural dynamics of sup -nm, whose regulatable amyloid formation is believed to have functional significance in yeast. using a combination of single-molecule fret as a molecular ruler, coincidence to interrogate intermolecular interactions, and correlation analysis to probe conformational dynamics, we showed that the monomeric protein populates an ensemble of compact and rapidly interconverting conformations. a particularly interesting feature of intrinsically disordered proteins is that they are relatively unstructured in isolation, but can gain stable structure by interaction with binding partners. in this context, we used single-molecule fluorescence to characterize the complex folding pathway for the parkinson's-related idp alpha-synuclein induced by binding to a lipid-mimic. this combined single-molecule fluorescence methodology provides a powerful approach for detailed studies of the coupling of folding and dynamics with interactions and biology of this important class of proteins. m. ito , j. johansson , r. stromberg , l. nilsson department of biosciences and nutrirtion, karolinska institutet, huddinge, sweden, department of anatomy, physiology and biochemistry, swedish university of agricultural sciences, the biomedical centre, uppsala, sweden amyloid β-peptide (aβ) is a - amino acid polypeptide and known to aggregate and form insoluble amyloid fibril, which is regarded as a primary cause of alzheimer's disease (ad). the discovery of practical and effective treatments and drugs for ad has been waited eagerly. in a recent experimental study, it was suggested that stabilization of the helical conformation of the aβ middle region, which strongly favors collapsed coil formation in the extracellular environment, would reduce the aβ fibril formation. based on the experimental evidence, inhibition of the unfolding of the aβ α-helix can be a forceful strategy to repress the aβ fibril formation resulting in prevention of ad. however, the detailed mechanism of the unfolding of the aβ α-helix has remained unclear, because the x-ray or the nmr structure of the aβ α-helix in aqueous solution has not been reported due to its instability. the aim of this study is to find effective ways to inhibit the unfolding of the aβ middle region, which is a prerequisite for the aβ fibril formation. in this study, we attempted to elucidate the molecular mechanism of the aβ unfolding by molecular modeling and molecular dynamics (md) simulations. the md simulations were performed for α-helical structures of a wild-type aβ( - ) model and a mutant aβ( - ) model. linker average hydrophilicity as a tool to discriminate between extended and non-extended calcium binding proteins a. isvoran , e. quiniou , c. craescu , l. mouawad west university of timisoara, department of chemistry, pestalozzi , timisoara, romania, inserm u , centre universitaire paris-sud, bâtiment , orsay, france the ef-hand calcium binding proteins (cabps) may exist either in an extended or a compact conformation, sometimes correlated with their functions. for the cabps with know structure and function, calcium sensors are usually extended and calcium buffers compact, hence the interest to predict the form of the protein starting from its sequence. in this study we used two different procedures, the sosuidumbbell algorithm and a novel procedure that is based on the linker average hydrophilicity (lah). the two procedures were tested on known-structure cabps and then applied to unknown-structure centrins. the so-suidumbbell algorithm yielded the right conformations for of the known-structure proteins and predicted that all centrins should are compact. the lah procedure discriminated well between the extended and non-extended forms of all the known-structure cabps and it reflected well the phylogenetic classification of centrins being a simple and powerful means to discriminate between extended and nonextended forms of cabps. only few residues that constitute the linker are responsible for the form of the cabp, showing that this form is mainly governed by short-range interactions. (http://u .curie.u-psud.fr/modelisation/lah) lipid and protein organization of hepatitis b antigen characterized by fluorescence spectroscopy v. greiner , c. egelé , s. oncul , f. ronzon , c. manin , a. klymchenko , y. mély laboratoire de biophotonique et pharmacologie, umr cnrs, faculté de pharmacie, université de strasbourg, france, sanofi pasteur, av. marcel mérieux, marcy l'étoile, france. hepatitis b surface antigen (hbsag) particles are nm lipoprotein particles, mainly composed of the major s surface viral protein containing trp residues and yeast-derived lipids. since the structure of these particles is still missing, we further characterized them by fluorescence techniques. fcs indicated that the particles diffuse mainly as monomers and contain about proteins per particle. fluorescence quenching and time-resolved fluorescence experiments showed that the fluorescence signal is largely dominated by the trp residues at the protein surface. moreover, time-resolved anisotropy measurements indicate that the protein motion is restricted and that the surface trp residues exhibit both local and segmental motions. the lipid part of the particles has been studied by environment sensitive -hydroxyflavone probes and viscosity-sensitive dphbased probes, and compared to lipid bilayers and low density lipoproteins (ldls), taken as models. the results suggest that the lipid part of hbsag is closer to ldls than to model lipid bilayers. we present an extensive calorimetric study of bovine alphalactalbumin for various ca++ content. equilibrium dsc raw data are analyzed and the melting temperature tm, the specific heat jump deltacp, the heat of unfolding deltahm are directly extracted. the binding of calcium on the native (n) state greatly stabilizes the protein, essentially by the enthalpic difference between the unfolded (u) and n states. we show that subsequent addition of calcium in the mm range stabilizes further the n state. the equilibrium calorimetric measurements are completed with out of equilibrium stopped flow refolding kinetics by cd spectroscopy performed at different temperature and ca++ concentrations. we discuss the possible stabilization mechanisms compatible with our measurements. protein unfolding/refolding in cellular compartments: application of luciferase constructs our studies show that a reporter enzyme, firefly luciferase, can be used for evaluation of the stress-induced proteotoxicity within different cellular compartments such as the nucleus, cytoplasm or mitochondria. in transfected mammalian cells, firefly luciferase is localized in microsomes. we engineered plasmid constructs encoding luciferase with inserted specific sequences that ensure its cytoplasmic or intranuclear, or intramitochondrial localization. in addition, we fused luciferase to the green fluorescent protein (gfp) that enables to visualize patterns of the compartment-targeted product. using such gfp-labeled constructs we had a possibility to monitor protein unfolding, aggregation and refolding in the cytoplasm, nucleus and mitochondria of transfectants exposed to either stressful conditions. gfp-luciferase expressed in mammalian cells behaves as a relatively labile protein which can undergo reversible unfolding and aggregation in response to heat shock, atp depletion or action of toxic agents. in the case of cell recovery, refolding of denatured luciferase is carried out at the chaperone machine. we explored unfolding/refolding of gfp-luciferase in the cytoplasm, nucleus and mitochondria of ischemia-stressed rat cardiac cells and in several cancer cell lines treated with hyperthermia or some chemotherapeutic drugs. the results obtained have revealed intriguing correlations between the proteotoxic impact within either compartment and the viability of treated cells. amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid bilayers s. jha , d. sellin , r. seidel , r. winter biophysical chemistry, department of chemistry, tu dortmund university, otto-hahn str. , d- , dortmund, max-planck-institute for molecular physiology, otto-hahn str. , d- , dortmund, germany the islet amyloid polypeptide (iapp), which is considered as the primary culprit for β-cell loss in type diabetes mellitus patients, is synthesized in the β-cells of the pancreas from its precursor, the pro-islet amyloid polypeptide (proiapp). proiapp is co-processed in the secretory granules and co-secreted to the extracellular matrix together with insulin as iapp. here, we compare the amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid membranes, which have been discussed as loci of initiation of the fibrillation reaction. the two peptides show an enhanced amyloidogenic propensity in the presence of negatively charged membranes and similar secondary structural properties. however, proiapp shows a much less amyloidogenic propensity, probably due to the increased net charge on proiapp, compared to iapp. unlike iapp, proiapp forms small oligomeric structures at the lipid interface, having heights of ∼ . nm. this morphological distinction can be attributed to the presence of the pro-region, flanking the amyloidogenic iapp. the addition of proiapp to iapp marginally delays iapp fibrillation, probably by interfering with the interaction between amyloidogenic iapp cores of distinct iapp molecules. thus, it appears reasonable to speculate that the pro-region of the proiapp could serve to delay the fibrillogenesis of iapp at negatively charged lipid bilayers. the role of transmembrane domain interactions in the kinetics and folding of cpt z. a. jenei , k. borthwick , v. a. zammit , a. m. dixon chemistry dept., univ. of warwick, coventry, uk, clinicalȃsciencesȃres.ȃinst., warwick univ., coventry, uk carnitine palmitoyltransferase i (cpt ) enzymes are polytopic integral membrane proteins in the outer membrane of mitochondria (omm). cpt controls the rate of entry of long-chain fatty acids into the mitochondrial matrix for βoxidation. the two catalytically active isoforms, cpt a and cpt b, are different in their inhibitor binding kinetics and structure (interaction between n-and c-segments, interactions of transmembrane domains (tmd)). it has been suggested that inter-and intramolecular tmds interactions are important for cpt a, but not for cpt b function. cpt a has also been implicated in formation of oligomeric complexes through its tm segments. the study of tm helix-helix interactions in cpt isoforms could lead to a better understanding of their function and inhibitor binding kinetics, and will contribute towards the design of pharmacological strategies aimed at modulating the activities of cpt enzymes in conditions such as diabetes. to investigate the ability of the tmd in cpt to self-associate and the order of any oligomers formed, several biochemical and biophysical techniques have been used. we found the self-association of rcpt a tmds (tm , tm ) to be different as measured using the in vivo tox-cat assay. chemical cross-linking and analytical ultracentrifugation studies demonstrated formation of both trimers and hexamers by the rcpt a tm peptide. these results provide further evidence that tm plays role in formation of a channel in the omm. self-assembly of transmembrane domains in cpt : role of gxxxg(a) motif in possible channel formation z. a. jenei , k. borthwick , v. a. zammit , a. m. dixon department of chemistry and, warwick medical school, university of warwick, coventry, uk carnitine palmitoyltransferase a (cpt a), a membrane protein that controls the rate of oxidation of long-chain fatty acids, is of key importance in diabetes and has recently been reported to exist as an oligomer in vivo. we have investigated full-length cpt a and find that the protein exists as a hexamer in liver mitochondria. using mutants of cpt a expressed in yeast mitochondria, we have localised key protein interactions in the hexamer to the transmembrane (tm) domains of the protein. detailed study of the tm domains in isolation, in both e.coli membranes and detergent micelles, demonstrated that while tm shows little self-assembly, tm displayed significant self-association. biophysical analyses of a tm -derived synthetic peptide revealed oligomerization behaviour identical to native cpt a in mitochondria, providing a strong link between tm helixhelix interactions and cpt a hexamer formation. this is significant in light of a recent suggestion that cpt a oligomerization may lead to formation of a channel in the mitochondrial outer membrane through which acylcarnitine gains access to the inter-membrane space. our data supports this new theory, and we go on to demonstrate experimentally the structural determinants of hexamer (channel) formation, specifically gxxxg(a) motifs in the tm domain which pack favourably in the hexamer and stabilize the oligomer. investigation of flexible loop role in structure and thermodynamic stability of firefly luciferase p. maghami, b. ranjbar, s. hosseinkhani department of biochemistry and biophysics, faculty of basic sciences, tarbiat modares university, tehran, iran protein folding, like any chemical process, consists of two fundamental components, thermodynamics and kinetics, which determine the stability of the folded state and the pathway of folding, respectively. these processes are currently too difficult to be solved de novo by purely computational methods. experimental evidence is required to simplify the problem via protein engineering .in this research, the wild type firefly luciferase (photinus pyralis) and some of its mutants were over expressed and purified. then their unfolding thermodynamics were examined, using circular dichroism and conventional fluorescence measurements. the unfolding equilibrium constant were measured over a complete rang of denaturant conditions. the measurements were shown structural and physico-chemical changes between wild-type and mutant proteins. exploring intrinsic disorder of unstructured membrane proteins by surface polymer physics intrinsically unstructured proteins (iups) are considered as a separate class within the protein world because they lack a well-defined folded structure. because iup's function is indeed directly linked to structural disorder, they are assumed to be natively unfolded. several physicochemical techniques are available to discriminate the degree of disorder. however a clear structural classification is still lacking. in this context, polymer physics emerges as a powerful tool for getting inside on the conformational abilities directly related to structural disordered of iups. in the present contribution, surface pressure and ellipsometry experiments in conjunction with polymer physics have been used to infer structural data in terms of molecular conformation and flexibility of a membrane protein essential for bacterial division, zipa. this protein has been pointed to posses a high molecular flexibility and to adopt a random coil conformation. folding dynamics of peptides studied by timeresolved infrared spectroscopy c. krejtschi , o. ridderbusch , r. huang , l. wu , t. a. keiderling , k. hauser institute of biophysics, university of frankfurt, germany, department of chemistry, university of illinois at chicago, usa peptides are ideal model systems to study protein folding mechanisms. ir techniques provide both the necessary time resolution as well as the structural sensitivity. we initiate rapid heating by laser-excited ns temperature jumps (∼ • c) and study fast ns-to-µs relaxation dynamics [ ] . the dynamics of the α-helix to random coil transition of polyglutamic acid was analyzed under reversible folding/refolding ph-conditions. the observed relaxation kinetics allowed separation of the folding and unfolding process with additional use of ftir measurements in thermal equilibrium. sitespecific dynamics were monitored by use of isotopic labeling for a β-hairpin peptide whose conformation is stabilized by a hydrophobic core. various single and cross-strand isotopically labeled variants were analyzed. the isotope-edited kinetics show variations in local structural stability of the hairpin backbone. our data support a multistate dynamic behavior, and the site-specific kinetics are consistent with a hydrophobic collapse hypothesis for hairpin folding [ ] . small heat shock protein hsp was predicted to belong to the family of intrinsically disordered proteins. one of the features of these proteins is that they do not demonstrate cooperative thermal transitions on heating. we applied different methods (dsc, ftir and intrinsic tryptophan fluorescence) to investigate the thermal unfolding of hsp . dsc results have shown that thermal denaturation of hsp begins from o c and occurs, with very low cooperativity, within a broad temperature region (up to o c and above). the thermal unfolding of hsp is fully reversible. the ftir data show that heating of hsp from to o c results in complete disappearance of α-helices (from - % to ) and the decrease in β-sheets content from to %. studies on the temperature dependences of tryptophan fluorescence have shown a significant red shift of the spectrum. these changes occurred within temperature region from to o c with midpoint at ∼ o c. probably, this transition can be explained by destruction of β-sheets around trp , the only trp residue of the α-crystallin domain of hsp (other trp residues of hsp are localized in the n-terminal domain). the data obtained confirm the suggestion that hsp is a protein, whose significant part is intrinsically disordered. we propose that, on heating, the α-crystallin domain containing β-sheets melts at higher temperature than the n-terminal domain containing the most of α-helices. t. rosenkranz , a. katranidis , d. atta , j. enderlein , i. gregor , m. grzelakowski , p. rigler , w. meier , j. fitter isb- : molecular biophysics, research centre jülich, germany, institute of physics, biophysics/complex systems, georg august university, göttingen, germany, institut für physikalische chemie; universität basel, basel, swizerland the protein folding mechanism is the missing link in the biological flow of information from the dna to its specific function. since most of proteins within a cell consist of more than one domain studies on this protein class are of major importance. it is a common feature of multidomain proteins to aggregate under refolding conditions, which hinders a refolding. molecular encapsulation of single molecules prevents aggregation. by immobilizing the nanocapsule the observation period in a wide field microscope will be extended, so that slow or rare folding events can be detected. a major goal of this study is to investigate polymeric vesicles with respect to their suitability for protein folding studies [ ] . polymer vesicles maintain their structural integrity even under harsh unfolding conditions. furthermore the nanocontainer proved to be permeable to guanidinium hydrochloride. using encapsulated phoshoglycerate kinase, labeled with atto- , a dye which experiences fluorescence quenching by photo-induced electron transfer (pet) with tryptophans, we demonstrate the remarkable properties of polymeric nanocontainers. applying pet as a folding probe we detected multiple unfolding/refolding transitions of single proteins. proteins frequently become irreversibly modified by carbonylation, a process of introducing the carbonyl group (carbon monoxide) in a reaction with reactive oxygen species (ros) such as superoxide, peroxide or ozone. the main targets for carbonylation in proteins are amino-acid side chains of lysine, arginine and proline. products of carbonylation are aminoadipic semialdehyde from lysine (asa) and glutamic semialdehyde (gsa) from arginine and proline. importantly, carbonylated proteins are marked for proteolysis by the proteasome, but can escape degradation and form aggregates that can be cytotoxic. carbonylation increases with the age of cells and it is associated with ageing and age related disorders such as alzheimer's disease, parkinson's disease and cancer. we have used the molecular dynamics method to study the stability of carbonylated proteins villin headpiece and ubiquitin. simulations were run after mutations of arginine, proline and lysine into gsa and asa had been performed. in addition, we have used thermodynamic integration on lysine, arginine, proline, asa and gsa residues in order to estimate their solvation free energy (related to relative hydrophobicity and hydrophilicity). our results suggest that carbonylation markedly decreases the overall stability of proteins, and that one potential reason for that may be a disruption of the balance between hydrophilic and hydrophobic regions in the protein. a. martino, d. crane, i. m. feavers, b. bolgiano division of bacteriology, national institute for biological standards and control, potters bar, uk the sensitivity to protein's secondary structure and progress in computational calculations have made circular dichroism (cd) an attractive technique to explore the optical properties of three promising vaccine candidates to neisseria meningitidis. clinical batches of a c-term deleted form of nada (genome-derived neisseria antigen -gna ) and the fusion proteins gna - (fp- ) and gna - (fp- ) were therefore studied. increases in temperature and denaturant concentration on secondary structures and folding/unfolding profile were monitored by cd in the far and near uv regions and complemented by trp fluorescence spectroscopy data. furthermore, epitope conformational changes on binding activities to immune-sera were investigated. the calculated secondary structure content was in broad agreement with the available predicted or solved protein structures. upon temperature incubation, a structural transition from a highly α-helical nada to a more unordered conformation, with a mid point at ∼ • c, was observed. fp- and fp- maintained their conformation up to • c or m guhcl in the case of fp- . unfolding was not always reversible. reductions in binding to monoclonal ab titrated along with increasing unfolding. folding/unfolding studies have proven useful in better understanding the solution behaviour and extent of folding of proteins. cold denaturation of yfh offers the clue to understand the effect of alcohols on protein stability s. r. martin , v. esposito , p. de los rios , a. pastore , p. a. temussi national institute for medical research, the ridgeway, nw aa london, u.k., laboratoire de biophysique statistique, sb/itp, ecole polytechnique fédérale de lausanne (epfl), ch- , lausanne, switzerland, dipartimento di chimica, università di napoli federico ii, via cinthia, i- napoli, italy although alcohols are well known to be protein denaturants when present at high concentrations, their effect on proteins at low concentrations is much less well characterized. here we present a study of the effects of alcohols on protein stability using yfh . exploiting the unusual property of this protein of undergoing cold denaturation around • c without any ad hoc destabilization, we determined the stability curve on the basis of both high and low temperature unfolding in the presence of three commonly used alcohols: trifluoroethanol,ethanol methanol. in all cases, we observed an extended temperature range of protein stability as determined by a modest increase of the high temperature of unfolding but an appreciable decrease in the low temperature of unfolding. we suggest that alcohols, at low concentration and physiological ph, stabilize proteins by greatly widening the range of temperatures over which the protein is stable. our results also clarify the molecular mechanism of the interaction and validate the current theoretical interpretation of the mechanism of cold denaturation. biomolecular sciences and biotechnology tor vergata moro , rome, italy cholesterol plays an important role in regulating the structural properties of phospholipid and non-phospholipid membranes. in this study we have applied in situ energy dispersive x-ray diffraction (edxd) to investigate the effect of cholesterol on the structure of different phospholipid and non-phospholipid oriented membranes. in detail, phosphatidylcholine (pc) bilayers and niosomal membranes, made of a non-ionic surfactant centre for bioactive chemistry, department of chemistry department of chemistry this process is initiated at nuclear envelope remnants (ners) in the presence of atp and gtp. the mvs can be divided in two main populations: mv and mv . mv has a classical lipid composition while mv is enriched in phosphoinositides (pips: pi, pip, pip and pip ). ners have an unusual lipid composition, enriched both in cholesterol and pips. physicochemical properties of the pips were investigated as a function of ph and temperature (t) using nmr, saxs and dls to map out their phase state. pips-water dispersions are observed in lamellar, hexagonal or isotropic phases depending on t and ph. in parallel, model membranes mimicking mv and ners lipid composition were studied by h and p nmr. mv modelling shows a complex behaviour of pips on pc membranes: they order or disorder membranes, whereas the order of pc/pi/pip/pip membrane is lower than that of pc or pc/pi membranes c. manzo, t. s. van zanten, m. f. garcia-parajo bionanophotonics group, ibec-institut de bioenginyeria de catalunya, barcelona, spain membrane proteins play a fundamental role in intra-and inter-cellular functions. in particular, the proteins lateral mobility in the fluid membrane environment is crucial for the regulation of several mechanisms, as receptor-mediated signal transduction and establishment of immunological synapses. these mechanisms are controlled through protein crowding and reduced lateral diffusion, which induce macromolecular associations and limit the application of conventional single molecule fluorescence techniques. to measure proteins mobility on living cells membrane, we developed a fluorescent correlation spectroscopy (fcs) setup in which the sample illumination is obtained through near-field scanning optical microscopy (nsom) probes. the use of nsom probes is particularly suited for the observation of dynamics on the cell membrane and overcomes the drawbacks of other techniques. in fact, through a shear-force-based position control, the probe is kept at a fixed distance from the membrane and its sub-wavelength aperture (∼ nm) reduces the illumination area, allowing the observation of highly crowded regions of the membrane. on the basis of preliminary results, the nsom-fcs is expected to provide an additional insight on the proteins trafficking at the membrane level. the technique also presents several potential developments, as the further reduction of the illumination area and two-colors correlation. single molecule fluorescence microscopy of the store-operated calcium channel subunit orai j. madl, j. weghuber, d. bergmair, m. fahrner, m. muik, c. romanin, g. j. schütz johannes kepler university, institute for biophysics, linz, austria store-operated calcium entry (soce) is essential for many cellular signalling processes. the essential pore forming subunit of soce channels in the plasma membrane is orai . here we present single molecule fluorescence microscopy of orai which was performed in order to directly visualize the stoichiometry of mobile orai pores. the protein was fluorescently labeled with monomeric gfp. a novel single molecule fluorescence approach, toccsl (thinning out clusters while conserving the stoichiometry of labeling), was used for the determination of the stoichiometry. this technique allows reducing the density of fluorescently labeled molecules without affecting the stoichiometry of labeling. density reduction is achieved by completely photobleaching a defined area within the plasma membrane; nonbleached gfp-orai aggregates enter the bleached region subsequently by diffusion. our data indicate that there are different populations of orai present in the cell: most of orai is located in the plasma membrane. a second population of orai -mgfp was found to be localized in intracellular vesicles. a significant fraction of the plasma membrane orai exhibits a diffusive movement. we found by analyzing the bleaching characteristics of single orai -mgfp aggregates that in resting cells mobile orai is predominantly dimeric. a. kobitski , a. nierth , m. helm , a. jäschke , g. u. nienhaus university of ulm, germany, university of heidelberg, germany, university of karlsruhe, germany rna molecules have attracted enormous attention in recent years, and various novel roles were revealed for rna in biological processes. ribozymes are a class of rna molecules capable of catalyzing chemical reactions. we have studied a diels-alderase (dase) ribozyme, a small artificial -mer ribozyme, which is capable of catalyzing carbon-carbon bond formation between an anthracene diene and a maleimide dienophile in multiple turnovers. single-molecule fluorescence resonance energy transfer was employed to investigate the intramolecular dynamics of this rna molecule as a function of mg + ion concentration. folding into a functional state occurs via an intermediate state, and continuous fluctuations between these two states were observed on the ms time-scale at the midpoint concentration of mg + ions. an effect of substrates binding on the folding and catalytic reaction of the dase ribozyme is in the focus of our recent research with the ultimate goal to obtain a detailed structural view of the single-molecule conformational changes that accompany the catalytic reaction. a. katranidis , r. schlesinger , k. nierhaus , i. gregor , m. gerrits , g. bueldt , j. numerous studies showed that protein folding and maturation can differ substantially between de novo synthesized proteins and in vitro refolded proteins. here we present an approach employing a two color single molecule sensitive fluorescence wide-field microscope in order to visualize surface tethered fluorescently labeled ribosomes and de novo synthesized gfp molecules in real time [ ] . fluorescence of co-translational folded proteins was observed from mature fluorescent gfp molecules which carry additional amino acids at the c terminus remaining linked to the ribosome. thus it was possible to co-localize fluorescence from labeled ribosomes and from gfp molecules. we demonstrate that the green fluorescence protein mutant gfp emerald is produced with a characteristic time of five minutes. the fastest gfp molecules appeared already within one minute. processes precedent to chromophore formation, such as polypeptide synthesis and protein folding, are fast and last not longer than one minute. in fluorescence spectroscopy, photobleaching is a process which leads to irreversible loss of fluorescent properties of a dye molecule, usually due to photochemical reactions. it is especially important for fcs experiments on slow-diffusion systems since for high excitation intensities it can have a strong impact on fluorescence intensity correlation function. usually it is observed as apparent shortening of the mean diffusion time of the dye molecules. the behavior of tmr-labeled fd-virus rods in water solution under various excitation conditions was investigated. the experiments were conducted for low ( : ) and high ( : ) tmr:virus ratios and for increasing laser intensities. the correlation function was measured in the experiments. the results were fitted using origin software to estimate the influence of photobleaching, and compared with computer simulations. a strong effect of photobleaching was visible for rods labeled with a single dye molecule, while rods labeled with tmr molecules showed little to no bleaching. a prolongation of characteristic diffusion times for highly labeled virus rods in comparison to low-labeled ones was also observed. k. toth , a. gansen , a. valeri , v. böhm , c. a. seidel , j. langowski abt. biophysik der makromoleküle, deutsches krebsforschungszentrum, heidelberg, germany, lehrstuhl für molekulare physikalische chemie, heinrich heine universität, düsseldorf, germanythe nucleosome has a central role in the compaction of genomic dna and the control of dna accessibility for transcription and replication. we studied the effect of dna sequence and selective histone acetylation on the structure, stability and disassembly of the mononucleosomes. quantitative single molecule fret measurements between dyes attached to different parts of the nucleosome permitted us to detect the equilibrium between several subpopulations of reconstituted nucleosomes in solution. we obtained that the heterogeneity and stability of the samples are correlated with each other and influenced both by the dna sequence and the histone acetylation. the path of the linker dna is more sensitive to all studied effects than the dna on the core. intermediates of the disassembly pathway were identified and characterized. j. strömqvist , s. johansson , y. ohsugi , k. andersson , l. xu , m. kinjo , p. höglund , j. widengren experimental biomolecular physics, kth, stockholm, sweden, department of microbiology and cell biology, karolinska institutet, stockholm, sweden, laboratory of molecular cell dynamics, hokkaido university, sapporo, japan dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined.here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. dynamic multiple-target tracing probes spatiotemporal cartography of cell membranes in order to decipher the non random and non homogeneity of the plasma membrane organization, we had performed fluorescence correlation spectroscopy measurements on live cells. this allowed us to establish the presence of nanoscale confining structures and to demonstrate their implication in signaling process . complementing these studies, we present here a new analytical method, namely multiple-target tracing (mtt) which takes advantage of the high resolution provided by singlemolecule sensitivity to generate dynamic maps at high densities of tracked particles. introducing deflation by subtracting detected peaks allows detecting peaks of lower intensity. we achieved an exhaustive detection of particles with performances reaching theoretical limits, and a reconnection of trajectories integrating the statistical information from past trajectories. we demonstrate the potential of this new method of analysis by applying it to the epidermal growth factor receptor labeled with quantum dots, in the plasma membrane of live cells. this has allowed us to build up a global representation of molecular dynamics in cell membranes. dual polarisation interferometry (dpi) is a surface analytical technique capable of dynamically measuring biophysical parameters of conformational change in biomolecular interactions. the technique measures three key parameters, namely layer thickness, layer density (ri) and mass, thereby enabling the resolution of conformational changes involved during binding. a number of different applications are presented. protein-protein interactions: understanding the biophysical nature of protein interactions can deliver insights into the mechanisms by which proteins interact, thereby elucidating protein function. dpi enables correlation between binding affinity and conformational change, greatly enhancing the study of structure-function relationships. lipid layers: the birefringence mode of dpi can be used to study the formation of lipid bilayers and biomolecular self-assembly. it is possible to use a combination of bilayer refringence and mass to study phase transitions associated with protein or peptide binding to the lipid bilayer. the individual stages of adsorption, absorption and micellisation can be distinguished. carbohydrate interactions: dpi uses a planar glass surface and a wide range of coupling chemistries. a carbohydratespecific surface can be used to study a wide range of biomolecular interactions, such as lectins, acidic proteins, extracellular matrix signaling and interactions with complex membranes. the experimental characterization of the elementary conformational steps constituting the protein folding pathway remains a big challenge. quenching of the triplet state of tryptophan by close contact with cysteine has been shown to provide a new tool for measuring the rate of intramolecular contact formation -one of the most elementary events in the folding process -in peptides and proteins using only natural probes. here we show an extensive study on a stabilized mutant of the second beta-hairpin of gb domain. steady state fluorescence and kinetics of contact formation between a natural tryptophan and a cysteine added to the c-terminal are measured for different temperatures and solvent conditions. we separately address the contributions of different structural elements to the overall stability of the hairpin. we extract kinetics parameter for contact formations in the unfolded state, formation of the hydrophobic core and tails pairing in the folded state. by means of a fragment peptide terminated with a tryptophan and a cysteine, we also estimate the structural propensity of the turn region. the data coherently combine with a simple model previously developed to describe the dynamics of unstructured chain [biophys. j. , ( )], here modified with the addition of attractive interactions between specific residues. catalytic power of partially denatured enzymes: implementation of molten-globule-like states m. shushanyan , d. e. khoshtariya , m. makharadze , t. tretyakova , r. van eldik institute of molecular biology and biophysics, gotua , tbilisi, georgia, department of physics, i. javakhishvili tbilisi state university, tbilisi, georgia, department of chemistry and pharmacy, university of erlangen-nürnberg, germany impact of nonspecific moderate denaturants, urea and dmso, on the kinetic (functional) and thermodynamic (stability) patterns of a hydrolytic enzyme, α-chymotrypsin (α-ct) has been investigated. furthermore, an impact of urea and guhcl in combination with of temperature on the kinetic pattern of carboxypeptidase a has been examined. for the case of α-ct, in particular, we have observed about tenfold increase of the apparent mickaelis constant when going from to m urea ( o c), whereas the value of catalytic constant remained almost unchanged, indicating that the protein is not unfolded even under those severe conditions. the matching microcalorimetric experiments revealed that both the temperature-induced melting temperature and transition enthalpy decrease gradually with the increase of the additive concentration. with m urea the peak-shaped calorimetric feature disappears totally. however, catalytic power of α-ct was preserved owing to its catalytic constant. for other studied enzyme/substrate/denaturant arrays diverse kinetic peculiarities due to mgls have been observed. rubredoxins are a class of iron-containing proteins whose biological role on electron transfer processes and metal binding is still unclear. the unfolding dynamics of the rubredoxin mutant rda c from the mesophile desulfovibrio vulgaris (dv) was studied on the temperature range from • c to • c and along time at • c. resonance energy transfer (ret) was used to determine the donor (d; trp ) to acceptor (a; , -iaedans) distance. from • c to • c the d-a distance increased Å. however the random coil expected d-a distance was only achieved after heating the protein solution during . hours at • c. from uv-vis absorption data it's clear that this protein is capable of maintaining its iron-sulfur center at • c. by melting the protein at the same temperature all iron-centers disintegrate and the protein unfold after . hour. the trp fluorescence also shifts nm to the red reflecting the partial exposition of the indole ring to the solvent. from fluorescence and anisotropy decay curves a breathing type movement of the protein structure was observed between • c to • c without lost of significant secondary structure. this structure flexibility should play an important role on the thermal stability of the dvrd the antimicrobial peptide novicidin (nc) -modified from the sheep self defense peptide smap- , for reduced mammalian cytotoxicity -is a cationic peptide (net charge +∼ . ) that adopts random coil structure in solution, but an α-helix in the presence of lipid vesicles. the conformation of nc in presence of the lipids dlpc, dlpg, dmpc, dmpg, dopc, dopg, dope, and dops in different combinations reveal the lipid selectivity, affinity, and phase dependent changes with varying l/p ratios and temperatures, observed from cd spectroscopy. it is understood that the conformational change is dependent on chain length and head group of the lipid. apart from the in vivo results on the nc activity, studies using qcm-d, dual polarisation interferometry, and calcein release assay reveal the kinetics and concentration dependent activity of nc in lipid bilayers and vesicles. preliminary studies on orientation of nc in various lipid environments using ssnmr, lsnmr, and molecular dynamics simulations apparently suggest that nc may form toroidal pores/detergent effects depending on the chain length of the lipids. further experiments on nc in presence of lipids using dsc, itc, ssnmr, oriented cd, ld, and confocal microscopy to determine the structure, thermal stability, orientation in lipid bilayers, and thereof the action of nc will help in proposing a comprehensive model for its mechanism of action in model membranes. dielectric method for measuring glass transition and denaturation temperatures of hydrated proteins g. e. thomas , s. bone , g. drago institute for bioelectronic and molecular microsystems, bangor university, gwynedd, uk., applied enzyme technology, pontypool, uk.the flexibility of protein structures is important in allowing the variety of motions, covering a wide range of magnitudes and frequencies, essential to biological activity. high frequency dielectric measurements can be used to study the flexibility of proteins by probing the relaxation of dipolar constituents of their structures. hydrated proteins exhibit a broad dielectric loss extending over the frequency range from mhz to ghz which can be decomposed into a number of constituent dispersions. one of these dispersions, with a relaxation time of ∼ ns, has been attributed to the relaxation of protein backbone peptide groups in the protein interior. in the work reported here, this dielectric dispersion was investigated as a function of temperature for the enzyme glucose oxidase. two critical temperatures were identified as the glass transition and denaturation temperatures, both of which were found to decrease with increasing protein water content. the results were consistent with a scheme in which the hydrated glassy protein undergoes a change in structural mobility at the glass transition temperature and experiences an irreversible change in conformation at a higher denaturation temperature. both glass transition and denaturation temperatures are key indicators of protein stability and are important in the production and storage of protein based pharmaceuticals. a. szymańska, k. kacprzyk, g.Ślósarek department of molecular biophysics, a. mickiewicz university, umultowska , - poznań, poland aggregation dynamics of proteins plays an important role in molecular biology and medicine as it permits explanation of several disease states. an interesting problem is to find out in which conditions the interactions of the protein molecules lead to formation of ordered structures and in which to disordered ones. in this study, dynamic light scattering, circular dichroism and also congo red dye experiments were performed to analyze various structural states of lysozyme induced under different concentration of ethanol solvent. at low ethanol concentration the attractive interaction between the protein macromolecules dominate. after addition of more ethanol solvent, the translational diffusion coefficient was much smaller than that for lysozyme solution at zero ethanol concentration. it can be explained by the structural transformation of the polypeptide chain leading to a partially folded conformation needed for oligomerization and fibrillation process. on the basis of the cd experiments we concluded that the ethanol solvent induces changes in secondary structure of lysozyme solution. on addition of % v/v ethanol solution the intramolecular hydrogen bonds were destabilized. above this ethanol concentration, β -sheet were the dominant secondary structure of lysozyme in solution. the phase diagram illustrating the formation of: monomers, oligomers at various structural states, protofilament formation state, protofilament and amyloid fibrils was constructed. key: cord- -o hr mox authors: nan title: proceedings of réanimation , the french intensive care society international congress date: - - journal: ann intensive care doi: . /s - - - sha: doc_id: cord_uid: o hr mox nan rationale: expiratory muscles has recently been stated as the «neglected component» in mechanically ventilated patient. several authors stated these muscles importance in cough capacity, contractile efficiency of the diaphragm or reduction of hyperinflation. however, few studies reported potential factors leading to expiratory muscle weakness and its importance on weaning success or survival after mechanical ventilation. patients and methods: this study is a secondary analysis of our previously described cohort of patients ventilated for at least h assessed for respiratory muscles function. maximal expiratory pressure (mep) measurement was carried out during spontaneous breathing trial using a manometer with an unidirectional valve. mep diagnostic accuracy to predict icu-aw (icu acquired weakness), weaning success and sursvival within days were assessed using expiratory muscle strength as absolute values (cmh o), as %predicted values and as %lower limit of normal. results: due to the paucity of data reporting threshold value for expiratory muscle weakness, we considered our median value ( cmh o (iqr )) as the threshold value for expiratory muscle weakness group (mep ≤ cmh o) and normal expiratory muscle group (mep > cmh o). patients with low mep received more catecholamines (p = . ) and a higher duration of mechanical ventilation (p = . ). inversely, higher body mass index was associated with higher mep. patients with low mep presented more icu-aw compared to normal mep patients ( % vs. %; p = . ). no other outcomes were different between groups. mep was statistically able to predict icu-aw but area under (auc) receiving operating curves showed weak predictive ability (auc: . ( % ic . - . ; p < . ) for a threshold value ≤ cmh o. expiratory muscle weakness was unable to predict critical outcomes when adjusting mep to the %predicted or lower limit of normal. discussion: possible explanation is that contrary to inspiratory muscle weakness, cough inefficacy after weaning from mechanical ventilation could be managed with cough supplementation techniques (i.e. mechanical in-exsufflation). conclusion: in our cohort, mep was not associated with mechanical ventilation weaning or death. despite our results, different clinical techniques for quantifying expiratory muscle weakness may provide more beneficial results. compliance with ethics regulations: yes rationale: venoarterial extracorporeal membrane oxygenation (va-ecmo) is used to support tissue perfusion during extracorporeal cardiopulmonary resuscitation (e-cpr). shock, resuscitation and the extracorporeal circuit may trigger a capillary leakage and a vasoplegic shock. currently, in these situations, high doses of norepinephrine (ne) are required. because high ne doses may have significant cardiovascular side effects, alternative options to support arterial blood pressure are needed. in recent years, several approaches to decrease the administration of high ne doses have been tested, one of them is the administration of vasopressin (avp). randomized trials have shown that avp infusion increases arterial pressure and systemic vascular resistance, decreases catecholamine requirements in patients with or at high risk of vasoplegic syndrome and attenuates vascular dysfunction. currently, no data are available for the study of the effects of avp in shock state in post refractory cardiac arrest. patients and methods: pigs were randomized into two groups, in order to receive avp or ne. a refractory cardiac arrest of ischemic origin was surgically created and va-ecmo was started after a min period of cardio-pulmonary resuscitation. then, resuscitation lasted h in each randomization group. the evolution of the consequences of the shock was evaluated by lactatemia and microcirculation (sdf and nirs) at baseline hour, h (when ecmo starts), h and h . renal and hepatic functions were assessed. results: experimental conditions were met for animals (avp, n = ; ne, n = ). the groups were comparable on the shock impact and its severity. no significant differences were found between populations for ecmo flow and map. there was a significant difference on fluid volume resuscitation amount ( [ . - . ] ml in the ne group versus ml in the avp group, p < . ) (fig. ). no significant difference between the ne and avp groups for lactate clearance between h and h ( . [− . to . ]% vs . [ . - . ]%, p = . ). we did not find any significant for sublingual microcirculation indices and nirs values. renal and liver function evolution were similar in the two groups during the protocol. conclusion: avp administration in refractory cardiac arrest resuscitated by va-ecmo when compared to ne is associated with less fluid volume for similar global and regional hemodynamic effects. compliance with ethics regulations: yes. patients and methods: a single-center prospective study. patients younger than months with severe bronchiolitis and supported by niv or hfnc were included. niv/hfnc was discontinued according to the local practices and no protocol existed. exceptt the principal investigator, the attending team was blinded to the study. weaning failure was defined as the need to reinstate niv/hfnc in the h after discontinuation. ethical approval was not necessary for this study in accordance with the french data protection autority methodology reference number mr- . results: a total of patients (median age days, ( %) males) were included. respectively, ( %) and patients ( %) were supported by niv and hfnc at admission (fig. ) . regarding the mode of niv, a bilevel mode was used in patients ( %) (fig. ). in patients supported by hfnc, the ventilatory support was discontinued progressively by decreasing air flow in patients ( %) while it was stopped abruptly in ( %). in patients supported by niv, the respiratory support was stopped abruptly in ( %) of them while hfnc was used as a weaning method for ( %) patients. a total of ( %) patients experienced a weaning failure. patients supported by niv/ hfnc who experienced a prompt weaning had a lower pediatric intensive care unit (picu) length of stay as compared to patients in whom hfnc was used as a weaning method ( ± h versus ± h, p = . ). however, the hospital length of stay was similar according to the weaning method ( ± days versus ± days for prompt and progressive methods respectively, p = . ). the duration of the weaning process did not differ according to the bed-availability in picu. in patients with severe bronchiolitis, a prompt weaning from niv/hfnc was associated with a lower length of stay in picu. however, the hospital length of stay was similar according to the weaning method. we suggest that a prompt weaning should be preferred in order to reduce the risk of picu related complications. compliance with ethics regulations: yes. information and incitation to open a twitter account and to follow critical care journal feeds) or group (control group). ict were interrogated on their recent medical literature knowledge at and month on trials published in pre-selected journals. results: during the study period, on the french ict contacted, agree to participate: were already on twitter, were randomized to twitter incitation and to control group. at month, there were who answered electronic questionnaire. self-declaration of article knowledge was not different between groups (p = . ). knowledge of primary outcome of each trial was not significantly better in groups (p = . ). in per-protocol analysis of ict on twitter or not, knowledge of article and primary outcome were also not significantly different (respectively p = . and p = . ). short incitation to open a twitter account and follow major medical journals with specific focus on cardiac arrest did not improve knowledge of medical literature by intensive care trainees at month. further trials are needed to better imply intensive care trainees in scientific medical literature. compliance with ethics regulations: yes. - . ] ; p = . ) as independently associated with in-hospital mortality ( fig. ). discussion: triple therapy is the recommended first-line treatment of caps. however, herein, it was not significantly associated with better survival in critically ill, thrombotic aps patients. for the subgroup of "definite/probable caps" patients, double and triple regimens were associated with survival. but the bivariable analyses including the day- saps ii showed that survival was linked to in-icu anticoagulation and corticosteroids-not ivig or plasmapheresis. our findings indicate that corticosteroids should probably be added to in-icu anticoagulation to treat "definite/probable caps". frequent fever and elevated c-reactive protein in all thrombotic aps patients suggest a marked inflammatory state that could explain corticosteroid efficacy. neither plasmapheresis nor ivig impacted the prognosis of "definite/ probable caps", but that finding could be explained by a lack of power compared to caps registry data. conclusion: in-icu anticoagulation was the only aps-specific treatment independently associated with survival for all patients. doublebut not triple-therapy was independently associated with better survival of "definite/probable caps" patients. in these patients, double therapy should be used as first-line therapy while the role of triple therapy requires further evaluation. compliance with ethics regulations: yes. motor deficiency ( %) ( %) ( %) . cognitive impairment ( %) ( %) ( %) . intra-individual relationships between Δpdi and tfdi for mechanically ventilated (mv) patients (a) and healthy subjects (c). relationships between Δpdi and tfdi when breathing cycles were averaged for all participants during each condition for mv patients (b) and healthy subjects (d). − %: initial settings minus % inspiratory help, + %: initial settings plus % more inspiratory help, pep : zero positive end-expiratory pressure, sbt: spontaneous breathing trial. healthy subjects performed spontaneousbreathing (sb) and ventilation against inspiratory threshold at , , , and % of maximal inspiratorypressure (mip) groups. airway closure occurrence increased with bmi ( %, % and %, p = . ). when present, airway opening pressure was . cmh o ( . - . ) and similar between the groups. with increasing bmi, total peep increased from . to . cmh o between groups (p = . ). all values of esophageal pressure increased with bmi. endexpiratory esophageal pressure was strongly correlated with bmi (rho = . , p < . ), as illustrated in fig. . consequently end-expiratory transpulmonary pressure decreased from − . to − . cm h o with increasing bmi (p = . ). the ratio of eelv to predicted functional residual capacity was negatively correlated with end-expiratory pressure (rho = − . , p = . ), but not with bmi. driving pressure and elastance of the respiratory system, chest wall and lung were similar across all ranges of bmi. likewise, eelv was similar between groups. conclusion: in ards, increasing bmi is associated with increased occurrence of airway closure and increased values of esophageal pressure. conversely, chest wall elastance is not influenced by bmi, as well as lung elastance. including bmi in interpreting respiratory mechanics in ards patients can provide additional information for the clinical management. compliance with ethics regulations: yes. rationale: low tidal volume is the cornerstone of protective ventilation inthe initial phase of ards ( ) . whether such low tidal volume can still be achieved when the patient is allowed to breathe spontaneously under pressure support ventilation (psv) is unknown. in moderate-tosevere ards patients receiving neuromuscular blockade, we assessed the tidal volume and its potential association with the outcome during the "transition period" following neuromuscular blockade. patients and methods: retrospective observational study in two university intensive care units. patients fulfilling moderate-to-severe ards criteria less than h after intubation and receiving neuromuscular blockers were included upon entry in the "transition period". we defined the "transition period" as the h following neuromuscular blockers cessation. ventilatory and hemodynamic parameters were recorded every h during the "transition period". primary outcome was the association between mean tidal volume under pressure support ventilation (psv) during the "transition period" and the -day mortality after adjustment for confounding factors. data are reported as median [ st- rd quartile] or number (percentage). results: one hundred nine patients were included, with a pao /fio ratio of mmhg at intubation and mmhg at inclusion and a sofa score at [ . - ] . patients had been ventilated days [ - . ] before inclusion. during the "transition period", patients ( . %) were switched to psv. the median duration of psv was h . the mean tidal volume under psv was significantly lower in survivors than in non survivors at day ( . ml/kg [ . - . ] vs. . ml/kg [ . - . ] respectively, p = . ). by multivariate analysis (cox proportional hazards regression model), mean tidal volume during psv remained independently associated with the -day mortality after adjusting for sofa score and immunosuppression. patients with a mean tidal volume above ml/kg under psv during the "transition period" had a lower cumulative probability of survival at day as compared with others (log rank test, p = . ) (fig. ) . conclusion: in patients with moderate-to-severe ards, a higher tidal volume under psv within the h following neuromuscular blockers cessation is independently associated with the -day mortality.compliance with ethics regulations: yes. kaplan-meier estimate of the cumulative probability of survival according to the mean tidal volume (vt)-lower of higher than ml/ kg-under pressure support ventilation (psv) during the "transition period" transfusion is associated with adverse events, and equipoise remains on the optimal transfusion strategy in oncologic patients in surgical setting. patients and methods: this is a retrospective, single center study. all adults admitted to the intensive care unit (icu) after oncologic surgery from january to december were eligible. the following types of surgery for cancer or metastasis resection with a high risk of bleeding were eligible: thoracic, abdominal, neurosurgery, gynecologic, urologic, otorhinolaryngology or spinal surgery. the primary outcome was a composite outcome including post-operative complications (respiratory, cardiac, renal, thromboembolic, infectious and/or hemorrhagic) and/or hospital mortality. results: of the patients included, patients ( . %) had anemia (based on the who definition: hemoglobin level - . g/dl for female; hemoglobin level - . g/dl for male), patients ( %) had moderate anemia (hemoglobin level: - . g/dl) and patients ( . %) severe anemia (hemoglobin level < g/dl). fifty-six patients ( . %) received at least one rbc transfusion during their hospital stay. patients exposed to moderate and severe anemia required more often renal replacement therapy (rrt) for acute kidney injury (aki) ( . % vs. . %; p = . ), had more surgery-related infections ( . % vs. . %; p = . ). patients who received rbc had more often aki with rrt ( . % vs. . %; p < . ), thromboembolic events ( . % vs. . %; p = . ), sepsis ( . % vs. . %; p = . ), pneumonia ( . % vs. . %; p = . ), surgical site infections ( . % vs. . ; p < . ) and second surgery for infection ( % vs. . %; p = . ). the multivariate analysis found an association between moderate and severe anemia (moderate anemia: or . [ . - . ] ; severe anemia: or . [ . - . ]; p = . ) and severe post-operative complications (fig. a) . there was also an association between rbc transfusion and severe post-operative complications ]; p < . ) (fig. b) . conclusion: anemia was frequent in oncologic surgical patients. anemia, including moderate anemia, was independently associated to patient outcomes; however, rbc transfusion also negatively impacts on patients' prognosis. our study highlights the need for further research to identify the optimal hemoglobin threshold for rbc transfusion in surgical oncologic patients. compliance with ethics regulations: yes. rationale: right ventricular (rv) failure is a common complication in moderate to severe acute respiratory distress syndrome (ards). rv failure is exacerbated by hypercapnic acidosis and overdistension induced by mechanical ventilation. veno-venous extracorporeal co removal (ecco r) might allow ultraprotective mechanical ventilation strategy with a low tidal volume (vt) and plateau pressure (pplat). this study investigated if ecco r therapy could have beneficial effects on rv function. patients and methods: this prospective monocentric pilot study was conducted in a french icu from january to march . patients with moderate to severe ards with pao /fio ratio between to mmhg were enrolled. ventilation parameters, arterial blood gases, echocardiographic parameters performed by transthoracic echocardiography (tte), low-flow ecco r system operational characteristics, outcomes and adverse events were collected during the protocol. primary end point was evolution of rv echocardiographic parameters with ultraprotective ventilation strategy at ml/kg pbw during the -h following the start of ecco r. results: eighteen patients were included. efficacy of ecco r allowed an ultraprotective strategy in all patients. we observed a significant improvement of rv systolic function parameters assessed by tte (fig. ). tricuspid annular plane systolic excursion (tapse) increased significantly under ultraprotective ventilation compared to baseline (from . to . mm; p < . ). systolic excursion velocity (s') also increased after -day protocol (from . m/s to . m/s; p < . ). a significant improvement of aortic velocity time integral (vtiao) under ultraprotective ventilation settings was observed. there were no significant differences in the values of systolic pulmonary arterial pressure (spap). when patients were separated in two groups according to baseline paco level above or under mmhg, we showed the deleterious effect of hypercapnia on rv function, and observed in both groups a beneficial impact of an ultraprotective ventilation strategy on tapse. no severe adverse events directly related to ecco r were observed in our small cohort. conclusion: the low-flow ecco r allows ultraprotective ventilation strategy and improve rv function in moderate to severe ards patients. similarly to prone positioning, ecco r could become a strategy that enables to reconcile lung protective approach with rv protective approach in ards patients. large-scale clinical studies, including patients with severe rv dysfunction, will be required to confirm these results and to assess the overall benefits, in particular the best timing of beginning ecco r in ards patients. compliance with ethics regulations: yes. rationale: bronchoalveolar lavage (bal) is usually deemed to allow the diagnosis of a large array of pulmonary diseases and is usually considered as well tolerated in intensive care unit (icu) patients. however, recent data suggest that the diagnostic yield of bal could be rather low ( ) , and may question its innocuity ( ) . the present study aimed at assessing the benefit-to-risk balance of bal in icu patients. patients and methods: the study was approved by the appropriate ethics committee and registered with clinicaltrials.gov (nct ). in icus, from april to october , we prospectively collected adverse events (ae) during or within h after bal and assessed the bal input for decision-making in consecutive adult patients. aes were categorized in grades of increasing severity. the occurrence of a clinical ae at least of grade , i.e. sufficiently severe to need therapeutic action (s), including modification (s) in respiratory support, defined poor bal tolerance. the bal input for decision-making was declared satisfactory if it allowed to interrupt or initiate one or several treatments. results: we included bal in patients (age yrs ; female gender: [ . %]; simplified acute physiology score ii: ; immunosuppression [ . %], chronic pulmonary disease [ / ( . %)]). bal was performed either in non-intubated patients receiving standard o therapy (n = [ . %]), or noninvasive ventilation (n = [ . %]), or high-flow nasal cannula o therapy ( [ . %]), or in patients under invasive mechanical ventilation (n = [ . %]). a total of aes were observed in ( . %) patients. sixty-seven ( . %) patients reached the grade of ae or higher. the main predictor of poor bal tolerance identified by logistic regression was the association of a bal performed by a non-experienced physician (non-pulmonologist, or intensivist with less than years in the specialty or less than bal performed) in non-intubated patients (or: . [ % confidence interval . - . ] ; p < . ). ordinal regression also showed that when bal was performed by a non-experienced physician in a non-intubated patient, this was associated with an increased risk of ae of any grade (or: . [ . - . ]). a satisfactory bal input for decision-making was observed in ( . %) cases and was not predictable using logistic regression. conclusion: adverse events related to bal in icu patients are frequent, and sometimes serious. our findings call for an extreme caution when envisaging a bal in icu patients and for a mandatory accompaniment of the less experienced physicians. compliance with ethics regulations: yes. meningitis is a rare complication of critically ill patients with severe pneumococcal community-acquired pneumonia paul jaubert, julien charpentier, jean-daniel chiche, frédéric pene, alain cariou, guillaume savary, marine paul, jean-paul mira, mathieu jozwiak cochin, paris, france; mignot, versailles, france correspondence: paul jaubert (paul.jaubert@gmail.com) ann. intensive care , (suppl ): rationale: severe pneumococcal community-acquired pneumonia (pcap) is a frequent infection requiring intensive care unit (icu) admission. pneumococcal meningitis associated with pcap has been reported and could worsen the prognosis of patients. however, this complication is difficult to predict and lumbar puncture is not systematically performed, regardless the severity of pcap. thus, we investigated the characteristics of patients with pcap associated with pneumococcal meningitis. patients and methods: we retrospectively included all patients admitted for pcap in our icu between (inception of our electronic medical sheet) and the end of . community-acquired pneumonia was defined according to the criteria of the american thoracic society. we excluded all patients admitted in icu with initial suspicion of meningitis. variables regarding epidemiology, clinical and microbiological characteristics, management and prognosis of these patients were collected and analyzed. results: among the patients admitted for pcap ( ± years old, saps ii ± , % of men), % of the patients required mechanical ventilation and % vasopressors infusion. the icu mortality was %. s. pneumoniae was documented by a positive antigen test in % of the patient and/or by a positive sputum smear, tracheal aspirate or distal protected airway specimen in % of the patients, and/or by pleural aspirate in % of the patients and/or by positive blood culture in % (n = ) of the patients. a lumbar puncture was performed in % (n = ) of the patients with bacteriemia and in % (n = ) of the patients without bacteriemia, with a median delay of h [interquartile range: after the onset of antibiotherapy. alllumbar punctures (n = ) were performed for neurological signs: % of coma, % of confusion and % of seizures. when a lumbar puncture was performed, meningitis was diagnosed in % (n = ) of the patients with bacteriemia and in % (n = ) of the patients without bacteriemia (p < . ). the icu mortality ( % vs. %, respectively), age ( ± vs. ± years old, respectively), saps ii ( ± vs. ± , respectively) or icu length of stay ( ± vs. ± days, respectively) were not different between patients with and without meningitis (each p = ns). conclusion: meningitis is a rare complication of pcap and is more frequent in patients with bacteriemia. suprisingly, meningitis is not associated with higher icu mortality. further analyses are ongoing to identify independent risk factors of meningitis in patients with pcap. compliance with ethics regulations: yes. rationale: shock is the clinical expression of a circulatory failure that results in inadequate cellular oxygen utilization. whereas the host response to septic shock has been extensively described, knowledge of the pathogenesis of non-septic shocks remains limited. we aimed to characterize the systemic host response in shock related to non-septic conditions (nssh) as compared with septic shock (ssh). patients and methods: we performed a prospective study in two intensive care units (icus) in patients admitted for ssh (n = ) or nssh (n = ). immune responses were determined upon icu admission by measuring plasma biomarkers reflecting host response pathways implicated in the pathogenesis of critical illness (in ssh and nssh patients), and by applying genome-wide blood mrna expression profiling (in ssh and nssh patients). results: compared with nssh, patients with ssh had more chronic comorbidities, greater disease severity (apache iv score vs. , p < . ) and worse outcomes resulting in higher mortality rates up to one year after icu admission ( . % vs. . %, p < . ). plasma biomarker analysis revealed severely disturbed host responses in both ssh and nssh patients. however, ssh patients displayed more prominent inflammatory responses, endothelial cell activation, loss of vascular integrity and a more pro-coagulant state relative to nssh patients. blood leukocyte genomic responses were more than % common between ssh and nssh patients relative to health (fig. a) , comprising overexpression of innate pro-and anti-inflammatory pathways, and underexpression of lymphocyte and antigen-presentation gene sets. direct comparison of ssh to nssh patients matched for severity (fig. b) showed overexpression of genes involved in mitochondrial dysfunction and specific metabolic pathways, and underexpression of lymphocyte, nf-κb and cytokine pathways. conclusion: patients with ssh and nssh present with largely similar host response aberrations at icu admission; however, patients with septic shock show more dysregulated inflammatory and vascular host responses, as well as specific leukocyte transcriptome alterations consistent with greatermetabolic reprogrammingand more severe immune suppression. compliance with ethics regulations: yes. rationale: aki is associated with short and long term mortality and morbidity. although recovery has been demonstrated to be associated with outcome of critically ill patients, interpretation of available data is limited by time dependent nature of recovery and by competing risks. our objective was to describe renal recovery, pattern of recovery according to adqi definitions and risk factor of this later. monocenter retrospective cohort study. adult patients admitted in our icu from july to december were included. aki was defined according to kdigo criteria and recovery according to adqi definition. incidence of recovery at each time point was depicted using competing risk survival analysis. risk of transition between aki and no-aki was assessed by a semi-markov model. last, a trajectoire analysis was performed to depict most frequent recovery patterns. results are reported as n (%) or median (iqr). results: patients were included with a median age of ( - ). median sofa score at admission was [ ] [ ] [ ] [ ] [ ] [ ] . at icu admission, patients ( . %) had an aki stage , patients ( . %) an aki stage and patients ( . %) an aki stage . according to adqi criteria, aki was defined as rapidly reversed in patients ( . % of aki patients), persistent aki in patients ( . %) and as acute kidney disease (akd) in patients ( . %), remaining patients couldn't be classified (n = ). risk of recovery was of % per day until day then % per day (fig. a) . fine and gray model, taking into account death as competing risk, identified risk factors negatively associated with renal recovery, namely sofa score (shr = . per point; % ic = [ . - . ]), preexisting hypertension (shr = . ; % ic = [ . - . ]) and aki severity (stage vs. stage shr = . ; % ic = [ . - . ]). risk of de novo aki was maximal during the first days and ranged from to % per day. trajectoire model identified clusters of patients ( fig. b) , closely associated with patients' outcome: a) low patients' severity and no or mild aki (n = ; hospital mortality: %); b) moderate to severe aki but little associated organ dysfunction (n = , hospital mortality: . %); c) severe aki and multiple organ failure (n = ; hospital mortality: . %). conclusion: this study, assessing aki recovery patterns, is the first to our knowledge using adqi definition. despite the high rate of early recovery and of rapidly reversed aki, up to % of aki patients had not recovered at day and could therefore be classified has having akd. compliance with ethics regulations: yes. rationale: sepsis is the most frequent cause of acute kidney injury (aki). the "acute disease quality initiative workgroup" recently proposed new definitions for aki, classifying it as transient or persistent. we aimed to determine the incidence, attributable mortality and host response characteristics of transient and persistent aki in patients with sepsis. patients and methods: we performed a prospective observational study comprising consecutive admissions for sepsis in intensive care units (icus) in the netherlands, stratified according to the presence and evolution of aki. attributable mortality fraction (excess risk for dying with persistent aki relative to transient aki) was determined using a logistic regression model adjusting for confounding variables. in a subset of sepsis patients, plasma biomarkers indicative of major pathways involved in sepsis pathogenesis were measured. in a second subset of patients, whole-genome blood-leukocyte transcriptomes were analyzed. results: sepsis patients were included. aki occurred in . % (n = ), of which . % (n = ) was transient and . % (n = ) persistent. patients with persistent aki had higher disease severity scores on admission than patients with transient aki or without aki and more frequently had severe (injury of failure) rifle aki-stages on admission (n = , . %) than transient aki patients (n = , . %, p < . ). persistent aki, but not transient aki, was associated with increased mortality by day- (adjusted or . , % ci . - . ; p = . ) ( figure) and up to -year (adjusted or . , % ci . - . ;p = . ). the attributable mortality of persistent relative to transient aki by day- was . % ( % ci . - . %). persistent aki was associated with enhanced and sustained inflammatory and procoagulant responses during the first days, and a more severe loss of vascular integrity compared with transient aki. baseline blood gene expression showed minimal differences with respect to the presence or evolution of aki. conclusion: persistent aki is associated with higher sepsis severity, sustained inflammatory and procoagulant responses, and loss of vascular integrity as compared with transient aki, and independently contributes to sepsis mortality. compliance with ethics regulations: yes. rationale: to address the paucity of data on the epidemiology of patients admitted to intensive care units (icus) with in-hospital cardiac arrest (ihca), we examined key features, mortality and trends in mortality in a large cohort of patients admitted in french icus over the past years. patients and methods: from to database of the collège des utilisateurs de bases de données en réanimation (cub-réa), we determined temporal trends in the characteristics of ihca, patients' outcomes and predictors of icu mortality. results: of the icu admissions, ( . %) were cardiac arrests and were ihca ( . %). during the study period, the age of ihca patients increased by . years (p = . ) and patients presented more comorbidities (chronic heart disease, chronic kidney disease and cancer). patients were also more critically ill over the period as reflected by the increase of saps-ii by . % (p < . ). paradoxically, in-hospital management became lighter through the time with reduced respiratory support (p < . ), renal support (p < . ) and use of vasoactive drugs (p < . ). crude in-icu mortality decreased from % to . % over the past eighteen years (p < . ), fig. rationale: in surgery, prophylaxis antibiotic aims at preventing the occurrence of post-operative infections. for adults, it is currently recommended to only use prophylactic antibiotic therapy during the time of the intervention. but in pediatric cardiac surgery, there is no consensus around the optimal duration of use of antibiotic prophylaxis. the protocol was modified in in the icu and its time reduced to h. we aimed to determine whether h of post-sternotomy antibiotic prophylaxis was not less effective than h treatment to help prevent care-associated infections. patients and methods: after agreement of the ethics committee of our institution, we performed a retrospective non inferiority study, with an inferiority margin to %. the primary objective is to compare the incidence of care-related infections between a second-generation cephalosporin (c g) antibiotic prophylaxis during h and a -h protocols. the secondary objectives are to determine the infection's incidence, to identify the risk factors for nosocomial infections and to compare the incidence of multidrug-resistant infections. results: between january and july , children underwent cardiac surgeries with sternal opening. received h of c g antibiotic prophylaxis and received h of c g treatment. five previously infected children have been excluded. both groups were demographically and surgically similar. the median age was months (range a few hours of life to . years old) and the median weight was . kg. in the intent-to-treat analysis, incidence of care-related infections is at . % in the c g- h group and . % in the c g- h group. a multivariate analysis shows that the shorter -h time antibiotic prophylaxis is not inferior regarding infection prevention compared to h of antibiotic prophylaxis, p = . . as in the per protocole analysis, the c g- h group rate was . % and . % for the g g- h group. conclusion: it demonstrates that shortening the antibiotic prophylaxis treatment time to h does not affect or increase the rate of infections after a pediatric sternotomy surgery compared to -h protocole. prophylaxis in pediatric cardiac surgery should be short-lived. a multicenter prospective study would allow a consensus and confirm this decision. compliance with ethics regulations: yes. rationale: the use of "big data" is getting increasingly popular in the medical field, especially in intensive care where large amounts of data are continuously generated. however, big data can be misleading when essential clinical data are missing. the adequate adjustment for potential confounding factors (e.g., severity of respiratory distress) should be the key procedure in the big data analyses; however, it is challenging to capture the clinical severity within large electronic databases. bronchiolitis is one main reason for admission to pediatric intensive care unit (picu). the modified wood's clinical asthma score (mwcas) is widely used to assess the severity of bronchiolitis. the objective of the study is to build an automated mwcas (a-mwcas) to continuously assess the severity of respiratory distress in critically ill children. this retrospective study included all infants < years old with a clinical diagnosis of bronchiolitis, ventilated with non-invasive neurally adjusted ventilatory assist, in a canadian picu, between october and june . we developed an algorithm, using python . , which was directly connected to the electronic medical record. the components of the score were collected using structured query language (sql) queries and processed to derive the a-mwcas. for validation, the a-mwcas score was compared to the mwcas manually computed by a clinical expert (m-mwcas) . results: sixty-four infants were included in the study, for which of a-mwcas and m-mwcas were generated respectively. the cohen's kappa coefficient was applied to estimate the agreement between the two scores which was . ( % confidence interval) ( table ) which corresponds to . % of complete agreement. . % of the a-mwcas scores were within ± . of the m-mwcas. the kappa coefficient for the each score component were: . for the oxygen saturation, . for the expiratory wheezing, . for the inspiratory breath sounds, . for the use of accessories muscles and . for the mental status, respectively. discussion: the largest discrepancy was observed in the mental status, which clinical evaluation is relatively subjective and varies among care team members (doctor, nurse, respiratory therapist…). the automated score likely decreases this variability by consistently using the same source (respiratory therapist), but its validity should be confirmed in a prospective study. the a-mwcas provides a valid estimation of the mwcas that is fast and robust. after external prospective validation, it may help to add some clinical sense within large electronic databases, with improved assessment of the respiratory distress. compliance with ethics regulations: yes. rationale: in paediatric intensive care units (picu), survival rates have dramatically improved. this has been accompanied by increased morbidity, including psychological morbidity. these new impairments, that can affect the survivors and their families have been conceptualized under the frame of post-intensive care syndrome (pics) and picsfamily. the aim of this study was to explore the experience of critically ill children parent's during the stay in picu, and its impact on the family. patients and methods: we planned a prospective, single centre study for months. we collected qualitative written data from parents whose child had been admitted to the picu for the first time, for at least two nights. results: fifty-seven questionnaires were analysed from thirty-seven admissions. picu admissions were mostly unplanned. among parents % experienced very painful memories during admission and % have feared for their child's life. during the stay, noise has bothered % of parents, and many have described difficulties to rest at night. % had the sensation that their child was suffering, mostly from pain, tiredness, anxiety or fear. during picu stay, % of parents had to stop working, and siblings schooling was impacted in % of cases, % of parents considered themselves to be useful for their child and % have participated to nursing care. more than % were satisfied about information given and communication, % appreciated empathy and support from care givers. parents received support from family, friends, and also from other parents of hospitalized children. parents expressed relief ( %) and serenity ( %) to leave picu, % of them were in demand to meet picu staff again after discharge. conclusion: picu parent's experience is tough, and the impact on family is clear. these are known risks factors for pics. on a very positive note, parents seemed to be satisfied by family-centred care, and were able to preserve their parental role. however, there is still room for improvement of practices. compliance with ethics regulations: yes. the gut has been suspected to be involved in multiple organs dysfunction syndrome (mods) in the intensive care unit (icu). studies suggested a link between gastrointestinal dysfunction (gid) and outcomes. but these studies included very few patients and most of them were retrospective. patients and methods: this study is a secondary analysis of data from a previous study that included patients from french icus. gid is defined as the association of vomiting and constipation or diarrhea during the first week after icu admission. patients included were treated with vasopressors and mechanical ventilation. the first goal was to determine if gid is a risk factor of -day mortality in this population. secondary goals were to assess the impact of gid on nosocomial infections. results: among included patients, ( . %) had gid. by day- , ( %) of the patients with gid and ( %) of the patients without gid had died (odds ratio . [ . - . ]; p = . ). multivariable regression model did not show any association between gastrointestinal dysfunction and increased risk of -day mortality in patients (odds ratio . [ . - . ], p = . ). gastrointestinal dysfunction was strongly associated with other secondary outcomes ( table ). patients with gid had longer ventilation duration, icu length of stay and hospital length of stay. they also had more nosocomial infections, in particularly ventilator-associated pneumonia. this association still existed in a multivariable regression model for prediction of nosocomial infection including the same variables than the previous model (odds ratio . [ . - . ], p = . ). no association with day- mortality was observed. conclusion: gastrointestinal dysfunction was not a risk factor of day- mortality but was associated with an increased risk of nosocomial infection and an increased length of stay. this study is observational and no causality link can be done. however, our data suggest further studies on strategies aimed to limit gid. compliance with ethics regulations: yes. rationale: acute cholangitis (ac), a bacterial infection related to an obstruction of the biliary tree, may be responsible for life-threatening organ failure. however, little is known about the outcome and the predictive factors of mortality of critically ill patients admitted in icu for acute cholangitis. we aimed to describe characteristics of patients admitted in icu for ac and to analyze predictive factors of in-hospital mortality including the time to biliary drainage procedure. patients and methods: retrospective study of all cases of acute cholangitis admitted in french icus ( tertiary hospitals and non-ter- [ . ; . ] µg/l. % of patients (n = ) have positive blood culture, mostly gram negative bacilli ( %) and % producing extended spectrum beta lactamase enterobacteriaecae. at icu admission, persisting obstruction was frequent ( %) and therapeutic endoscopic retrograde cholangiopancreatography was performed in % of them. in a multivariable analysis, at icu admission, several factors were significantly associated with in-hospital mortality: sofa score (or = . [ % ic . ; . ] by point, p = . ), arterial lactate (or = . [ . ; . ] by mmol/l, p < . ), total serum bilirubin (or = . [ . ; . ] by umol/l, p < . ), obstruction nonrelated to gallstones (p < . ) and ac complications (liver abcess and/or pancreatitis) (or = . [ . ; . ] p = . ). in addition, time > h between icu admission and biliary drainage was associated to in-hospital mortality (adjusted or = . [ . ; . ] p = . ). conclusion: acute cholangitis is responsible for high mortality in icu. organ failure severity, causes and local complications of cholangitis are predictive factors of mortality as well as delayed biliary drainage. compliance with ethics regulations: yes. the united kingdom) were included (n = ). predictors of one-year mortality were retrospectively screened and tested on a single center training cohort. a predictive score was developed and tested on an independent multicenter cohort. results: four independent pre-transplantation risk factors were associated with one-year mortality after transplantation in the training cohort: age ≥ years (or = . , % ci = . - . , p = . ), pre-transplantation arterial lactate level ≥ mml/l (or = . , % ci = . - . , p = . ), mechanical ventilation with pao / fio ≤ mmhg (or = . , % ci = . - . , p = . ) and pretransplantation leukocyte count ≤ g/l (or = . , % ci = . - . , p = . ). a simplified version of the model was derived by assigning point to each risk factor: the transplantation for aclf- model (tam) score. a cut-off at points distinguished a high-risk group (score > ) from a low-risk group (score ≤ ) with one-year survival of . % vs. . % respectively (p < . ). the model and its simplified version were validated on the independent multicenter cohort. there was a significant difference between the high-risk and low-risk group with one-year survival of % vs. . % respectively (p < . ). conclusion: liver transplantation can be an effective treatment for critically ill cirrhotic patients with hepatic and extra hepatic organ failure provided patients are carefully selected and that they are transplanted at the optimal time in the intensive care. the tam score can help stratify post-transplantation survival and assist clinicians in the transplantation decision-making process at the bedside of aclf- patients. compliance with ethics regulations: yes. rationale: trans-thoracic echocardiography (tte) is commonly used in the initial management of patients with shock in icu. there is little published evidence for any mortality benefit. we compared the effect of echocardiography protocol versus standard care for survival and clinical outcomes. patients and methods: this randomized controlled trial included selected shocked patients (systolic blood pressure < mm hg and signs of organ hypoperfusion) randomized to early tte plus standard care versus standard care without tte. the primary outcome measure was survivalto days. secondary outcome measures included initial treatment and vasopressor weaning. results: consecutive subjects with circulatory shock (low systolic arterial blood pressure (sap) and signs of organ hypoperfusion) at the time of icu admission are included in the study. in the tte group: fluid prescription during the first h was significantly lower rationale: both the negative prognostic value and reversibility of left ventricular (lv) diastolic dysfunction in septic patients remain debated. the excess of mortality in septic shock patients with hyperdynamic profile has only been reported by small-size studies. accordingly, the primary objective of the prodiasys study was to assess the impact of lv diastolic dysfunction (and its severity) and of lv hyperkinesia echocardiographically identified during the initial phase of septic shock on -day survival. the secondary objective was to assess the potential link between lv diastolic dysfunction, cumulative water balance (on day ), and outcome. patients and methods: this was a multicenter, prospective, observational, cohort study. patients older than years hospitalized in icu for septic shock (sepsis- definition) were eligible. exclusion criteria were administration of inotropes, severe left valvular disease, constrictive pericarditis and moribund patients. in each patient, echocardiography was first performed within h after the diagnosis of septic shock and then daily until day , after vasopressor discontinuation, at icu discharge and on day or at hospital discharge, whichever occurred first. vital and biological parameters usually monitored for septic shock management were collected at each echocardiographic assessment. vital status was collected on day . associations between lv diastolic dysfunction or lv hyperkinesia and day- mortality were analyzed using a chi test. adjusted analyses were performed using logistic regression models, including variables known to be linked with the prognosis of septic shock (e.g., severity scores, delay of antibiotherapy). the relationship between the grade (i to iii) of lv diastolic dysfunction and -day survival were analyzed using a logistic regression model. the relationship between the presence of lv diastolic dysfunction and cumulated water balance on day were analyzed using a linear regression model adjusted on the body weight on admission. the relationship between the grade of lv diastolic dysfunction and cumulated water balance on day were analyzed using a linear regression model. diaphragm dysfunction and weaning induced pulmonary edema are two frequent causes of weaning failure but their coexistence and interaction have been poorly investigated. we hypothesized that diaphragm dysfunction may not induce a sufficient decrease in intra-thoracic pressure to increase venous return and generate a weaning induced pulmonary edema. we therefore investigated whether weaning induced pulmonary edema and diaphragm dysfunction are or not associated and evaluated the effect of diaphragm dysfunction on cardiac function and lung aeration during a spontaneous breathing trial (sbt). patients and methods: patients with readiness to wean criteria who had failed a first sbt were eligible. before and after a second sbt, diaphragm function was assessed by measuring the change in tracheal pressure induced by a bilateral phrenic nerve stimulation (ptr, stim), cardiac function (cardiac output, systolic pulmonary arterial pressure) was evaluated with echocardiography and lung aeration was estimated from the lung ultrasound score (lus). plasma protein concentration and hemoglobin were also sampled before and after the sbt. diaphragm dysfunction was defined by ptr, stim < − cmh o and weaning induced pulmonary edema was diagnosed in case of sbt failure associated with ) increase in plasma protein concentration or hemoglobin > % during the spontaneous breathing trial and/or ) early (e) over late peak diastolic velocity ratio > . or e over peak diastolic velocity ratio > . . results: fifty-three patients were included and / ( %) failed the sbt. diaphragm dysfunction was present in / ( %) of patients with weaning induced pulmonary edema, in / ( %) patients with sbt success and in / ( %) patients with other causes of sbt failure (p < . ). during the sbt, diaphragm dysfunction induced a significant increase in systolic pulmonary arterial pressure but no change in cardiac output. patients with diaphragm dysfunction had a higher lus as compared to their counterparts ( ± vs. ± , respectively, p < . ). conclusion: diaphragm dysfunction induces a loss of lung recruitment and a significant increase in systolic pulmonary arterial pressure during the sbt. coexistence of diaphragm dysfunction and weaning induced pulmonary edema is common in case of sbt failure but weaning induced pulmonary edema appears more likely to be involved than diaphragm dysfunction. compliance with ethics regulations: yes. rationale: diaphragmatic weakness in the intensive care unit (icu) is associated with poor outcome. prolonged mechanical ventilation is associated either with a decrease (atrophy) or an increase (supposed injury) in diaphragmatic thickness, both associated with prolonged weaning. shear wave elastography is a non-invasive technique that measures diaphragm shear modulus (sm), a surrogate of its mechanical properties. the aim of this study was to describe the diaphragm shear modulus during the icu stay and to describe its relation with diaphragm thickness. patients and methods: this prospective and monocentric study included all consecutive critically ill patients. ultrasound examination of the diaphragm (aixplorer; supersonic-imagine, aix-en-provence, france) was obtained by two investigatorsevery other day until icu discharge. demographics, diaphragm thickness, sm and outcomes were collected. a mixed model regression was used to study the relation between sm and diaphragm thickness. results: we enrolled patients from december st to june st, being invasively mechanically ventilated during the stay. diaphragm ultrasound evaluation was feasible in / ( %) patients. the duration of mechanical ventilation during the icu stay was [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] days with [ ] [ ] [ ] [ ] [ ] days spent on controlled mechanical ventilation. sm was . ± . kpa and diaphragm end-expiratory thickness was . ± . cm upon icu admission. increase and decrease ≥ % during icu stay occured in and percent of the patients respectively for diaphragmatic thickness, and in and percent of the patients respectively for diaphragmatic sm. diaphragm thickness over time was inversely correlated with diaphragm sm and with time spent under mechanical ventilation (table) . diaphragm sm over time was correlated with time spent under pressure support ventilation or under spontaneous breathing (compared to controlled ventilation) and with time spent under deep sedation. diaphragm sm was inversely correlated with age, sepsis, exposition to steroids (table) . no association was found between diaphragm sm and outcomes. discussion: our results are in line with the myotrauma concept, suggesting alteration in diaphragm mechanical properties associated with increased diaphragm thickness in critically ill patients. we hypothesize that this observation most likely reflects muscle injury and tissue infiltration with edema and inflammatory cells. conclusion: shear wave ultrasound elastography suggests that in critically ill patients, the increase in diaphragmatic mass is associated with an alteration in diaphragm mechanical properties as measured by sm. compliance with ethics regulations: yes. rationale: diaphragm dysfunction and intensive care unit (icu) acquired weakness (icu-aw) are associated with poor outcomes in the icu but their long term impact on prognosis and health-related quality of life (hrqol) is poorly established. this study sought to determine whether diaphragm dysfunction is associated with negative long-term outcomes and whether the coexistence of diaphragm dysfunction and icu-aw has a particular impact on two-year survival and hrqol. patients and methods: we used a previous cohort study conducted in our institution to follow up mechanically ventilated patients in whom diaphragm and limb muscle functions were investigated at the time of liberation from mechanical ventilation. diaphragm dysfunction was defined by tracheal pressure generated by phrenic nerve stimulation < cmh o and icu-acquired weakness was defined by medical research council (mrc) score < . hrqol was evaluated with the sf- questionnaire. results: sixty-nine of the patients enrolled in the original study were included in the survival analysis and were interviewed. overall two-year survival was % ( / ): % ( / ) in patients with diaphragm dysfunction, % ( / ) in patients without diaphragm dysfunction, % ( / ) in patients with icu-acquired weakness and % ( / ) in patients without icu-acquired weakness. patients with concomitant diaphragm dysfunction and icu-acquired weakness had a poorer outcome with a -year survival rate of % ( / ) compared to patients without diaphragm function and icu-acquired weakness ( % ( / ) (p < . )). hrqol was not influenced by the presence of icu-acquired weakness, diaphragm dysfunction or their coexistence. conclusion: icu-acquired weakness but not diaphragm dysfunction has a strong negative impact on two-year survival of critically ill patients. the presence of diaphragm dysfunction appears more likely to be a determinant of early prognosis and does not appear to have a significant impact on long-term survival. compliance with ethics regulations: yes. rationale: influenza can lead to severe condition with acute respiratory failure and acute respiratory distress syndrome due to a massive pulmonary inflammatory in response to the viral invasion. lung bacteriobiota has been described to be associated with pulmonary inflammation in chronic respiratory diseases such as chronic obstructive pulmonary disease or cystic fibrosis. lung mycobiota has been poorly investigated despite the well-known role for fungi in numerous respiratory diseases. the aim of our study was to assess the prognostic value of lung bacteriobiota and mycobiota among critically ill influenza patients. patients and methods: we prospectively included influenza patients admitted to icu. sputum were stored a - °c. bacterial and fungal dna were extracted thanks to qiaamp ® powerfecal ® pro dna kit. s rrna gene v -v regions and its regions were amplified by pcr and sequenced on illumina miseq ® . taxonomic assignation was obtained by dada pipeline and microbiota analysis were performed according to day- mortality by the mean of phyloseq package on r . . software. results: thirty-nine patients were admitted to icu for influenza with sputa available and finally dna samples available after extraction. bacteriobiota alpha diversity was significantly lower among non-survivors than survivors when expressed by the mean of shannon index, simpson index or evenness (respectively p = . , p = . , p = . ). area under the curve to predict day- mortality was . , ci [ . ; . ] for shannon index, . ci [ . ; . ] for simpson index and . ci [ . ; . ] for evenness. β-diversity analysis also demonstrated significant differences between survivors and non-survivors (adjusted permutational multivariate anova, p = . ). nonsurvivors had a higher abundance of staphylococcus, haemophilus, streptococcus and moraxella. none of the fungal alpha-diversity index nor beta-diversity were significantively different between survivors and non-survivors. non-survivors had a higher proportion of candida albicans and malassezia but not of aspergillus. conclusion: the lung bacteriobiota profile, but not the mycobiota one, of critically ill influenza patients is associated with day- mortality and may be used to identify subjects with a poor prognosis at the time of admission. compliance with ethics regulations: yes. that takes into account the interaction between multiple cellular pathways. the pathway profiles between moderate and severe influenza were then compared to delineate the biological mechanisms underpinning the progression from moderate to severe influenza. results: patients ( severe and moderate influenza patients) and healthy control subjects were included in the study. severe influenza was associated with upregulation in several neutrophilrelated pathways, including pathways involved in neutrophil differentiation, migration, degranulation and neutrophil extracellular trap (net) formation. the degree of upregulation in neutrophil-related pathways was significantly higher in severely infected patients compared to moderately infected patients. severe influenza was also associated with downregulation in immune response pathways, including pathways involved in antigen presentation, cd + t-cell co-stimulation, cd + t cell and natural killer (nk) cells effector functions. apoptosis pathways were also downregulated in severe influenza patients compared to moderate and healthy controls. conclusion: these findings showed that there are changes in gene expression profile that may highlight distinct pathogenic mechanisms associated with progression from moderate to severe influenza infection. compliance with ethics regulations: yes. rationale: herpesviridae reactivation among non-immunocompromised critically ill patients is associated with impaired prognosis, especially during acute respiratory distress syndrome (ards). however, few is known about herpes simplex virus (hsv) and cytomegalovirus (cmv) reactivation occurring in patients with severe ards under venovenous extracorporeal membrane oxygenation (ecmo). we tried to determine the frequency of herpesviridae reactivation and its impact on patients'prognosis during ecmo for severe ards. patients and methods: we conducted an observational, retrospective study in a medical icu (ards and ecmo referee center) between and . patients with a severe ards requiring a venovenous ecmo for days or more were included. hsv and/or cmv reactivation occurring after ecmo insertion was screened for these patients. patients with immunosuppression, antiviral therapy against hsv and/ or cmv prior to inclusion, or hsv/cmv reactivation known at the time of ecmo insertion were excluded. hsv reactivation was defined by a positive qualitative throat sample (virocult ® ) pcr or positive bronchoalveolar lavage (bal) pcr. cmv reactivation was defined by a positive quantitative blood or bal pcr. results: during a five-year period, non-immunocompromised patients with a severe ards necessitating a veno-venous ecmo were included. sixty-seven ( %) experienced hsv and/or cmv reactivation during ecmo course ( viral co-infection, hsv alone and cmv alone). hsv reactivation occurred earlier than cmv after the beginning of mv ( ( - ) vs. ( - ) days; p < . ) and after ecmo implementation ( ( - ) vs. ( - ) days; p < . ). in univariate analysis, hsv/cmv reactivation was associated with a longer duration of mechanical ventilation ( ( - . ) vs. . ( - ) days; p < . ), a longer duration of . ) vs. ( - ) days;p < . ), and a prolonged vs. ( - ) days; p < . ) and hospital stay ( ( - . ) vs. ( - ) days; p < . ). however, in multivariate analysis, viral reactivation remained associated with prolonged mv only. when comparing patients having cmv (alone or combined with hsv) vs. hsv reactivation alone, cmv positive patients had a longer mechanical ventilation duration and fewer ventilator-free days at day- and a longer icu and hospital length of stay. conclusion: herpesviridae reactivation is frequent among patients with sevre ards under veno-venous ecmo and is associated with a longer duration of mechanical ventilation. cmv seems to have a proper negative role on pulmonary fiunction as compared to hsv alone. hsv and cmv deserve to be researched in severe ards patients under ecmo. compliance with ethics regulations: yes. charlotte vandueren , benjamin zuber , eve garrigues , antoine gros , nicolas epaillard , guillaume voiriot , yacine tandjaoui rationale: respiratory syncytial virus (rsv) is a common cause of pediatric bronchiolitis and influenza-like illness in adults. its involvement in severe infections in adults remains unclear. the captif study aimed at comparing characteristics and prognosis of icu patients infected with rsv and influenza, assuming that, based on the limited evidence, the mortality of rsv infection would be lower than the influenza related one. patients and methods: multicenter franco-belgian retrospective study. adults admitted to icus between /nov/ and / apr/ with respiratory rsv infection were included and matched : to influenza patients on center and icu admission date. patients' characteristics, clinical presentation, and outcome were compared between groups using univariate and multivariable analyses. results: we report here the results for the first cases among included patients. mean age was . ( . ) years and saps- score was ( ), not different between groups. compared to influenza patients, rsv patients more frequently had chronic respiratory failure ( % vs %, p < . ) or immune suppression ( vs %, p = . ). frequencies of cardiac, renal and hepatic chronic diseases were similar. almost all patients had respiratory symptoms (> %), extrarespiratory symptoms were more frequent in influenza patients ( vs %, = . ). rsv patients more frequently had bronchospasm ( vs %, p = . ). clinical presentation such as ards ( %), shock ( %) and pulmonary coinfection ( %) were similar, however sofa score was higher in rsv patients ( . ( . ) vs . ( ), p = . ). the p/f ratio was around mmhg in both groups, paco was higher in rsv patients ( vs mmhg, < . ). respiratory assistance at diagnosis tended to differ (p = . ), rsv patients receiving more non invasive ventilation ( vs %) and less high flow oxygen therapy ( vs %) but invasive ventilation was required similarly ( vs %). during icu stay, ards was more frequent in rsv patients ( vs %, p = . ), accordingly prone position ( . vs . %) and ecmo ( . vs . %) were more frequently needed. length of mechanical ventilation ( days ( - ) ) and icu los ( days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ) were not different. icu mortality was similar in rsv and influenza patients ( . % and . %), the multivariate analysis did not find an association between type of virus and mortality. conclusion: rsv infection is frequent in adult icu patients. it presents more frequently than influenza as an acute on chronic respiratory failure with bronchospasm. despite difference in case mix and clinical presentation, vrs severity and burden appear similar to influenza justifying effort to prevent and treat it. compliance with ethics regulations: yes. rationale: mortality in acute stroke patients requiring mechanical ventilation ranges from to % at year. studies evaluating indicators of outcome in these patients have limitations, including singlecenter, retrospective designs and no adjustment for withholding/ withdrawal of life-sustaining treatments (wlst). our objective was to identify factors associated with -year survival in acute stroke patients requiring mechanical ventilation. patients and methods: retrospective analysis of a prospective multicenter database between and . icu stroke patients entered in the database and requiring mechanical ventilation within h were included. were excluded patients with stroke of traumatic origin, subdural hematoma or venous cerebral thrombosis. factors associated with -year survival were identified using a cox model stratified on inclusion center, adjusted on wflst occurring during the first h. data are presented as median [q -q ] or percentages. cox model results are presented as hazard ratios (hr) and % confidence intervals (ci). results: we identified patients from icus, aged [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] years and % males. on admission, the glasgow coma score (gcs) was [ ] [ ] [ ] [ ] [ ] [ ] and the saps score was . types of strokes were ischemic ( %), hemorrhagic ( %) and subarachnoid hemorrhage (sah) ( %). ischemic stroke patients received thrombolysis or thromboaspiration in / ( %) cases, and hemorrhagic stroke/ sah patients received neurosurgery or embolization in / ( %) cases. reasons for endotracheal intubation were coma ( %), acute respiratory failure ( %), seizures ( %), cardiac arrest ( %) and elective procedure ( %). sixty-five ( %) patients received a decision of wflst in the first h. one-year survival year was %. variables independently associated with -year survival were stroke type (ischemic as reference, hemorrhagic hr . (fig. ) . inclusion period ( inclusion period ( - inclusion period ( / inclusion period ( - inclusion period ( / inclusion period ( - or having a stroke unit on site was not associated with -year survival. conclusion: in acute stroke patients requiring mechanical ventilation, the reason for intubation and the opportunity to receive a specific stroke therapy are independently associated with long-term survival. these variables should be integrated in the decision process regarding initiation of mv in acute stroke patients. compliance with ethics regulations: yes. rationale: international guidelines recommend targeted temperature management (ttm) between ° and °c for out-of-hospital cardiac arrest (ca) patients. however, it is unknown if this treatment is effective whatever the severity of the insult. we aimed to examine the association between ttm and long-term neurological outcome according to the risk evaluated at time of admission in intensive care unit (icu) using a dedicated and validated score. patients and methods: we used data prospectively collected in the sudden death expert center (sdec) registry (great paris area, france) between may and december and in the resuscitation outcome consortium-continuous chest compression (roc-ccc) between june and may . we used a modified version of the cardiac arrest hospital prognosis (mcahp) score to assess the risk of poor outcome at icu admission in each of datasets. we finally studied the association between ttm use and long-term neurological prognosis according to mcahp score at icu admission divided into tertiles of severity in each of the datasets. results: there were patients analyzed in the french dataset and in the north-american dataset. the mcahp identified categories: low risk (score < points, % of unfavourable outcome), medium risk ( ≤ score < , % of unfavourable outcome) and high-risk group (score > , % of unfavourable outcome). according to the mcahp score at icu admission, ttm was associated with a better long-term neurological prognosis in patients with low risk (aor = . [ . - . rationale: acute ischaemic stroke is associated with a high risk of mortality, morbidity and healthcare-related costs. over the last decades new treatments, such as thrombolysis and thrombectomy, have been introduced. because of their further improvement, complications have been decreasing. this also led to extending indications for treatment to patients who were previously not eligible. the impact of this evolution on long-term outcome and cost-effectiveness has mainly been assessed in clinical trials and simulation studies. patients and methods: this single-centre retrospective study included patients treated for stroke between january and february . functional outcome at days was assessed by the modified rankin scale (mrs). cost data were retrieved from individual invoices of patients. undiscounted total healthcare costs were calculated for the index hospital stay, capped at days. contribution of cost categories to total costs was analysed. mrs at days was used as a proxy for utilities to define quality-adjusted life years (qalys). multivariate analysis was done for gender, age, charlson comorbidity index, pre-stroke mrs, stroke severity (nihss) and treatment modality (thrombectomy, thrombolysis, thrombectomy + thrombolysis, no intervention). incremental cost-effectiveness ratios (icers), associated to each treatment modality, were calculated. results: no intervention was done in patients ( . %). patients ( . %) required thrombolysis, ( . %) thrombectomy and ( . %) the combination. total costs were mean , eur ) . hospitalisation costs (mean , eur, iqr - , ) represented % of total costs, compared with drug costs ( eur, iqr - ), procedural costs ( eur, iqr - ), honoraria ( eur, iqr - ), lab ( eur, iqr - ) and imaging ( eur, iqr - ). mean total costs differed between treatment modalities: , (iqr - , ) eur for no intervention, , ) eur for thrombolysis, , (iqr , ) eur for thrombectomy and , (iqr , ) eur for the combination (p < . ). drivers for total costs were treatment modality (p < . ) and nihss-stroke severity (p < . ). utility scores were . rationale: emergency endotracheal intubation (eti) in the intensive care unit (icu) often concerns hypoxemic patients with hemodynamic instability. a cardiovascular collapse (cvc) after eti is a life-threatening complication. french guidelines suggested systematic fluid loading prior to eti. our study aimed to predict cvc after eti, while using echocardiography, and to evaluate the impact of fluid loading. patients and methods: a prospective study of consecutive intubations was performed from june to november in three icus. patients were selected if mean blood pressure measurements ≥ mmhg before eti. cvc was defined as mean blood pressure < mmhg within min following eti. four echocardiographic examinations were performed: - min before and - min after eti (or when a cvc occurred); -after passive leg raising; - h following eti. patients were classified as fluid responders when the left ventricular outflow tract velocity-time integral increased by at least % compared with baseline. results: echocardiographic examinations were performed. cvc occurred in / procedures ( %). in cvc group, mean dose of diprivan, used for fast sequence induction, was higher ( . ± mg/kg vs . ± . mg/kg, p = . ). in the cvc group, fluid responsiveness was considered in % patients and left ventricular (lv) systolic dysfunction %. lv diastolic dysfunction did not concern any patient in the cvc group. systolic blood pressure (sbp) < mmhg was the sole independent risk factor for cvc occurrence in multivariate analysis: or . ci % . - . , p = . . fluid responsiveness independent risk factors for cvc patients was sbp < mmhg (or . , ci % . rationale: the autonomic nervous system is highly adaptable and allows the organism to maintain its balance when experiencing stress. heart rate variability (hrv) is a mean to evaluate cardiac effects of autonomic nervous system activity and a relation between hrv and outcome has been proposed in various types of patients. we attempted to evaluate the best determinants of such variation in survival prediction using a physiological data-warehousing program (reastoc clinicaltrials identifier nct ). patients and methods: physiological tracings were recorded at hz from the standard monitoring system (intelliview philips mp ) using the synapse software (ltsi inserm umr ), for a h period, during the h following icu admission. all measurements were recorded while patients were laying in bed, with the head at ° and without any medical intervention. physiological data were associated with metadata collection by a dedicated research assistant. hrv was derived using kubios hrv, in either temporal ( (sdnn), (rmssd) and triangular index (ti)), frequency ( (lf), (hf)), non-linear domains (poincaré plotting) and entropy. results: consecutive patients were recorded between may and april . a lower lf/hf (< . ) and sd /sd (< . ) ratios on admission were associated with a higher icu mortality. multivariate analysis enabled to develop a mortality predictive model (bicus) associating spo /fio and hrv parameters (lf/hf and shannon entropy) with an auc = . (p < . ) for a bicus value > (fig. ) . conclusion: hrv measured on admission enables to predict prognosis in the icu, independently of the admission diagnosis, treatment and mv requirements. bicus may help predict prognosis on a real time basis, using parameters derived from standard routine monitoring. compliance with ethics regulations: yes. rationale: stroke, in the context of type diabetes (t d) is associated with a worse outcome than in non-diabetic conditions, reflected by an increased ischemic volume and more intracerebral hemorrhage. an unbalanced diet is one of major risk for developing t d. we aimed at creating a reproducible mouse model of stroke in impaired glucose tolerance condition induced by high fat diet. patients and methods: adult c bl mice ( male and female) were fed for months with either high fat diet (hfd, % lipids, % proteins, % carbohydrates) or a normal diet (nd, . % lipids, . % proteins, . % carbohydrates) . we used a model of middle cerebral artery occlusion (mcao) by a monofilament for min. oral glucose tolerance test and insulin tolerance test were used for evaluating the pre-diabetic state. mice were euthanized h after reperfusion. systemic inflammation, cerebral infarct volume and hemorrhagic transformation were determined. results: hfd was associated with an increased glycaemia following the oral glucose tolerance test. plasma leptinlevels in stroke conditions were significantly higher in hfd vs nd group. the hfd group presented a significant increase of infarct volume (hfd: . ± . mm vs nd: . ± . mm p = . ) and hemorrhagic transformation (hfd: . ± . vs nd: . ± . p = . ) (fig. ) compared to nd group. discussion: in humans, one of the mechanisms leading to insulin resistance is low-grade inflammation. hfd increases gut permeability, which leads microbiota dysbiosis, thereby promoting metabolic endotoxaemia and a low-grade inflammation state. experimental mouse models available for diabetes studies use leptin receptor deficient mice which develop t d or destruction of pancreatic beta cells by streptozotocine injection (t d). studies using diet-induced insulin resistance models generally feed the mice for weeks or more. however, metabolic disorders could appear earlier such as increase inflammatory markers. in our model, a short exposition to hfd ( weeks) leads to an increase of the pro-inflammatory markers as plasma leptin and a more severe stroke status (infarct and hemorrhagic transformation). conclusion: two months of hfd in adult mice altered hyperglycemia control. this metabolic disorder was associated with significantly higher leptin production, increased infarct volume and hemorrhagic complications than in normal-fed mice. this new model is particularly relevant to study stroke under pre-diabetic conditions induced by hfd. compliance with ethics regulations: yes. eight weeks of hfd increase ischemic volume and hemorrhagic transformation. (a)-infarct volume (v) h after reperfusion, all value are mean ± sem, hfd: v = . ± . mm , n = , nd: v = . ± . mm , n = , *p = . (b)-hemorrhage transformation (ht) score h after mcao. all value are mean ± sem hfd: ht score = . ± . , n = , nd: ht score = . +/+ . , n = *p = . rationale: cardiac arrest (ca), as massive ischemia reperfusion (ir), is an universal health issue. medication taken at the time of the ca could have prognosis consequences. no medication has proven its benefit on ca prognosis. pharmacological pre-or postconditioning aims to reduce ir injury but with disappointing results. metformin (met) is a worldwide-prescribed antidiabetic drug, and several clinical reports plead for a potential protective effect in various settings of sterile and non sterile inflammation, including ir. our hypothesis is that met act as a preconditioning drug against ca-induced ir. patients and methods: retrospective single academic medical center survival study (french west indies) on resuscitated ca in icu (institutional ethical committee approval). data were extracted from medical charts, pmsi, and laboratory dbsynergy ™ software. anonymized data were entered on a excel ™ and transferred to ibm ® -spss ® software (v . . . ) for analysis. univariate study (chi- , fisher exact tests, student-t test, mann-whitney u-test if required) was followed by a multivariate model (odd ratio or and % ic: kaplan-meier estimator and non parametric logrank test-mantel cox model). assuming an overall in-hospital mortality for ca in icu of % with an expected mortality decrease of % by met, the number of patients to be included is . results: the inclusion period was to , with included patients ( diabetic patients among whom took met). the d mortality was % in met+ patients (n = ) versus % in nomet patients (n = ), p < . . comparing alive (n = ) versus deceased (n = ) at d in univariate then multivariate analysis, asystole on the first ekg, number of iterative cardiac arrest,sofa, no-flow, lactate, low-flow and sapsii appear as independent criteria associated with d mortality.conversely, met intake showed up as a protective criterion (or . , ci . - . ). the survival curve, including strata of low-flow duration at the cut-off min, is reported on the fig. . among diabetic patients (n = ), the mortality of patients in the met+ (n = ) was % versus % in the nomet (n = ), p = . . conclusion: in diabetic patients suffering of massive ir related to resuscitated ca, a current treatment by met is associated with a better survival. these results support a protective effect of met and are important to initiate prospective evaluations, because of millions diabetic people around the world and the potential benefit of met. the potential benefit in non diabetic patients and in sterile as well as non sterile inflammation should be addressed. compliance with ethics regulations: yes. rationale: during systemic inflammation, the accumulation of misfolded proteins in the endoplasmic reticulum (er) induces er stress (ers). in animal models, the inhibition of ers reduces inflammatory response and organ failure. cardiopulmonary bypass (cpb) induces a significant systemic inflammatory response but ers expression has never been described in cardiac surgery patients. our objective was to describe the variations of the glucose related protein of kda (grp ), the final effector of the ers, during cpb. patients and methods: we conducted a prospective monocenter study including patients undergoing cardiac surgery with cpb. two samples (paxgene ® tube + edta tube) were taken at three times: before cpb, h after the end of cpb (h -cpb) and h after (h -cpb). after rna isolation and reverse transcription, we performed a quantitative polymerase chain reaction to evaluate the expression of gene encoding for grp and determined the plasma level of grp using enzyme-linked immunosorbent assay. our main objective was to study the variation of grp between pre-cpb and h -cpb samples. our secondary objectives were to evaluate the association of ers with morbi-mortality: organ failure at h (catecholamines and/or invasive ventilation and/or acute renal failure), troponinemia and pao /fio ratio (lung damage control). fig. ). we found an inverse correlation between grp plasma level and troponinemia at h (r = − . ; % ci[− . ; − . ]; p = . ) and a correlation between the pao /fio ratio and grp plasma level at h (r = . ; % ci[ . ; . ]; p = . ). we showed a significant relationship between the variation in plasma concentration of grp and post-operative organ failure after cpb. further studies are needed to better understand the molecular mechanisms of ers in acute inflammatory organ failure in humans. compliance with ethics regulations: yes. patients and methods: in a retrospective monocentric study ( / - / ) conducted in cardio-vascular surgical intensive care unit (icu) in henri mondor teaching hospital, all consecutive adult patients who underwent peripheral va-ecmo were included, with exclusion of those dying in the first h. diagnosis of acute mesenteric ischemia was performed using digestive endoscopy, abdominal ct-scan or fist-line laparotomy. significative results in the univariate analysis were analyzed in a multivariate analysis using logistic regression. results: va-ecmo were implanted. median age was ( - ) years and median . va-ecmo was implanted after a cardiotomy in % of the cases and for a medical reason in % of the cases including % of refractory cardiac arrest. patients characteristics are reported in the table. acute mesenteric ischemia was suspected in patients, with a delay of ( - ) days after ecmo implantation. digestive endoscopy was performed in patients, ctscan in five patients and first-line laparotomy in three patients. acute mesenteric ischemia was confirmed in patients, i.e. an incidence of %. laparotomy was performed in six of the patients, two having a stage i colitis ischemitis with stable conditions and being considered too severe to undergo futile surgery. overall mortality was %. all the patients with acute mesenteric ischemia died in the icu. independent risk factors of developing acute mesenteric ischemia were renal replacement therapy , p = . )) and onset of a second shock state within the first days of icu stay (or . ( % ic . - . , p = . )). conversely, early enteral nutrition was negatively associated with acute mesenteric ischemia (or . ( % ic . - . ), p . ). conclusion: acute mesenteric ischemia is a relatively frequent condition among patients under va-ecmo for cardiogenic shock. its extremely poor prognosis requires low threshold of suspicion. compliance with ethics regulations: yes. ( ). it allows the computation of trans-pulmonary pressure ( ) and can be used to set positive end-expiratory pressure (peep) ( . ) . prone position(pp) can reduce mortality in patients with acute respiratory distress syndrome (ards), but peep selection in pp is controversial. in human ards end-expiratory pes at zero flow (peept,es) was not different between supine (sp) and pp at same peep ( ). as no study measured ppl in sp and pp in ards we aimed at comparing peept,es and end-expiratory ppl at zero flow (peept,ppl) in this condition. our hypothesis was that peept,es was close to dorsal peept,ppl (peept,ppldorsal) in sp and to ventral peept,ppl (peept,pplventral) in pp. in eight female pigs of kgs intubated, sedated, paralyzed and mechanically ventilated, ards was induced by repeated saline lavage until pao /fio < mmhg under fio and peep cmh o. pes was measured by nutrivent catheter. ppl was measured by custom-made pouch sensors inserted surgically into the right anterior and posterior sixth intercostal space. ppl sensors were filled with air. after ards induction animals were randomly assigned to sp or pp. in each position, a recruitment manoeuver was performed and peep decreased from to cmh o by steps of cmh o lasting min each, then the animals were crossed over into the alternate position where the same procedure was done. at the end of each step nonstressed volume and correct position (baydur maneuver) were determined for pes and ppl sensors, then a -s end-expiratory occlusion was performed and pes and ppl recorded. linear mixed model was used to compare the value of pes and ppl at each peep and position. results: box-and-whisker plots of pes and ppl in sp and pp are shown in fig. . there is marked dorsal-to-ventral gradient in ppl at each peep in sp, which is reverted in pp at peep and only. there was no interaction between pressures and peep or position. with increasing peep pes increased significantly from peep in sp and pp. peept,pplventral was significantly lower than peept,es in sp but not in pp. (medtronic) , carescape (ge)) were set in pressure support cmh o, peep cmh o, fio % and equipped with the same double limb ventilator circuit (intersurgical) without any humidification device. asl bench model was set with inspiratory/expiratory resistance (r) and compliance (c) combinations: r / -c , r / -c and r / -c mimicking normal, ards and copd conditions, respectively ( ) . inspiratory effort generated by asl consisted of consecutive breaths obtained from the esophageal pressure in a real patient at the time of a spontaneous breathing trial. for each icu ventilator and rc combination, two steps were performed: in the first, atc was not activated and ventilator attached to asl without ett (atc-ett-); in the second, atc was set on at % compensation for an ett mm id and such an ett (shiley hi contour, covidien) joined icu ventilator to asl (atc+ ett+). the null hypothesis is that vtatc+ ett+ minus vtatc-ett-is . primary end point was the breath by breath paired difference betwen atc+ ett+ and atc-ett-. it was tested to zero for each ventilator in each rc condition. results: median vt was ml. table displays mean (± sd) difference in vt (ml) between atc+ ett+ and atc-ett-: a negative value means that atc under delivers and a positive value that atc over delivers vt for a given patient's inspiratory effort and rc. in four ventilators (c , s , elisa and ) atc almost systematically under delivered vt. in several instances under compensation was greater than % median vt. by contrast atc performed better with the other three ventilators (evita xl, v and carescape ). conclusion: atc tended to under deliver vt. furthermore, there were marked differences between icu ventilators the clinician should be aware of when using the atc option. compliance with ethics regulations: na. rationale: during the last decades, identification of factors associated with ventilation-induced lung injury has led to improved survival in patients with ards. the mechanical power of ventilation is the total energy transmitted from the ventilator to the respiratory system per unit of time and comprises three different components: elastic related to peep, elastic related to tidal volume and resistive. this integrative variable has been recently proposed as an useful predictor of ventilationinduced lung injury and death among ventilated patients. our goal was to determine the respective impact of the total mechanical power and its three components on the outcome of patients with ards. patients and methods: we performed a post hoc analysis of a randomized, controlled study of patients with ards with a pao /fio ratio < . themechanical power at inclusion and averaged on the first days after inclusion (total and its three different components) was computed according to the following equation: powerrs (j/ min) = . respiratory rate tidal volume [peep ( ) + ½ driving pressure ( ) + (peak pressure-plateau pressure) ( )], where the ( ), ( ) and ( ) parts correspond respectively to the elastic related to peep, elastic related to tidal volume and resistive components. the association between each of these four types of mechanical power evaluated during the first days after inclusion and mortality at d was assessed one after the other through multiple logistic regression, allowing control for potential confounding variables at inclusion (age, igs score without age, group of randomization, pao /fio , arterial ph). results: data from patients were analyzed, among which ( . %) died before d . there was no difference concerning the mechanical power at inclusion between survivors and non survivors (either total or its three components). among the four different types of mechanical power tested during the first days after inclusion, the elastic component related to tidal volume was the only one that was independently associated with mortality at d (or . ; % ci . - . ; p = . ) (figure) . conclusion: our study shows that only the elastic component of the mechanical power related to tidal volume independently predicted mortality at d among patients with ards, whereas the total mechanical power, its elastic component related to peep and its resistive component did not. further studies are needed to better define how the mechanical power of ventilation could be useful to synthetize the risk of ventilation-induced lung injury. compliance with ethics regulations: yes. probability of death at d as a factor of mean value (on d -d ) of the elastic component related to tidal volume of the mechanical power. to examine the effect of early-stage mechanical ventilation (mv) on diaphragmatic contractility. in the nd step, if a diaphragmatic dysfunction was detected, we assessed its influence on the weaning from ventilator. patients and methods: we measured prospectively the ultrasounddiaphragmatic thickening fraction (dtf) between groups: a study group versus a control group (n = for each). the study group included all adult patients receiving mv, in whom, the dtf was measured within a minimum of h and a maximum of days of mv. for the control group, were enrolled after their approval for participation, adult volunteers in spontaneous ventilation (sv). patients with factors affecting the diaphragmatic contractility (neuromuscular disease, severe obesity, and neuromuscular blockers…) were excluded. the ultrasound measurements were obtained at the zone of apposition of the right hemithorax. teleinspiratory and telexpiratory diameters (tid/ ted) were taken on the medio-axillary lines: posterior, median and anterior. the dtf was calculated as following: dtf = (tid-ted/ted) x . at the st step, the dtfs were compared and at the nd step: the relationship between dtf and weaning was analysed. results: our groups were comparable in corpulence and co morbidities. the sv group was younger ( vs. years, p < . ) with a predominant female composition. the diaphragmatic exploration concluded that in the mv group, the mean tid tended to be higher but without significant difference ( . + versus . + mm, p = . ), the mean ted was significantly higher ( . + versus . + . mm, p = . ) and dtf was significantly lower ( . + . % versus + . %, p = . ). the ventilation mode had no effect on dtf ( . + % for control volume vs. . + % for psv mode, p = . ). fourteen among ventilated patients had a successful weaning with a mean duration of days. a negative correlation was found close to significance between dtf and weaning duration (rho = − . and p = . ). a dtf value > % wasassociated with weaning success (or = , % ci = [ . - . ] and p = . ) with sensitivity = . %, specificity = %, ppv = % and npv = %. conclusion: the diaphragmatic contractile function was altered from the first days of mv. weaning duration seemed to be negatively correlated with dtf, and a dtf at the first days of mv greater than % was predictive of weaning success. compliance with ethics regulations: yes. rationale: mechanical ventilation is a life-saving treatment that is however associated with lung injury and/or diaphragm dysfunction. the optimal ventilator settings to provide lung protective ventilation while maintaining safe diaphragm activity are difficult to determine. a noninvasive and bedside evaluation of the diaphragm activity could be helpful in this context. the present study investigated whether changes in diaphragm shear modulus (i.e. stiffness, Δsmdi) assessed by ultrasound shear wave elastography (swe) may be used as a surrogate of changes in transdiaphragmatic pressure (Δpdi) in mechanically ventilated patients. patients and methods: patients had to be ventilated for at least h without contraindications for the placement of an oeso-gastric catheter. pdi was monitored continuously and smdi was measured at the zone of apposition of the right hemi-diaphragm, at hz sampling rate. measurements were performed twice under initial ventilator settings and at the end of a weaning trial. pearson correlation coefficients (r) were computed to determine within-individual correlations between pdi and smdi and changes in pdi and in smdi occurring between initial ventilator settings and the end of the sbt were compared by a paired test. results: twenty-five patients were enrolled and displayed a significant correlation between Δsmdi and Δpdi (mean r = . , range = . - . , all p < . ) (fig. a ). compared to their counterparts, patients with significant within correlations had a lower respiratory rate ( . ± . vs . ± . breath/min. respectively; p < . ) and a significant increase in Δsmdi ( . ± . kpa vs . ± . kpa. p < . ) between initial ventilator settings and the sbt. patients without Δsmdi-Δpdi correlation only displayed an increase in Δpdi ( . ± . vs . ± . cmh o, p < . ) at the end of the sbt with no concomitant significant increase in Δsmdi ( . ± . kpa vs . ± . kpa, p > . ). (fig. b) . conclusion: smdi obtained by swe appears as a promising technique to assess diaphragm activity in mechanically ventilated patients but technological improvements are necessary to increase swe sampling rate before enabling its generalization in the icu. compliance with ethics regulations: yes. rationale: end-inspiratory (eip) and end-expiratory (eep) pauses are commonly used during volume assist control ventilation to assess plateau pressure and total positive end-expiratory pressure (peeptot). they can also be used during assisted ventilation (av) for muscle pressure assessment. it requires ventilators able to perform eip during av. plateau pressure (pplat) usually increases in av during eip due to "hidden" inspiratory effort. pressure muscular index (pmi) is equal to pplat minus the sum of peeptot (measured during an eep) and set pressure support (ps); it theoretically reflects patient's effort without esophageal pressure (pes) monitoring. pes is the gold standard method to assess inspiratory muscle pressure (pmus, difference of pes drop at neural end-inspiration and correction factor for chest wall elastance and tidal volume). we aimed to illustrate the feasibility of measuring pmi using a standard icu ventilator at the bedside and study the correlation between pmus and pmi. patients and methods: measurements were recorded in icu patients. pes was measured using an nasogastric probe (equipped with an esophageal balloon) inserted for advanced monitoring (severe acute respiratory distress syndrome-ards) or for a study protocol (difficult weaning after copd exacerbation). recorded eip, eep and pes were used for post hoc analyses. results reported as ranges and median [iqr] . correlation between pmus and pmi tested with spearman correlation test. results: out of eip and eep duos could be analyzed ( -esophageal spasm/ -calibration error). ventilator mode was pressure support ventilation (ps - cmh o). cmh o, pmus = . [ . - . ] cmh o, pmi = . [ . - . ]. for all recordings, spearman r coefficient between pmus and pmi was . (p = . ). conclusion: muscular effort can be assessed in av using eip and eep using icu ventilators. however, recordings can be influenced by expiratory muscles contraction. patient's ability to follow directions during the maneuvers is an important factor to obtain reliable values. there seem to be a correlation in our small sample between muscular pressure assessed without and with pes. compliance with ethics regulations: yes. rationale: severe pneumonia can culminate in acute respiratory distress syndrome (ards). an uncontrolled inflammatory response is a key feature favoring transition towards ards. however, the underlying mechanisms remain poorly understood. in this context, the contribution of "innate t cells" (itc) -a family of non-peptide reactive t cells comprising nkt cells, mucosal associated invariant t (mait) cells and γδt cells-has never been explored. itc have emerged as key players in orchestration of the host response during infections and inflammation processes. for these reasons, these cells are already seen as potential therapeutic targets in other medical fields (especially oncology). here, we hypothesized that a tight regulation of their functions could be paramount to control the inflammatory response and to prevent ards development. patients and methods: to explore this, we combined a murinemodel of influenza a virus (iav) infection mimicking ardssymptoms and a clinical study recruiting patients admitted in icu for severe pneumonia. using flow-cytometry approaches, we investigated ( ) the abundance and dynamics of itc in various compartments, ( ) their pattern of activation/regulation markers (respectively cd and pd- ) and ( ) their cytokine production. results: during experimental iav pneumonia, itc were transiently recruited into the airways. unlike γδt and nkt, mait cells phenotype was largely changed, displaying a progressive cd overexpression and increased il- a production. during the resolution phase, up to % of pulmonary maits expressed pd- (versus < % in controls), which can suggest emergence of regulatory functions. last, using gene-targeted mice, we suggested that mait cells confer a protective effect during pneumonia. in the ongoing clinical study, the proportion of circulating mait cells in patients was markedly decreased compared to controls ( . ± . % versus . ± . % of t cells), but not for nkt or γδt cells. notably, some patients with severe ards presented detectable levels of maits in their respiratory fluids. in addition, circulating mait cells in patients overexpressed cd and pd- ( . % and % respectively), but with a reduced proportion able to produce il- and ifnγ, compared to healthy controls. lastly, proportion of activated (cd +) mait cells significantly decreased with clinical improvement. conclusion: this translational approach combining in vivo animal experiments and clinical samples with ex vivo experiments indicates a preferential modulation in mait cells functions during severe pneumonia. these data justify an in-depth analysis of mait cells activation mechanisms and functions in this context, in order to further explore a potential use as a disease-progression marker and -in a long term perspective-as a potential therapeutic target. compliance with ethics regulations: yes. representative flow-cytometry dot-plots of mait cells labelling using fluorophore-conjugated mr tetramers loaded with -op-ru from lungs of an infected mouse (a) and blood sample of a patient with pneumonia (b). c: frequency of mait cells, proportion of cd and pd- + mait cells in bronchoalveolar lavage during experimental murine pneumonia. d: blood frequency of mait cells in patients with pneumonia compared with healthy controls (as % of total t cells) rationale: immune paralysis following hyperinflammatory states increases the risk of secondary infections and death. reversing t-cells exhaustion using recombinant il or immune checkpoints inhibitors may improve the prognosis of patients with sepsis admitted to the icu. however, there is an unmet need to better characterize the state of t-cells exhaustion in these patients, its reproducibility and its correlation with the outcomes before implementing immunotherapy in the therapeutic armamentarium against sepsis. patients and methods: prospective observational cohort study performed in two tertiary-care icus in a university hospital. peripheral blood mononuclear cells were collected at day in adult patients with sepsis admitted to the icu. the level of cd + and cd + t-cells exhaustion was quantified using multi-color flux cytometry targeting the following exhaustion markers: pd- , b and cd . cd + regulatory t-cells (cd + cd + cd hi cd lo cells) were also assessed. results: the patients included in the study could be split in five clusters according to their dominant pattern of exhaustion markers on cd + t-cell (i.e. no markers, pd- +, b +, b + cd + and b + pd- +) and independently of their underlying morbidities. no patients harbored a fully exhausted triple-positive pattern. by multivariate analysis, saps gravity score at day (p = . ), a dominant b and/or pd- cd + pattern (p = . ) and lung sepsis (p = . ) where associated with the risk of death at day , whereas hemoglobin level was associated with survival (p = . ). no cd + or cd + exhaustion pattern independently predicted the risk of secondary infections. neither the level of cd + regulatory t-cells nor the dominant cd + exhaustion pattern was associated with the outcomes. rationale: there is growing use of multiplex polymerase chain reaction (mpcr) for respiratory virus testing in patients with communityacquired pneumonia (cap). data on one-year outcomes in patients with severe cap of bacterial, viral and unidentified etiology are scarce. patients and methods: a single-center retrospective study was performed in intensive care unit (icu) patients with known one-year survival status who had undergone respiratory virus testing for cap by mpcr. one year after icu admission, mortality rates and functional status were compared in patients with cap of bacterial, viral or unidentified etiology. results: there were ( . %) patients in the bacterial group, ( . %) in the viral group and ( . %) with unidentified etiology. one-year mortality was . % (n = / ), % (n = / ) and . % (n = / ), respectively (p = . ). in multivariate analysis, one-year mortality was higher in the bacterial group than in the viral group (hr . , % ic . - . , p = . ), had a trend to be higher in the bacterial group compared to the unidentified etiology group (hr . , % ic . - . , p = . ) and was not different between the viral and unidentified etiology groups (hr . , % ic . - . , p = . ). severe dyspnea (mmrc score = or death), major adverse respiratory events (new homecare ventilatory support or death) and severe autonomy deficiencies (adl katz score ≤ ordeath) were observed in / ( . %), / ( . %) and / ( . %) patients, respectively, with no difference between groups. conclusion: cap of bacterial origin was associated with a poorer prognosis than viral or unidentified etiology. impaired functional status was observed in a substantial proportion at one-year, irrespective of the causative microorganisms involved. compliance with ethics regulations: yes. interest of unyvero multiplex pcr (curetis) for bal rapid microbiologic and antibiotic susceptibility documentations in immunocompromised patients under antibiotic therapy jean-luc baudel , jacques tankovic , redouane dahoumane , salah gallah , laurent benzerara , jean-remy lavillegrand , razach abdallah , geoffroy hariri , naike bige , hafid ait-oufella , nicolas veziris , eric maury , bertrand guidet rationale: our aim was to evaluate the interest of the unyvero rapid ( . h) multiplex pcr assay (performed on bronchoalveolar lavage [bal] samples) for the management of immunocompromised patients already treated with antibiotics and diagnosed with pneumonia (according to clinical and radiological findings). we thus performed an observational study that compared the results (and the length of time to obtain them) of routine microbiological evaluation and unyvero assay. patients and methods: from july to january and from april to august , we examined bal samples from immunocompromised patients (coming from hematology, oncology, hepatology, gastroenterology, internal medicine, and neurology units) diagnosed with pneumonia (based on clinical and radiological findings), and already receiving antibiotic treatment. the following data were collected: age, gender, saps score, lung ct scan ( %) or x-ray ( %) results, duration and content of prior antibiotic therapy, direct examination, culture, antibiogram and unyvero results, secondary confirmation of pneumonia or not, possible changes in antibiotic therapy that could have been made after obtention of unyvero results. informed consent was obtained from all patients. results: bal samples were analyzed in immunocompromised patients (m/f ratio . , saps . ± . ) mostly with hematologic ( %) or oncologic ( %) diseases. the patients received either corticosteroids ( %), or chemotherapy ( %), or immunotherapy ( %). % of the patients were under mechanical ventilation, % under optiflow. % presented a shock, % had aplasia or neutropenia, % were allografted, % were autografted. the duration of prior antibiotic therapy at the time of bal were . ± . days. direct examination was positive in . % of the cases, culture (both above and under the classical threshold of cfu/ml) in %, unyvero in . %. a retrospective analysis of all the cases confirmed the initial diagnosis of pneumonia in only % of the cases. compared to culture, the sensitivity of unyvero was %, its specificity %. unyvero could permit to rapidly deescalate antibiotic therapy in % of the cases and to rapidly stop it in %. the unyvero assay on bal samples is useful in this specific population for rapid obtention of microbiological results and also for confirmation of the negativity of cultures and thus permits a better management of antibiotic therapy, leading to a reduction of antibiotic resistance selection pressure in the icu. compliance with ethics regulations: yes. do not underestimate rsv pneumonia among critically ill patients erwan begot , suzanne champion , charline sazio , benjamin clouzeau , alexandre boyer , hoang-nam bui , marie-edith lafon , camille ciccone , julia dina , didier gruson , renaud prével chu bordeaux, medical intensive care unit, bordeaux, france; chu bordeaux, virology laboratory, bordeaux, france; national reference center for measles mumps and rubella, chu de caen, caen, france correspondence: erwan begot (erwan.begot@chu-bordeaux.fr) ann. intensive care , (suppl ):f- rationale: respiratory syncitial virus (rsv) is a well-known cause of respiratory failure among neonates but its pathogenicity in adults is now emerging as a potential cause of viral pneumonia. data are limited with conflicting results regarding rsv pneumonia severity in adults. data are lacking about critically ill rsv patients' characteristics and outcomes. the aim of this study is to compare rsv patients' characteristics, care and outcomes to influenza patients' ones. patients and methods: patients diagnosed with rsv and influenza pneumonia admitted to our medical icu were included. data were retrospectively recorded. quantitative data are expressed by median and interquartile range and compared by use of mann-whitney test. qualitative data are expressed by number and percentages and compared by use of fischer exact t-test. rsv strains were prospectively collected. results: eighteen critically ill patients with rsv pneumonia and with influenza pneumonia were included. rsv and influenza patients had the same characteristics at admission except for age (respectively yo [ ; ] and acute respiratory distress syndrome rates (respectively / ( %) vs / ( %), p = . ). they received similar treatment as suggested by oro-tracheal intubation rates (respectively / ( %) vs / ( %), p: . ) and antibiotics prescription (respectively / ( %) vs / ( %), p: . ). rsv and influenza patients also had the same rates of bacterial co-infections ( / ( %) vs ( %), p: . ). invasive aspergillosis remained a rare event but also occurred among rsv patients ( / ( %) vs / ( %), p: . ). acute coronary syndromes were as frequent in both groups (respectively / ( %) vs / ( %), p = . ). day- mortality was similar between rsv and influenza patients (respectively / ( %) rationale: respiratory distress from seawater drowning is commonly considered multifactorial. etiologies are debatable and include heart failure, infection and acute respiratory distress syndrome (ards). documented bacterial infections seems mostly related to the site of drowning. data in this regard are scarce with prospective studies lacking. the objective of our study was to describe prospectively the characteristics and determinants of respiratory distress from seawater drowning. patients and methods: all patients admitted for seawater drowning to seven intensive care units (icu) on the french riviera in the summers of and were prospectively included. recorded data included clinical features on examination, personal history, chest x-rays, echocardiography and biological results obtained within the first h. a paired student's t-test was used to study statistical differences between quantitative variables on admission and during early evaluation (i.e. first h). results: forty-eight patients were admitted to seven centers of which ( %) were diagnosed as having ards, ( %) early pneumonia and ( %) acute cardiogenic pulmonary edema. twenty-one ( %) respiratory samples were collected but bacterial culture was positive in only cases. multidrug-resistant bacteria were not observed, and amoxicillin-clavulanate as first-line treatment was effective in all cases. echocardiography performed in ( %) patients was normal and unable to identify specific patient profiles. the median clinical pulmonary infection score (cpis) on admission was (iqr, - ) and decreased rapidly and significantly (p < . ) within h to (iqr, - ) (fig. ) . conclusion: data from this multicenter cohort suggest that respiratory distress following seawater drowning can mimic bacterial pneumonia during the first h with subsequent rapid clinical improvement in patients admitted to the icu. probabilistic antibacterial therapy should therefore be limited to the most severe patients. isolate ards is often the only etiology found and is resolutive within h. this prospective cohort is the largest of its kind and gives a better insight into the limited impact of cardiogenic and infectious processes on sea drowning-related respiratory distress. compliance with ethics regulations: yes. rationale: patients treated with "extracorporeal membrane oxygenation" (ecmo) are at a higher risk of developing nosocomial infections and they are consequently often treated with beta-lactams. french guidelines recommend obtaining beta-lactam trough concentrations above four times the minimal inhibitory concentration (mic) of the causative bacteria. the ecmo device may alter the pharmacokinetics of these medications, which may result in underexposure to beta-lactam antibiotics. patients and methods: this observational, prospective, multicenter, case-control study was performed in the intensive care units of two tertiary care hospitals in france. ecmo patients with sepsis treated with piperacillin-tazobactam were enrolled. control patients were matched according to sofa score and creatinine clearance. the pharmacokinetics of piperacillin was described based on a population pharmacokinetic model, allowing to calculate the time spent above × the mic breakpoint for pseudomonas aeruginosa susceptibility after the first dose and at steady state between two piperacillin infusions. results: forty-two patients were included. the median age was years [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , the sofa score was [ ] [ ] [ ] [ ] [ ] [ ] , and median creatinine clearance was ml/min . there was no significant difference in the time above x mic in patients treated with ecmo and controls during the first administration (p = . ) and at steady state (p = . ). there was no significant difference between the trough at steady state (p = . ), with / patients ( %) exhibiting concentrations of piperacillin lower than x mic. ecmo support was not associated with a steady state trough concentration below x mic (or = . [ . - . ], p = . ). the only variable independently associated with this risk was a creatinine clearance ≥ ml/min, (or = . [ . - . ], p = . ). conclusion: ecmo support has no significant impact on piperacillin exposure. intensive care unit patients with sepsis are, however, frequently underexposed with piperacillin, which suggest that therapeutic drug monitoring should be strongly recommended for severe infections. impact of a visual support dedicated to prognosis of patients on symptoms of stress of family members rationale: family members commonly have inaccurate expectations of patient's prognosis. adding to classic oral information a visual support, depicting day by day the evolution of the condition of the patient, improves the concordance in prognosis estimate between physicians and family members. the objective of this study was to evaluate the impact of this support on symptoms of anxiety/depression of family members. patients and methods: we conducted a bi-center prospective beforeafter study. all consecutive patients admitted in the two icus were eligible. in the before period ( months), family members received classic oral information. in the after period ( months) , in addition to classic oral information, the visual support ( fig. ) was available for family members in the patient's room from the day of admission until discharge from the icu. at day and from admission, symptoms of anxiety/depression of referent family member were evaluated by hospital anxiety and depression scale (hads). results: patients and their referent family members were included ( in period before and after). characteristics of patients of the two groups were similar regarding age, reason for admission, saps ii at admission and sofa score at day . also characteristics of referent family members were comparable in terms of age, sex ratio, type of relationship with the patient and number of visits since admission. at day , total had score was [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in the group before without the support and [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in the group after with the support (p = . ). the prevalence of symptoms of anxiety (had-a score > ) and depression (had-d score > ) was similar in the two groups (respectively . % and . % in the group before, and . % and . % in the group after (ns)). at day , total had score was in the group before [ - ] and [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in the group after (p = . ). by multivariate analysis the following factors were significantly associated with total had score > at day : age of patient ]), number of visits of referent ) and previous or current treatment of referent for anxiety or depression . ]). conclusion: in this study, the use of a visual support dedicated to prognosis of patients did not modify the level of stress of family members. compliance with ethics regulations: yes. rationale: the use of sedation and opioids at the end of life is a topic of considerable ethical debate. incidence of discomfort during the end-of-life of icu patients and impact of sedation on discomfort are poorly known. patients and methods: post-hoc analysis of an observational prospective multicenter study comparing terminal weaning vs. immediate extubation for end-of-life in icu patients, aimed at assessing the incidence of discomfort events according to levels of sedation. discomforts including gasps, significant bronchial obstruction or high behavioral pain scale score, were prospectively assessed by nurses from mechanical ventilation withdrawal until death. level of sedation was assessed using the richmond agitation sedation scale (rass). results: among the patients included in the original study, ( %) experienced discomfort after mechanical ventilation withdrawal. patients with discomfort received lower doses of midazolam and equivalent morphine, and less frequently had deep sedation (rass - ) than patients without discomfort ( % vs %, p < . ). after multivariate logistic regression, immediate extubation was the only factor associated with discomfort whereas deep sedation and administrations of vasoactive drugs were two factors independently associated with no discomfort. death occurred less rapidly in patient with discomfort than in those without discomfort ( . h [ . - . ] vs . [ . - . ], p < . ) (figure) . long-term evaluation of psychological disorders in family members of dead patients did not differ between those with discomfort and the others. discussion: despite the theoretically expected anticipatory titrated doses of opioids and benzodiazepines to alleviate any discomfort after withdrawal of mechanical ventilation, half of the patients did not receive sedation or opiate when the decision to withdraw mechanical ventilation was taken. a major point that could interfere with the continuous deep sedation practice until death is the fear of potentially hastening death, and there is much controversy regarding its proper use in end-of-life care. conclusion: discomfort was frequent during end-of-life of icu patients and was mainly associated with terminal extubation and less profound sedation. compliance with ethics regulations: yes. rationale: bereavement in intensive care unit (icu) is associated with psychiatric disorders on relatives called post-intensive care syndrome family (pics-f). no isolated intervention (such as condolence letter) has shown a positive effect on these disorders, despite a well acceptance by relatives. we thought that a more integrated bereavement program should be considered. the goal of this study is to evaluate a combined psychologist-physician post-death meeting (pdm) in a bereavement program to evaluate needs and adhesion of relatives, and the effect on symptoms of anxiety and depression. patients and methods: monocentric, prospective study focused on relatives of patient admitted > h and deceased in icu. during patient's stay, relatives' presence was allowed on a h-basis and they could meet a clinician psychologist. formal meeting between relatives and the staff was realized at patient's admission and after important decision-making treatment. two weeks after patient's death, the psychologist called relatives to offer emotional support and to invite to a pdm. pdm occurs weeks after patient's death with the psychologist and the physician in charge of the patient. the objectives of the meeting were to provide emotional support, to answer medical question, and to detect symptoms of anxiety and/or depression with the hospital anxiety and depression scale (hads). we hypothesized that pmd would be able to alleviate pics-f at months. we aimed to enroll families to detect a % lowering of hads. results: the rate of pdm acceptance was lower than expected. after inclusions, only relatives accepted the pdm, whereas the phone call was well perceived ( %). main association with acceptance of pmd was a short duration of icu stay ( . days [ - . ] vs . days [ . - . ] p = . ) and icu admission for acute respiratory failure ( . % vs . %, p = . ) ( table ) . we found no relation between the number of in icu meeting (psychologist of medical staff) and pmd acceptance. for relatives who accept pmd we found a high proportion of symptoms of anxiety and depression ( % and %) with a hads at . [ - . ] (median, iqr). no evaluation was performed at months. conclusion: post death contact appears well perceived by relatives but pmd quite useless. this result may be explained by the inclusion of only late death (> h) where psychologist and medical staff had the opportunity to support relatives. further study should focus on early death (< h). compliance with ethics regulations: yes. rationale: pediatric intensivists frequently question themselves on the issue of limitation or termination of life-sustaining treatments (llst) carried out on children. such a decision comes under the claeys-leonetti law which forbids doctors from applying unreasonable treatment however, every so often, parents oppose themselves to a collegial llst decision that the medical and paramedical team had taken. such cases can even end up in court. in order to sort out this problem, this study focused on the factors that underlie the disagreement and the solution brought forward by pediatricians whenever parents demand to persue treatments although considered as unreasonable obstinacy. patients and methods: we carried out a qualitative study involving three multipurpose pediatric critical care unit. all pediatricians operating within these units were contacted. those who volonteered were met individually for a semi-directed interview. every interview was recorded and entitled to a complete hand-written retranscription. the interviews were analysed following the phenomenological interpretive analysis method and were subject to dual listing. results: pediatricians out of took part in the study. / claimed they would increase treatments or carry out cardiopulmonary resuscitation acts if asked to do so by parents, even if this went against the initial collegial decision. / claimed they would persue treatments although not beyond the current level. / said they would oppose themselves to parents concerning blood transfusion for comfort reasons. several key factors were identified as leading a doctor to the non-application of a llst decision: the certainty regarding the child's death on a short or mid-term basis ( / ), the litigiousness risk ( / ), the apprehension of mediatic pressure ( / ), the fear of a violent reaction from parents ( / ), other self-interest positions within the medical team ( / ), empathy towards parents ( / ), the uncertainty concerning the neurological prognosis ( / ), the lapse of time needed to fully accept the application in force of a decision ( / ). pediatricians out of admitted their own-suffering when confronted to the situation. conclusion: this study points out that pediatricians tend to follow parents' position when confronted to parental opposition. in such situations, pediatricians go against their own decision in order to safeguard the parental alliance even if it leads to unreasonable obstinacy, thus conflicting with medical deontological code obligations. compliance with ethics regulations: yes. rationale: end-of-life management strategies are clearly a worldwide issue of major importance that intensivists have to deal with on a daily basis. advance directives may be the solution sought to guide physicians to take such difficult decisions. yet, health care directives are not legislated in tunisia. the objective of this project was to draw a general descriptive overview to assess patients' wishes in tunisia. patients and methods: data were collected from a -item-questionnaire based on the french intensive care society's form for advance directives which was filled by people of general population in tunisia, including doctors and paramedics, from may to mid-september . all people included were or older and well informed of the form's utility. results: a total of participants were included. the mean age was . ± . years with extremes of and and a sex ratio of . . fourty-one ( . %) were either doctors or nurses and ( %) did suffer from a severe medical condition. among all the participants, ( . %) thought that end-of-life decisions were up to the doctor. for the rest, they willingly chose to be hospitalized in an icu, to undergo cardiopulmonary rescuscitation and to have ventilation support with orotracheal intubation or tracheostomy respectively in ( . %), ( . %) and ( . %) of the cases. only ( . %) refused temporary dialysis. when asked about sequelae they can live with, participants accepted hemiplegia in . % and paraplegia in . % of the cases. on the contrary, ( . %) refused to live in permanent coma and ( . %) disagreed to undergo tracheostomy and ventilation for life. moreover, ( . %) found that serious un aesthetic sequelae was a fatal consequence they could not survive. as well, only ( . %) consented to live with deep intellectual deficiency. regarding palliative care, ( . %) participants wished to be profoundly sedated until death, ( . %) prefered to die home over ( . %) in hospital. sixtytwo ( . %) desired to see a representative of their religion. furthermore, ( %) were for organ donnation. gender, being a health care professional and age under versus equal or over were not significant in dependent factors (p > . ). conclusion: it is our duty ashealth care professionals to spread advance directives awareness and education. nevertheless, the law should keep the pace with ethics evolution. compliance with ethics regulations: yes. rationale: adapted organ support techniques are needed to enhance reliability of preclinical animal experiments in the intensive care setting (guillon, annals of intensive care- ). a few renal replacement therapy (rrt) models have already been developed in rats, mostly hemodialysis in chronic kidney disease models or hemofiltration techniques in sepsis experiments. mounting evidence from clinical (gaudry, nejm- ) and histopathological studies suggest that rrt for acute kidney injury (aki) could impair renal recovery by acting as a 'second hit' leading to a maladaptive repair of tubular epithelium. we aimed to study this hypothesis in a hemodialysis model in rats with septic aki. patients and methods: on day , sprague-dawley rats were injected with lipopolysaccharide or placebo (nacl . %) intraperitoneally. on day , anesthetized rats underwent femoral artery catheterization for hemodynamic parameters monitoring. at the same time, one femoral vein and one carotid artery were catheterized for arterio-venous sterile extracorporeal circulation with or without passing through a miniature sterile polyester sulfone hemodialyzer ( cm surface, kda pores, microkros ® ) filled with dialyzate liquid in the outer compartment (table ) . vessels were ligated after the procedure and rats allowed to awaken. on day , rats were sacrificed. results: all rats injected with lipopolysaccharides o :b mg/kg survived at day . anesthesia was much challenging: ketamine + xylazine and tiletamine-zolazepam + xylazine required induction and maintenance intraperitoneal injections. these medications induced important hemodynamic parameters fluctuations and high mortality. isoflurane gas inhalation enabled better stability, less hypothermia and quick awakening. adequate temperature was controlled with a heating pad during the procedure and an incubator after. supine position was maintained. the whole circuit was anticoagulated with ml of heparinized saline ui/ml, since clots occurred in the absence of anticoagulation and bleeding when higher dosing was used. circuit (< . ml including dialyzer) was filled with saline solution before initiation, and total restitution of blood at the end of the experiment prevented any blood transfusion requirement. hematocrit was determined at beginning ( %) and end of experiment ( %). a peristaltic pump provided a blood flow rate of . ml/min, (higher rate was not tolerated) for h. of note, rats who underwent sham procedure (vessels ligature only) survived and did not display aki. circulation of a counterflow dialysate in the dialyzer is planned but has not been performed yet. conclusion: this hemodialysis system for rats is feasible at a reasonable price and might help research involving rrt in either ckd or aki. compliance with ethics regulations: yes. there were no significant relationship between rri and past medical history or severity score. we observed a significant negative correlation between rri and diastolic arterial pressure (p = . ) and heart rate (p = . ) as it could be expected by rri formula. an increased rri was associated with higher potassium (p = . ) and higher creatinine levels (p = . ). although not significant, we found a higher rate of subsequent rrt in the high rri group ( % vs %, p = . ). over the first days, fluid balance was significantly different between groups ( ml vs - ml respectively for low and high rri group, p = . ). since standard of care were similar, this suggests different fluid volume status between the two groups. in the low rri group, the cause of aki could predominantly be prerenal since positive fluid balance was not explained by more severe aki with refractory oliguria as shown by the low rrt rate. nevertheless, we did not observed any relationship between rri and the evolution of serum urea or creatinine levels, nor with the presumed aetiology of aki. conclusion: when focussing on the first rri measurement once stage aki was reached, rri ≤ . seems to be in favour of prerenal and transient renal dysfunction even if this is not supported by creatinine serum evolution. compliance with ethics regulations: yes. rationale: critically ill patients are at higher risk of bleeding but also dialysis filter clotting (inflammatory state). intermittent hemodialysis with calcium-free citrate-containing ( . mmol/l) dialysate (cafcit-ihd) recently emerged as a new safe and simple alternative to continuous renal replacement therapy allowing heparin-free extended dialysis sessions (> h). in this study, we aimed to answer to two issues still unresolved: (i) can citrate contained in the dialysate accumulate and lead to citrate intoxication in patients with liver disorders, and (ii) can citrate be avoided using citrate-and calcium-free dialysate (ccf-ihd)? patients and methods: monocentric retrospective study. among the sessions performed with cafcit-ihd, the ihd sessions ( critically ill patients) with citrate measurement available before and after the dialysis filter were reviewed. estimation of the liver clearance was performed using the picco lemon ® system (pulsion). in addition, sessions performed using ccf-ihd were reviewed. results: all the patients had liver disorders (post-liver transplantation period n = ; cirrhosis with child > a ). among the eighteen cafcit-ihd patients, fifteen ( %) and six ( %) received mechanical ventilation or vasopressive drugs, respectively. the median time of the dialysis session was h [ ] [ ] [ ] [ ] , with hourly ultrafiltration rate of ml (one premature termination not related to dysfunctional catheter). in all patients, ionized calcium (ica) decreased below . mmol/l after the filter, whereas post-filter calcium reinjection according to ionic dialysance led to a stable pre-filter (i.e. patient) ica. median citrate concentrations were all below . mmol/l after the filter (minimal concentration to obtain anticoagulation mmol/l) and all except one below the normal value (< µmol/l) before the filter. during all the sessions, ionized to total calcium ratio was below . and the strong ionized gap decreased. when available (n = ), no correlation could be identified between serum citrate concentration and liver clearance. last, in ccf-ihd sessions performed in critically ill patients, no premature termination occurred (median time of the sessions h) and post-filter ica also decreased below . mmol/l. no citrate accumulation could be identified in critically ill patients (even with liver disorders) and receiving extended dialysis sessions ( h or more) using calcium-free citrate containing-ihd. interestingly, we demonstrated that citrate is not required to obtain optimal regional anticoagulation (i.e. post-filter ica < . mmol/l), and a citrate-and calcium-free dialysate could be a safe alternative. compliance with ethics regulations: yes. rationale: ventilator induced diaphragmatic dysfunction is highly prevalent in adult critical care and associated with worse outcomes. specificities in pediatric respiratory physiology suggest that critically ill children may be at high risk of developing this complication, but no study has described the evolution of diaphragmatic function in critically ill children undergoing mechanical ventilation. this study aims to validate a method to quantify diaphragmatic function in mechanically ventilated children. in this prospective single-center observational study, children between week and years old intubated for elective ent surgery and without pre-existing neuromuscular disease or recent muscle paralysis were recruited. immediately after intubation, diaphragmatic function was evaluated using brief airway occlusion maneuvers during which airway pressure at the endotracheal tube (paw) and electrical activity of the diaphragm (eadi) were simultaneously measured for consecutive spontaneous breaths, while the endotracheal tube was occluded with a specific valve. occlusion maneuvers were repeated times. in order to account for central respiratory drive and sedation use, we recorded the neuromechanical efficiency ratio (nme, paw/eadi), in addition to the maximal inspiratory force (mif). in order to determine the optimal measure of nme during an occlusion, the variability over the three occlusion maneuvers of different variables (first breath, last breath, breath with maximal paw deflection, breath with maximal nme value, and median nme value) was assessed using coefficients of variation and repeatability coefficients. results: patients had a median age of . years (interquartile range . - . ), a median weight of kg ( - ), and were male ( %). the median evolution of paw, eadi, and nme ratio over the occluded breaths are represented on fig. . nme values corresponding to the last breath and the breath with maximal paw deflection were the least variable, with median coefficient of variation of % and % and repeatability coefficients of . and . , respectively. conclusion: brief airway occlusions can be used to assess diaphragmatic function in intubated children through both mif and nme ratio, and the latter should ideally be computed on the last breath or the breath with the largest pressure deflection to improve repeatability and decrease variation. compliance with ethics regulations: yes. epidemiology is poorly understood due to the rare use of validated diagnostic tools. the main objective of the study was to determine, by systematically calculating the wat- score, the incidence of ws in our surgical picu. the secondary objective was to analyze the risk factors, consequences and management modalities of ws. patients and methods: following institutional review board approval, we conducted a prospective monocentric study between july and january . all consecutive mechanically ventilated children admitted in our surgical picu with sedation/analgesia by continuous intra-venous (iv) benzodiazepines (bzd) and/or opioids for at least h were included. as soon as sedation was decreased and during h following their total discontinuation, wat- score was assessed twice a day. ws was defined by a wat- score > . the search for risk factors and consequences associated with ws was performed by univariate analysis (mann-whitney and chi test). ethical standards were satisfied and the lack of opposition from patients and their parents was systematically checked. results: the incidence of ws was % among the patients of our cohort including % of children admitted postoperatively and % after severe traumatic brain injury (tbi). significant results are reported in table . our results show that even for sedation time less than days, children could develop ws ( / patients). on the other hand, age, severity (pelod score), number of previous surgeries and severe tbi were not associated with ws. our study also demonstrated that cessation of sedation and prevention of ws was not uniform in our unit. the high incidence of withdrawal syndrome in our study, even in children sedated for less than days, and its consequences require thinking about prevention. we suggest a systematic monitoring of the occurrence of this adverse event using a validated score, from days of continuous iv sedation/analgesia. compliance with ethics regulations: yes. rationale: severe traumatic brain injury (tbi) is a major healthcare problem. amplitude and duration of intracranial hypertension is highly associated with patient outcome. the intracranial pressure (icp) is therefore one key parameter to monitor in the acute phase. when icp is monitored with an external ventricular drain, the pressure recorded by the monitor does not always correspond to the real icp, depending on the status (open/closed) of the -way tap. misleading values could therefore be sent to the patient medical record. our hypothesis is that a machine-learning algorithm will be able to identify automatically and in real time the reliable and non-reliable values of the icp signal. we retrospectively studied pediatric patients having an external ventricular drain between july and july , in a single pediatric intensive care unit. the icp signals were extracted from a high-frequency database ( hz) and pre-processed adequately. to train the algorithms, an annotated database was manually created with two classes: reliable icp vs. non-reliable icp (drain system opened to allow cerebrospinal fluid removal). eleven signal characteristics were compared between the two classes (mann-whitney test), and significantly differing variables were tested in the algorithms. we compared the performance of two machine-learning algorithms: the k-nearest neighbors (knn) and the support vector machine (svm). using -fold cross-validation method, % of the data was used to train the algorithms and % was used for testing. the best classifier was further validated by simulating a real-time icp analysis, using a s sliding-window approach with % overlap. the study was approved by the localresearch ethics committee. results: sixteen patients were included in the study. the training database created from patients, contained segments (of s duration) per class and per patient. eight signal variables were identified and kept to define the segments. the knn algorithm, with k = , led to the best performance, with a mean of % (mean ± sd: % ± . %). the knn was then visually validated on icp signals from the remaining two patients ( figure) . by simulating a real-time icp extraction, our algorithm was able to efficiently identify the reliable icp segments, and to display a mean value only for valid segments. university hospital picu (paris). all consecutive children ( month- years) admitted for acute encephalitis were included and diagnosis was confirmed using the consensus conference criteria's. data regarding clinical, biological and radiological presentations were collected as well as data on the therapeutics used and outcomes at discharge and at the last medical consultation. results: patients were included with a mean age of . years (range . to years old). infectious causes were identified in % (n = ), autoimmune causes in % (n = ) and acute demyelinating encephalomyelitis in % (n = ) of cases. etiology remained undetermined in % of cases (n = ). the most common pathogens were, in order of frequency, influenzae virus, mycoplasma pneumoniae and epstein-bar virus. the main clinical features were fever ( % n = ); epileptic seizures ( % n = ) and coma ( % n = ). regarding therapeutics, % of patients required mechanical ventilation and % of patients required hemodynamic support. % received corticosteroids, % intravenous immunoglobulins and % plasmatic exchanges. the use of these specific treatments was heterogeneous, especially in infectious and undetermined encephalitis, where respectively % and % received boluses of corticoids. the mean length of stay in picu was . days (range - days). the mortality rate was % and the overall rate of sequelae at discharge was % and % at distance, with % considered as severe (gose-ped score > ). the use of mechanical ventilation and young age at diagnosis were risk factors associated with poor prognosis at discharge. the etiology of acute encephalitis remains indeterminate in more than % cases with a clear predominance of infectious causes when an etiology is found. this is a severe pathology responsible for significant mortality and morbidity requiring long-term follow-up. compliance with ethics regulations: yes. rationale: preserving neurological outcome of children under extracorporeal membrane oxygenation (ecmo) remains challenging. acute brain injury (abi) is a frequent complication of ecmo that could be prevented by continuous neuromonitoring. cerebral near infrared spectroscopy (nirs) is routinely used for detecting cerebral complications of cardiac surgery. in adults and infants under prolonged ecmo, cerebral hypoxia is associated with poor neurological outcome. the aim of this study was to assess the value of an impaired cerebral oxygenation on mortality and occurrence of an abi in children under ecmo. patients and methods: children under years old were included in this observational retrospective monocentric study if they needed veno-venous (v-v) or veno-arterial (v-a) ecmo for respiratory and/ or circulatory failure and had concomittant nirs monitoring. cerebral desaturation was defined as a rsco value under % or under % from the baseline; cerebral hyperoxia was defined as a rsco value above %. proportion of time in cerebral desaturation and hyperoxia were recorded. neurological lesions were identified on imaging (mri or scan) by blinded radiologist and classified as major or minor. abi was defined as any hemorragic or ischemic lesion on cerebral imaging, including brain death. results: patients were included. ecmo duration was [ ; ] days. the mortality rate was ( . %), and the proportion of abi was ( %) including brain deaths, ( . %) major lesions, and ( . %) minor lesions. mean rsco was ± % in the right hemisphere, and ± % in the left hemisphere. there was no significant difference in cerebral hypoxia between survivors and non survivors, and between patients with and without an abi. cerebral hyperoxia was associated with a better survival (p = . in the right hemisphere, and p = . in the left hemisphere). in v-v ecmo and at the right conclusion: in our study, cerebral hypoxia was not associated with poor neurological outcome, but cerebral hyperoxia seems to be protective especially in v-v ecmo. this is the first study assessing the value of cerebral oxymetry in all age ranges pediatric ecmo. in this population, multimodal monitoring might be better than nirs alone to predict neurological impairment. further prospective studies are needed to assess first the feasibility, then the impact of such a monitoring. compliance with ethics regulations: yes. cerebral autoregulation impairment is associated with acute neurological events during pediatric extracorporeal membrane rationale: children supported by extracorporeal membrane oxygenation (ecmo) present a high risk of adverse neurological complications. as some animal studies have shown, cerebral autoregulation (ca) impairment after exposure to ecmo, may be a key factor. our main objective was to investigate the feasibility of ca continuous monitoring during ecmo treatment. the second objective was to analyze the relationship between ca impairment and neurological outcome. patients and methods: an observational prospective study including children treated by ecmo in centers was conducted. a correlation coefficient between the variations of regional cerebral oxygen saturation (rsco ) and the variations of mean arterial blood pressure(map) was calculated as an index of ca (cerebral oxygenation reactivity index, cox) during ecmo. a cox > . was considered as indicative for dysautoregulation. cox values were averaged inside mmhg-map bins, allowing determining optimal map (mapopt) and lower (lla) and upper (ula) limits of autoregulation in -h periods. neurological outcome was assessed by the onset of an acute neurologic event (ane) defined by occurrence of hemorrhagic or ischemic stroke and/ or clinical or electrical seizure and/or brain death during the ecmo treatment. rationale: myocardial ischemia reperfusion (ir) injury is the leading cause of perioperative morbi-mortality. protective effect of pharmacologic preconditioning such as anesthetic preconditioning (apc) with sevoflurane (sev) has been widely demonstrated in animal and human models. apc seems to protect myocardial cells from apoptosis, a programmed process of cell death tightly controlled by bcl- family proteins. however, the involved mechanisms in apc have yet to be characterized. we hypothesized that apc protects against myocardial apoptotic cell death by regulating bcl- anti-apoptotic members. to study the sev-induced apc mechanisms against myocardial ir, we used a validated in vitro model reproducing ir injury. rat cardiomyoblast cells h c were cultivated in . % o hypoxia in the presence of ischemia-mimicking medium. after min of ischemia, the reperfusion injuries are induced by replacing the culture medium with a krebs-henseleit normoxic medium for min. apc was performed by adding sev directly into the culture medium at an initial concentration of mm, prior to ischemia, for min. we then used another preconditioning agent, metformin (met), to explore the same signaling pathways. apoptotic cell death was measured by caspase activity assay and western blotting (expression of cleaved caspase ) under ir and apc conditions. results: our model faithfully reproduced the protective effect of apc which results in a significant decreased apoptosis under ir ( % reduction of the caspase enzymatic activity, correlated with a decrease of caspase cleavage). we showed that sev induces overexpression of the anti-apoptotic protein bcl-xl, which is responsible for the protective effect of apc. furthermore, these observations were confirmed in vivo in mouse heart lysates. we demonstrated that bcl-xl overexpression was due to the activation of the protein kinase akt. interestingly, we were able to show that preconditioning with met reproduces the protective effect of sev by inducing an akt-dependent bcl-xl overexpression. indeed, sev and met, which are both complex inhibitors of mitochondrial respiratory chain, seem to share a common reactive oxygenated species-dependent protective mechanism responsible for bcl-xl protein regulation. rationale: despite early endovascular treatment with successful recanalization, % of acute ischemic stroke (ais) patients experience a poor functional outcome after a large vessel occlusion. sepsis is frequent at the acute phase of stroke and is associated with poorer short and long term outcomes. we aimed to investigate the cerebral consequences of sepsis after recanalized ais and explore possible mechanisms involved. patients and methods: male c bl mice were randomly assigned to a x factorial plan to one of the following groups: ) a -minute middle cerebral artery (t-mcao) transient occlusion under inhaled general anesthesia, followed min after recanalization by intraperitoneal (i.p.) sepsis (lps, µg/g diluted in µl of nacl . %), (tmcao/ lps group); ) t-mcao followed by i.p. placebo ( µl of nacl . %) (tmcao/placebo group); ) sham operation (cervicotomy without carotid catheterization) followed by i.p. lps. (sham/lps group); ) sham operation followed by i.p. placebo, (sham/placebo group). in all groups, animals received subcutaneous fluid resuscitation ( µl nacl . %) immediately after the procedure and h later. twenty-four hours after recanalization, animals were scored for sepsis features and neurological deficit (on the modified neurological severity scale), (mnss) before sacrifice. the primary outcome measurement was a composite of death and hemorrhagic transformation at h. secondary outcome measurements included neurological deficit, sepsis features, neutrophil activation reflected by plasmatic myeloperoxydase (mpo) levels, stroke volume, and microglial activation in brain parenchyma (infarct core, perilesional area, controlateral hemisphere). results: t-mcao/lps animals had higher mnss ( . fold, p = . ) and sepsis ( fold, p = . ) scores at h with increased plasma mpo levels at h ( . fold, p < . ) and h ( . fold, p < . ), as well as, lower temperature ( . °c reduction, p = . ) and glycemia ( . g/l reduction, p = . ) as compared to tmcao/placebo animals. t-mcao/lps animals had a higher risk of unfavorable outcome at h ( -group comparison: p = . ; x analysis: t-mcao/lps, / − %vs. t-mcao/placebo / - %-, p < . ), whereas stroke volumes were not significantly different between groups. detailed results are presented in table . compared to t-mcao/placebo group, t-mcao/ lps animals had . fold increase (p = . ) in the mean number of microglial cells in the hemisphere controlateral to t-mcao, whereas no significant difference was observed in infarct core or peri-infarct parenchyma. conclusion: early sepsis after experimental ais worsens outcome and neurological deficit, without impacting stroke volume. early sepsisinduced systemic activation of neutrophils and increased microglial activation in the hemisphere contralateral to ischemia may have an important role on neurological outcomes observed in this setting. compliance with ethics regulations: yes. rationale: extracellular vesicles (evs) regulate diverse cellular and biological processes via facilitating intercellular cross-talk. several studies have suggested an association between lung injury and the generation of evs derived from platelets, neutrophils, monocytes, lymphocytes, red blood cells, endothelial cells, and epithelial cells. every year more than , patients require cardiac surgery with cardiopulmonary bypass (cpb). this cpb allows a substitution of the heart pump function and an oxygenation of the blood permitting a stop of the mechanical ventilation (mv). stopping mv during cpb is responsible for lung damage, leading to postoperative systemic inflammation while maintaining mv with positive expiratory pressure (peep) diminished the occurrence of atelectasis and the postoperative inflammatory response. in addition, this surgery is marked by immune dysfunction, leading to real immunosuppression of patients in postoperative care. a link between pulmonary injury and postoperative immunosuppression has been established, however, the mechanisms underlying this association are not fully known and evs may have a role in this post-operative immunosuppression. the purpose of this study is to investigate whether lung injury induced during cardiac surgery with cpb lead to the emergence of evs. the effect of mv during cpb on the production of these evs has also been studied. patients and methods: patients were prospectively divided into two groups: without mv during cpb and dead space mv with positive end-expiratory pressure during cpb. pao (arterial oxygen tension)/ fio (inspired oxygen fraction) ratio, biological markers of lung injury (cxcl , ccl , tnf-α, il- β, il- , rage, il- ) and blood cell count were collected before, h and days after surgery. the quantification of plasma evs was performed using turnable resistive pulse sensing and characterization of evs was performed using flow cytometry before, h and days after surgery. rationale: the benefit of prone positioning (pp) during moderate to severe acute respiratory distress syndrome (ards) may be related to its impact on the inflammatory response to ventilator-induced lung injuries. [ c]-pk is a positron emission tomography (pet) radiotracer that allows the non-invasive quantification of macrophages. we aimed to evaluate the effects of pp on [ c]-pk lung uptake in animals with experimental ards. patients and methods: experimental ards (by hydrochloric acid) was induced in pigs in supine position (sp), to obtain a pao / fio < mmhg. animals were under general anesthesia, neuromuscular blockade, and ventilated with a ml kg − tidal volume, and cmh o of positive end-expiratory pressure (peep). immediately after experimental ards, animals were randomized to be prone positioned, or to remain in sp. pet and computerized tomography (ct) were acquired h after randomization (h ). [ c]-pk uptake was measured on the whole lungs, and by dividing the lungs into regions or slices-of-interest (soi) along the ventro-dorsal axis, and was quantified by the standardized uptake value (suv), corrected for lung tissue density. results: pp was performed in animals, and sp in . after ards induction, pao /fio was [iqr, [ . - . ] in sp animals (p = . ). in pp animals, [ c]-pk suv was significantly lower in ventral soi, compared to sp, and significantly increased in dorsal soi ( fig. , *: p < . between groups in a given soi). in univariate analysis, [ c]-pk regional suv was positively associated with regional ct-measured peep-related increase in gas volume, and negatively with peep-related lung recruitment, but not with regional tidal volume. conclusion: during experimental ards, pp redistributed lung macrophage recruitment estimated by [ c]-pk uptake from ventral lung regions to dorsal regions, without affecting global macrophage influx. the intensity of macrophage recruitment was associated with peep-related lung inflation. compliance with ethics regulations: yes. rationale: acute respiratory distress syndrome (ards) is a pleiomorphic disease characterized by a severe respiratory failure associated with an increased mortality. nowadays, predicting clinical outcome of patients suffering from ards remains difficult. therefore, identifying new biomarkers to predict patient outcome, to evaluate response to therapy and to identify new potential pathways of interest are highly needed. exosomes are extracellular vesicles involved in cell-cell communication by transferring micrornas (mirnas) from donor to recipient cells. thus, exosomal mirnas can significantly affect biological pathways within recipient cells resulting in alterations of cellular function and the development of a pathological state. as biomarkers are highly needed in the particular field of ards, we realized a monocentric and prospective study to identify a new potential biomarker of interest. therefore, a prospective plasma sampling at the diagnosis of moderate to severe ards according to the definition of "berlin" has been performed. we analysed mirna content of exosomes from plasma ards patients compared to healthy subjects (hs) in order to identify new potential predictive biomarkers in ards. during one-year period, patients hospitalized in the icu of chu sart tilman suffering from infectious moderate-to-severe ards have been included. the ethical committee review boards of the hospital approved the research protocol (b , ref: / ), and informed consents were obtained. exosomes were isolated from plasma samples of ards patients and hs with standard ultracentrifugation protocol. exosomal mirna content was analyzed using small rna sequencing method, and diseases/biological processes associated to altered mirs were determined by bioinformatic analysis. results: for the first time, exosomal mirna expression modifications were studied in patients with moderate-to-severe infectious ards. we identified a new signature statistically significant composed of three up-regulated mirnas (mir- , mir- a and mir- ) and one downregulated (mir-let- b). conclusion: we identified potential biomarkers for ards from plasma exosomes. our findings may thus lead to predict ards outcome but also a better understanding about the roles of these mirs in the pathogenesis of ards and thus open new avenues for therapeutic approaches. in particular, exploit and develop the pro-fibrotic pathway induced by down-expression of mir-let- b. but also confirm in the future the current interest about mir- in its ability to restore pulmonary integrity after trauma. compliance with ethics regulations: yes. rationale: diabetic ketoacidosis (dka) is a life-threatening emergency. microvascular hyporeactivity was reported in these patients and was completely reversibly when ph was corrected with treatment: aggressive rehydration, electrolyte replacement and insulin therapy ( ) . red blood cell (rbc), a component of the microcirculation, showed alterations oftheir shape in diabetic patients ( ) but no data were available concerning the time course of the rbc deformability during treatment for dka. we aimed to assess the rbc deformability during dka treatment in icu patients. patients and methods: after approval by the ethics committee, rbcs deformability was assessed, in all icu patients admitted for dka and without infection, by ektacytometry technique (laser-assisted optical rotational red cell analyzer-lorrca): at icu admission, + h, + h and at the end of the icu stay ( - h). elongation index (ei) was defined as (l − w)/(l + w), where l is the length and w is the width. at °c, ei values were determined in the function of shear stress (ss) in a range of . - pa, based upon the laser diffraction pattern changes. a higher ei indicates greater rbc deformation. rbc deformability from patients with dka was compared at icu admission to healthy volunteers (v) and to diabetic patients followed in consultation (d). we also studied the evolution of deformability during treatment. results: icu dka patients compared to d and v were studied. as expected, glycemia and glycated hemoglobin were significantly higher in dka compared to d (respectively: glycemia: ( - ) vs ( - ) mg/dl and . % ( . - . ) vs . ( . - . ); all p < . ). dka patients received ( - ) ml of fluids and . ui/ kg bw ( . - . ) of insulin during their first h of icu stay. rbcs deformability from dka patients was significantly more altered at icu admission compared to others groups ( fig. ) and these alterations persists despite treatment. no correlations were observed between these alterations and quantity of fluids or insulin received, glycemia, glycated hemoglobin, ph, natremia, age or length of diabetes history. conclusion: in contrast of reversible microvascular hyporeactivity, rbc deformability from dka patients was already altered at icu admission and remains altered despite treatment. these alterations could contribute to the blood flow abnormalities observed in these patients. compliance with ethics regulations: yes. rationale: sepsis remains the first cause of acute circulatory failure in the emergency department (ed). standardized fluid resuscitation may not be adapted in certain patients, especially those with early sepsisinduced cardiac dysfunction in whom excessive fluid administration could be deleterious. information on early hemodynamic profile of septic patients in the ed are scarce. accordingly, we aimed at describing hemodynamic profiles encountered in septic patients assessed shortly after their ed admission using focused echocardiography. patients and methods: we prospectively enrolled adult patients with sepsis (qsofa score ≥ ) from january to july in the ed (nct ). focused echocardiography were performed by emergency physicians previously trained to ecmu level. each patient was evaluated according to a standardized protocol based on a limited number of simple binary clinical questions. investigators interpreted on-line the echocardiographic examination, determined the hemodynamic profile based on simple yet robust criteria (hypovolemia, left ventricular [lv] or right ventricular [rv] failure, vasoplegia with hyperdynamic state, tamponade, severe mitral or aortic regurgitation, or apparently normal profile), and recorded any substantial change in planned therapeutic management (surviving sepsis campaign ). data were digitally stored and validated off-line by an expert in critical care echocardiography. results: focused echocardiography were performed in patients (mean age: ± years; men: %; source of infection: pulmonary %, urinary %, abdominal %) after a median fluid loading of ml (iqr: - ml). according to sepsis- definition, patients had sepsis and sustained septic shock. mean sofa score was . ± . (hemodynamic failure %, respiratory failure %, renal failure %), mean lactate reached . ± . mmol/l, icu admission involved % of patients and overall -day mortality reached %. hemodynamic profile was hypovolemia in patients ( %), vasoplegia in patients ( %), cardiac failure in patients ( %) (lv failure: n = ; rv failure: n = ) and without relevant hemodynamic abnormality in patients ( %). ongoing therapy was altered based on early echocardiographic assessment in % of cases. mortality rate was not significantly different between groups (p = . ). conclusion: although hypovolemia was predominantly identified in patients presenting to the ed with sepsis during hemodynamic assessment, early ventricular dysfunction involved one-quarter of patients. these results suggest that early focused echocardiographic assessment promises to help the front-line physician tailoring the therapeutic management of septic patients in ed, especially regarding fluid resuscitation. compliance with ethics regulations: yes. right ventricular failure in septic shock characterization, incidence and impact on fluid-responsiveness guillaume geri , amélie prigent , xavier repessé , marine goudelin , gwenael prat , bruno evrard , cyril charron , philippe vignon , antoine vieillard-baron ambroise paré hospital, boulogne-billancourt, france; ambroise paré hospital, medical icu, aphp, boulogne-billancourt, france; chu limoges, limoges, france; chu brest, brest, france correspondence: guillaume geri (guillaume.geri@aphp.fr) ann. intensive care , (suppl ):f- rationale: right ventricular (rv) failure was defined by rv dilatation with systemic congestion. tricuspid annular plane systolic excursion (tapse) could be of limited value. we report the incidence of rv failure in patients with septic shock, its potential impact on the response to fluids, as well as tapse values. patients and methods: ancillary study of the hemopred prospective multicenter study including patients under mechanical ventilation with circulatory failure. with septic shock were analyzed. patients were classified in groups based on central venous pressure (cvp) and rv size (rv/lv end-diastolic area, eda). in group , patients had no rv dilatation (rv/lveda < . ). in group , patients had rv dilatation (rv/ lveda ≥ . ) with a cvp < mmhg (no venous congestion). rv failure was defined in group by rv dilatation and a cvp ≥ mmhg. passive leg raising (plr) was performed. results: % of patients were in group , % in group and % in group . in group and , rv/lv eda was higher than in group , . [ . ; . ] versus . [ . ; . ]. cvp was [ ; . ] mmhg in group . a correlation between rv size and cvp was only observed in group . higher rv size was associated with a lower response to plr (figure) . a large overlap of tapse values was observed between the groups. . % of patients with rv failure had an abnormal tapse. conclusion: rv failure is frequent in septic shock and alters fluid responsiveness. tapse was not accurate enough to diagnose rv failure. compliance with ethics regulations: yes. rationale: weaning-induced pulmonary oedema (wipo) is a leading cause of weaning failure in high-risk patients (heart failure, copd, obesity). we hypothesized that hypervolemia associated with positive fluid balance facilitates wipo in high-risk patients. patients and methods: in this prospective, observational, singlecenter study, patients with copd and/or heart failure with reduced ejection fraction (< %) were studied. exclusion criteria were nonsinus rhythm, severe mitral valve disease and inability to obtain adequate echocardiographic views. echocardiography was performed immediately before and during spontaneous breathing trial (sbt, -min t-tube). patients who failed sbt were treated according to echocardiographic results before undergoing a second sbt. fluid balance and body weight were collected at each sbt. shows interesting performance to predict fluid responsiveness in spontaneously breathing patients. nevertheless, measurement sites of inferior vena cava (ivc) diameters remain controversial for that purpose. the aim of the study was to test the accuracy of different measurement sites of civc to predict fluid responsiveness in spontaneously breathingpatients. this study is a post hoc analysis of two prospective cohorts. we included spontaneously breathing patients without mechanical ventilation presenting with sepsis-related acute circulatory failure and considered for volume expansion (ve). we assessed hemodynamic status at baseline and after a fluid challenge (fc) induced by a min-infusion of ml-gelatin %. the ivc diameters were measured off-line with ultrasonography using the bi-dimensional mode on a subcostal long-axis view. the civc was calculated as [ (expiratory-inspiratory)/expiratory] diameters during standardized (civc-st) and unstandardized breathing (civc-ns) conditions. breathing standardization consisted of a deep inspiration with concomitant control of buccal pressures and passive exhalation. patients were referred to be responders to fc (i.e. fluid responsive) when the stroke volume increased by ≥ %. results: among the patients included in the study, ( %) were responders to fc. the accuracy of civc-st and civc-ns before fc to predict fluid responsiveness differed significantly by measurement sites (interaction p value < . and < . , respectively). measuring ivc diameters cm from the junction of the ivc and the right atrium provided the best accuracy to predict fluid responsiveness ( fig. ). at cm caudal to the right atrium, civc-st was significantly better than civcns to predict fluid responsiveness: area under roc curve . ( % ci . - . ) versus . ( % ci . - . ), p < . . at cm, a civcst ≥ % and a civc-ns ≥ % predicted fluid responsiveness with sensitivity of % and %, and specificity of % and %, respectively. conclusion: accuracy of civc to predict fluid responsiveness in spontaneously breathing patients depends on both measurement sites of ivc diameters and breathing conditions. measuring ivc diameters during a standardized inspiration maneuver at cm caudal to the right atrium is the most relevant mean to optimize civc performance to guide ve. compliance with ethics regulations: yes. rationale: intermittent hemodialysis (ihd) is increasingly used in patients admitted to intensive care unit (icu) with acute kidney injury (aki) requiring renal replacement therapy (rrt). however, this technique is associated with nearly % of episodes of perdialytic hemodynamic instability (hi), a common cause of increased morbidity and mortality. at the same time, trans-thoracic echocardiography (tte) has become widely used in intensive care units and is now one of the hemodynamic monitoring methods used daily in the icu setting. patients and methods: search for one or more pre-dialysis tte criteria predictive of perdialytic hi, defined by a systolic blood pressure (sbp) lesser than mmhg or a suddain decrease in sbp of more than mmhg. prospective, observational study of standard care in a medical icu. collection of demographic, clinical and pre-dialysis echocardiographic data from included patients. results: twenty-five patients with a total of sessions of ihd between november and november were included in the study. tte was performed for each patient before each ihd session. hi occurred in hemodialysis sessions. in univariate analysis, the existence of prior heart disease ( % vs %, p = . ), a greater diameter of the left atrium ( . vs . cm, p = . ), a lower cardiac output ( . vs . l/min, p = . ), a right dysfunction assessed by lowered tapse and s-wave ( vs mm, p < . and . vs . cm/s, p = . , respectively) and an increase in paps ( vs mmhg, p = . ) were significantly associated with the occurrence of perdialytic hi (fig. rationale: several transthoracic echocardiography (tte) parameters of left (lv) and right ventricular (rv) systolic function are available. we compared the ability of these different parameters to track changes in lv or rv systolic function and to detect lv or rv systolic dysfunction in critically-ill patients. in patients ( mechanically ventilated and with atrial fibrillation), tte examinations were performed before and after i) infusion of -ml of saline (n = ), ii) changes in norepinephrine (n = ), iii) or in dobutamine (n = ) dosage. for the lv systolic function, we compared the mitral annular plane systolic excursion (mapse), the systolic (s') peak velocity of the lateral mitral annulus and the global longitudinal strain (glslv) to the lv ejection fraction (lvef), considered as the gold standard. for the rv systolic function, we compared the tricuspid annular plane systolic excursion (tapse), the systolic peak (s) velocity of the tricuspid annulus and the global longitudinal strain (glsrv) to the rv fractional area change (fac), considered as the gold standard. results: after pooling all values, lvef ( ± % at baseline) was better correlated to glslv (r = . ) than to mapse (r = . ) and s' wave (r = . ) (each p < . ). the concordance rate between changes (in %) in lvef and in the other parameters of lv systolic function was % for glslv, % for mapse and % for s' wave. both mapse and s' wave could not reliably detect moderate ( % ≤ lvef ≤ %) or severe (lvef < %) lv dysfunction. conversely, a glslv > − % predicted moderate lv dysfunction with a sensitivity of % ( % ic: - %) and a specificity of % ( % ic: - %) and a glslv > − . % predicted severe lv dysfunction with a sensitivity of % ( % ic: - %) and a specificity of % ( % ic: - %). after pooling all values, fac ( ± % at baseline) was better correlated to glsrv (r = . ) than to tapse (r = . ) and s wave (r = . ) (each p < . ). the concordance rate between changes (in %) in fac and in the other parameters of rv systolic function was % for glsrv, % for tapse and % for s wave.both tapse and s wave could detect rv dysfunction (fac ≤ %) with moderate reliability only. conversely, a glsrv > − % detected rv dysfunction with a sensitivity of % ( % ic: - %) and a specificity of % ( % ic: - %). in critically-ill patients, glslv and glsrv seem to be the best tte parameters of lv and rv systolic function. enrolments are still ongoing, which may allow further analysis. compliance with ethics regulations: yes. rationale: passive leg raising (plr), pulse pressure variation (ppv), and the -second end-expiratory occlusion test (eexpo) are frequently used to assess preload responsiveness. however, there are conditions in which they are not valid or feasible, which may preclude their applicability in the daily clinical practice. the aim of this study was to estimate the prevalence of such conditions in critically ill patients with acute circulatory failure. between january and april , all patients of a -bed medical icu were daily screened and those with acute circulatory failure, defined by norepinephrine infusion or fluid therapy > l during the previous h, were included. in each of them, we screened the criteria of validity/feasibility of ppv, plr and eexpo. results: eighty-four patients ( % with septic shock, % with cardiogenic shock, % with hypovolemic shock, % with non-septic vasoplegic shock) were enrolled in the study. among them, norepinephrine infusion was ongoing at the time of enrolment in % of the patients whilst % were under mechanical ventilation, and % with acute respiratory distress syndrome. plr was not applicable in % of cases. this was mainly due to venous compression stocking ( % of cases), intra-abdominal hypertension ( % of cases), and either an absence of cardiac output monitoring or impossibility to perform echocardiography ( % of cases). among the intubated patients, ppv was applicable in % of cases, including cases with high ppv under conditions generating false negatives (low tidal volume or lung compliance) or low ppv values under conditions generating false positives (spontaneous breathing, cardiac arrythmias). however, ppv was not interpretable in % of cases. this was mainly due to low tidal volume ventilation ( % of cases), spontaneous breathing activity ( % of cases), while the remaining non-interpretable cases ( %) had more than one reason. in the intubated patients, eexpo was not applicable in % of cases. this was due to impossibility for patients to sustain a -s hold of mechanical ventilation in % of cases, and either an absence of cardiac output monitoring or the impossibility to perform echocardiography in % of cases. plr and eexpo were both valid and feasible in % of the patients, and the three tests were all feasible in only % of patients. rationale: comorbid association between chronic respiratory diseases and sleep apnea syndrome (sas) revealed frequent with systematic search in icu following icu stay. this association carries prognosis impact depending whether specific treatment is implemented or not. nosas and stop bang scores are proposed for screening of sas in general population. the aim of the present study is to report the prevalence of sas in icu patients admitted for hypercapnic respiratory failure and compare association of nosas and stop bang score with sas severity. the study was conducted between january and september . patients consecutively admitted in the icu for hypercapnic respiratory failure had calculation of a no sas and stop bang scores at admission. in survivors nocturnal polygraphic records was performed to weeks following icu discharge. the association between the number of apnea-hypopnea episodes, bmi, and clinical variables suggestive of sas, was tested by poisson regression model. results: during the study-period, patients (mean age: ± years, ph . ± . , paco ± ) were admitted for hypercapnic respiratory failure. non invasive ventilation was used in % and death occurred in six patients. polygraphic records were performed in ( lost to follow-up) mean apnea-hypopnea index was ± with a minimum of and a maximum of . poisson logistic regression showed that no sas (p = . ) but not stop bang (p = . ) was associated with the level of apnea-hypopnea index. rationale: patients with severe acute exacerbations of chronic obstructive pulmonary disease (copd) may benefit from high-flow nasal oxygen regarding its physiological effects and good tolerance. bronchodilator vibrating mesh nebulization through high-flow nasal oxygen circuit has been described to induce similar effect to standard facial mask jet nebulization in stable copd patients. we aim to evaluate whether vibrating mesh nebulization of salbutamol through highflow nasal oxygen circuit is efficient in unstable patients with copd. patients and methods: we conducted a monocenter non-randomized physiological prospective cross-over study, between january and september , including icu patients with severe acute exacerbation of copd and respiratory acidosis treated by salbutamol nebulization. spirometry and airway resistances records were performed after a -h wash-out period without bronchodilator, before and after vibrating mesh nebulization of mg salbutamol through high-flow nasal oxygen circuit. the primary endpoint was forced expiratory volume in s after salbutamol nebulization. secondary endpoints included other spirometry parameters, clinical parameters, dyspnea assessed by a borg scale. results: fourteen consecutive patients were included, forced expiratory volume in s increased significantly after salbutamol nebulization through high-flow nasal oxygen ( ± ml, p = . ), as well as forced vital capacity ( ml ± , p = . ). airway resistances were not significantly changed after nebulization (− . ± . , p = . ) as well as peak expiratory flow (+ ml ± , p = . ). no difference was observed on borg scale (p = . ) and respiratory rate (p = . ) after salbutamol nebulization, while heart rate increased significantly (p = . ). discussion: salbutamol nebulization using vibrating mesh nebuliser placed on high-flow nasal oxygen circuit induces a significant but moderate bronchodilation in patients with severe acute exacerbation of copd. moreover, improvement of forced vital capacity after salbutamol nebulization suggests a reduction of dynamic hyperinflation. conclusion: salbutamol vibrating mesh nebulization through highflow nasal oxygen circuit increases significantly forced expiratory volume in s. compliance with ethics regulations: yes. t-piece versus sub-therapeutic pressure support for weaning from invasive mechanical ventilation in patients with chronic obstructive pulmonary disease: a comparative prospective study amira jamoussi, fatma jarraya, samia ayed, takoua merhabene, jalila ben khelil, mohamed besbes abderrahmen mami hospital, tunis, tunisia correspondence: amira jamoussi (dr.amira.jamoussi@gmail.com) ann. intensive care , (suppl ):f- rationale: the best weaning strategy for patients with chronic obstructive pulmonary disease (copd) remains unknown. the spontaneous breathing trial (sbt) represents a crucial step of weaning, but the choice between the t-piece (sv-tube) or the sub-therapeutic setting of the level of pressure support without positive expiratory pressure (psv) is still a matter of debate. we aimed to compare the success of extubation between two groups of copd patients according to the sbt type (vs-tube vs psv). patients and methods: it was a prospective and comparative study, from april to march , at the abderrahmen mami hospital's intensive care unit (icu). copd patients who underwent invasive mechanical ventilation (mv) for at least h and met the criteria for weaning were included and randomized to sv-tube or psv. a multivariate analysis was performed to determine the association between the sbt modality and the success of extubation (no re-intubation during the h following extubation). results: during the two years' study, patients were included. the mean age was ± years, the sex-ratio was . . weaning process was simple in patients ( %), difficult in patients ( %) and prolonged in patients ( %). fifteen and patients were respectively randomized to the sv-tube and psv groups. the mean duration of mv before randomization was comparable between the groups (sv-tube . ± . days vs psv . ± . days, p = . ). mean weaning time (days) was . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] for the sv-tube group and . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] for the psv group. the mean total mv duration (days) was higher in the sv-tube group than in the psv group ( . vs . , p = . ). the number of re-intubated patients within h following extubation was higher in the psv group ( / vs / , p = . ) as well as the overall reintubation rate ( . % vs %, p = . ). in multivariate analysis, the sbt's trial was independently associated to the success of extubation (or = . , ic [ . - . ], p = . ) in favor of sv-tube' modality. the median length of stay in intensive care was days [ ; ]. the mortality was higher in the psv group ( / vs / , p = . ). extubation failure was a factor associated with mortality (or = . , ci [ . , . ], p = . ). conclusion: ventilation weaning was easy in % of intubated copd patients. sv-tube as sbt modality was associated to success of extubation in patients with copd. mortality in intensive care was significantly higher in re-intubated patients. compliance with ethics regulations: yes. rationale: non-invasive ventilation has become the mainstay in hypercapnic respiratory failure. delaying intubation and invasive ventilation is associated with a worse outcome in these patients. although a predictive score of niv failure has been validated for hypoxemic respiratory failure no such score exists in hypercapnic respiratory failure. the aim of our study is to compare the performance of two scores in the predictive niv failure hypercapnic respiratory failure. patients and methods: consecutive patients admitted between january and july for hypercapnic respiratory failure, were included. hacor score and rox score were calculated in each patient at admission. in patients ventilated non-invasively, the outcome (niv success or failure) was noted. the area under curve (auc) and operative characteristics were computed for both scores. results: during the study-period, out of patients admitted for hypercapnic respiratory failure received niv as the primary ventilatory mode. these patients were mainly men ( / ), had a mean age of . ± years and had the following pulmonary disease: copd exacerbation . %, obesity-hypoventilation syndrome . %, bronchiectasis . %, and other diseases: . %. niv failure occurred in patients ( . %) and icu mortality in . %. mean hacor score and rox score were . ± . and . ± , respectively. the auc under roc was higher for hacor than rox ( . and . respectively) ( fig. ). the hacor score (cut-off ) had a sensitivity of . and specificity of . . conclusion: hacor score seems more accurate in predicting niv failure in hypercapnic respiratory failure. further prospective validation is needed. compliance with ethics regulations: na. rationale: published data on outcomes in respiratory weaning centers are limited and seem to depend on the organisation of healthcare systems and patient case-mix. the weaning center of our university hospital (post intensive care rehabilitation unit) admits for weaning and rehabilitation patients from medical and surgical intensive care units without severe neurological pathologies. the aim of this study was to describe patient's characteristics and outcome (weaning outcomes and survival) and to compare in subgroups according to the initial medical, surgical or cardiac surgical context. patients and methods: we conducted a monocentric retrospective observational study between / / and / / . «successful outcome» was defined by the association of survival and weaning from invasive ventilation. factors associated with evolution were investigated by uni-and multivariate analysis. survival after discharge was analysed according to the initial context and according to the type of ventilation at discharge. results: among patients included, ( . %) had a successful outcome with high use of non-invasive ventilation (niv) ( %). respiratory history (p = . ), female gender (p < . ), igs score at admission to the srpr (p = . ) and non-cardiac surgical setting (p < . ) were associated with an adverse course. the -month survival rate was % in discharged patients. the outcome was not different in the tree subgroups. niv rate at discharge was high in the subgroup of cardiac surgery patients. a multidisciplinary and personalised approach by a specialized weaning unit can provide a successful service model for patients who require liberation from prolonged invasive mechanical ventilation. compliance with ethics regulations: yes. rationale: high-dose insulin euglycemic therapy (hiet) is recommended as first line therapy for calcium channel blockers (ccbs) poisoning because of its inotropic effect. our first objective was to study its hemodynamic impact. we performed a retrospective cohort study of all consecutive patients admitted for ccbs poisoning treated with hiet, in one icu at the university hospital of lille between january and july . the hemodynamic impact was studied through mean arterial pressure (map), vasoactive-inotropic score (vis) and map/vis ratio during the h following hiet initiation. metabolic parameters were also collected. results: patients admitted for ccbs poisoning. patients treated with hiet in icu ( patients without circulatory shock, patients with shock after hiet and patients with shock at baseline before hiet). among shocked patients at baseline (n = ), no hemodynamic improvement was found except an increased map/vis ratio at h (p < . ). on the contrary, an initial worsening of vis ( [ rationale: ketamine is used in the induction and maintenance of general anesthesia. recently, there were concerns regarding its liver toxicity. we conducted a study to investigate the link between ketamine use and liver dysfunction (ld) in intensive care unit (icu) patients. patients and methods: data were extracted from the [anonymized] study, a randomized controlled trial designed to evaluate the effect of cisatracurium on -day mortality rate in moderate and severe acute respiratory distress syndrome (ards) patients. the main endpoint was the occurrence of a ld defined as a total serum bilirubin superior or equal to micromol/l. a matched case-control cohort was created: cases, receiving at least day of continuous ketamine infusion, were paired for with controls according to treatment with cisatracurium, hepatic and cardiovascular sofa sub-score, total serum bilirubin level at the time of inclusion, age, sex, ards from septic origin, shock anytime after inclusion. an analysis was also made on the whole cohort comparing the patients receiving at least day of continuous ketamine infusion to all patients who did not fulfill this criterion. results: cases were identified and matched to controls. in the ketamine group, the median ketamine duration was ( - ) days, and median total cumulative dose . ( . - . ) g. the occurrence of ld was higher in the ketamine group than in the matched control group ( . % versus . %, p = . , fig. ). the hazard ratio (hr) for ld in the ketamine group was . ( % ci . - . , p = . ). there was an increased risk of ld of . % per day of exposure to ketamine (hr . , % ci . - . p = . ) and of . % per gram of ketamine infused (hr . , % ci . - . , p = . ), with a risk starting to be statistically significant after days and gr. in multivariate analysis on the whole cohort, ketamine exposure (hr . , % ci . - . , p = . ), cumulative dose in gram (hr: . , % ic: . - . , p = . ) and ketamine exposure in days (hr: . , % ic: . - . , p < . ) remained independent risk factors for ld occurrence. conclusion: ketamine use in critically ill patients treated for ards is associated to a higher risk of liver dysfunction, assessed by total serum bilirubin. this risk is dose-dependent and increases with duration of treatment. the prescription of high doses or prolonged treatment with ketamine should probably be avoided in critically ill patients. compliance with ethics regulations: yes. rationale: ciguatera is one of the most common cases of marine poisoning associated with fish consumption in the world. the incidence of this intoxication is largely unreported. in martinique, the incidence of this intoxication seems constantly increasing. during the last years, numerous cases of large collective poisonings have been reported in martinique, especially during summer. the spectrum of clinical manifestations is large including gastrointestinal, neurological andcardiovascular symptoms. ciguatoxin, the toxin responsible for ciguatera fish poisoning is considered as a sodium channel agonist with cholinergic and adrenergic activity. it is rarely fatal and management of poisoned patients is essentially based on supportive care. the objective of this study was to describe the clinical characteristics and complications of ciguatera poisoning in martinique, focusing on the cardiovascular ones. observational, retrospective, single-center study covering six-year period from october to september , including all patients admitted to the emergency department of the university hospital of martinique (chu), and all patients who were declared to the regional health agency (ars) for ciguatera intoxication. results: one hundred and forty-nine patients ( ) who were ciguatera-affected were included. the incidence rate found was to be . cases per . patient-years in martinique over the period. about % of patients had gastrointestinal symptoms such as nausea, vomiting, diarrhea, or abdominal pain; % neurological disorders and % cardiovascular symptoms including, bradycardia, hypotension and interventricular block. ingestion of carangue fish was related to a major risk of chronic signs. conclusion: the incidence of ciguatera in martinique is increasing, with . cases/ . patient-years. the clinical presentation is defined mainly by digestive signs, followed by peripheral neurological disorders and cardiovascular symptoms. ciguatera fish poisoning in martinique presents similar clinical presentation to that of the other caribbean islands. there is no specific treatment. acute ciguatera poisoning is responsible for significant cardiovascular complications. physicians should be aware of the potential cardiovascular risk of ciguatera poisoning. compliance with ethics regulations: yes. rationale: pesticides have represented the most incriminated products in severe acute poisonings, in the developing countries, due to the availability of these products. organophosphate poisoning accounts for million poisonings/year worldwide. organophosphate (op) pesticides are used mainly as insecticides in agriculture. the moroccan anti-poison and pharmacovigilance centrer shows that op poisoning are responsible for % of all poisonings combined. the aim of our study: epidemiological, clinical, management and prognostic factors. patients and methods: a retrospective study was conducted on patients with op poisoning admitted to our nine-bed medical intensive care unit between january and december . inclusion criteria were: all patients over years of age and the exlusion criteria were: pesticide poisoning other than op, alcohol poisoning, drug poisoning, scorpionic poisoning and snake bites. statistical analysis was performed with spss software. results: forty patients were admitted for acute op poisoning. in morocco, organophosphores are available over-the-counter in several forms: rodentocides, malathion, cockroach trap, baygon insecticide ( fig. ). the average age was years with a female prévalence of . %. the intoxications were mostly intentional ( %). the symptomatology was determined by the three syndromes: central syndrome in %, muscarinic syndrome in %, nicotinic syndrome in %. rhythm disorders in %, and cardiovascular collapse in %. the symptomatic treatment was applied to all patients, antidotic treatment was administered in % of patients. the average length of hospitalization was days. conclusion: acute op poisoning is a real public health problem. its associated symptomatic treatment (respiratory and neurological resuscitation) and antidotic treatment. the mortality remains high in our context, therefore, we must attach great importance to the prevention. compliance with ethics regulations: yes. ( ). over an -month period, health officials in guadeloupe and martinique reported more than . such cases. assault of these brown algae represents not only an environmental and economic disaster, but also a threat for human health. after h on seashore, large amounts of toxic gas are produced by matter decomposition, including hydrogen sulfide (h s) and ammoniac (nh ). the acute effects on humans after exposure to high concentrations of h s are well described and of increasing severity with concentration, leading to potentially fatal hypoxic pulmonary, neurological and cardiovascular injuries (table ) ; however, the association of long-term exposure to sargassum and health events is unknown. although less documented, long term exposures may result in conjunctiva and upper airways irritation, headaches, vestibular syndrome, memory loss, and modification of learning abilities. in the absence of any available antidote, management of h s intoxication relies on supportive care and prevention using individual protection. the objective of this study was to evaluate the clinical characteristics and consequences of long-term exposure to sargassum among the local population. we conducted a prospective observational cohort study including all patients admitted to the emergency department at the university hospital of martinique from march to december due to exposure to sargassum. patients were managed according to the protocol established by the research group on sargassum in martinique. we assessed the patients exposure to sargassum and air pollutants using monitor located near of the patient's residence. demographics and clinical data (including cardiovascular, neurological and respiratory events) were collected. data are presented as mean ± sd or %.comparisons were performed using univariate analysis. results: in months, patients were included (age: ± years, m/ w, past history: hypertension (n = ), diabetes (n = ), asthma ( ). patients arrived with referral letter from their general practitioner ( %) and presented headaches ( %), developed gastrointestinal disturbances ( %), dizziness ( %), skin lesions ( %), cough ( %) and conjunctivitis ( %). not all patients were clinically symptomatic. in the patients presented in june ( %), symptoms more frequently occurred in the workplace or at home (p < . ). initial lung function tests were normal ( %). three patients were admitted in intensive care unit. conclusion: our study indicates that the magnitude of health effects following long-term exposure to sargassum may be larger than previously recognized. efforts to limit long-term exposure are mandatory. compliance with ethics regulations: yes. rationale: liver consequences of out-of-hospital cardiac arrest (ohca) have been poorly studied. the aim of this study was to describe the characteristics of ohca-induced acute liver dysfunction and its association with outcomes. we analyzed all consecutive ohca patients admitted to two academic centers between and . patients treated with vitamin k antagonist were not included. acute hepatocellular insufficiency (ahi), liver failure (lf) and hypoxic hepatitis (hh) were defined as a prothrombin (pt) ratio < %, a hepatic sofa sub-score > and an increase in transaminases > times the normal values, respectively. indocyanine green (icg) clearance was used as the reference measure of liver function in a subset of patients. multivariate logistic regression was used to identify potential risk factors for day mortality. rationale: neuron-specific-enolase (nse) is commonly used as a biomarker reflecting the extent of brain injury in different settings. in post-cardiac arrest patients, previous clinical studies reported that an increase in nse was predictive of a poor outcome but did not specifically focused on neurological outcome. in this prospective study, we aimed to determine the nse performance for prediction of severe brain damage in post-cardiac arrest patients. patients and methods: all consecutive patients admitted in our icu after cardiac arrest between january and february that were still comatose at h and had at least one measurement of serum nse were included. blood samples for nse measurement were serially collected at (h ) and h (h ) after cardiac arrest and serum nse levels were measured within h. we used the following criteria for the definition of severe brain damage (primary endpoint): cerebral performance categories (cpc) or level at discharge, brain death or withdrawal of life-sustaining treatments (wlst) based on neurological status. we also assessed the predictive value of serum nse using allcause mortality as a secondary endpoint. results: during the study period, patients were available for the analysis. they were mostly male ( . %), with an age of . years. among these patients, ( . %) had a good neurologic outcome (cpc - ) and patients were classified as having a severe brain damage ( wlst based on neurological status, brain deaths and survivors with . in univariate analysis, patients with severe brain damage less frequently received bystander cpr, had longer duration of no-flow, less initial shockable rhythm, more post-resuscitation shock and higher nse values: mean at h were . versus . ; and . versus . at h (p < . ). nse levels at h and h were strong predictors of severe brain damage (auc of . and . respectively, figure ) and also predicted all-cause mortality (auc of . and . respectively). to predict severe brain damage with % specificity, best nse cutoff values at h and h were . and . µg/l, with a sensitivity of . and . % respectively. conclusion: a high serum nse measured at h and h after cardiac arrest accurately predicted severe brain damage with a high specificity. our results support the use of nse for neuroprognostication after cardiac arrest, in combination with other predictors. compliance with ethics regulations: yes. rationale: the psychological care of patients, their relatives and of healthcare workers is a major issue in the intensive care unit (icu). psychologists may provide emotional support during trying times. the intervention of a psychologist may alleviate long term mental health issues such as post-traumatic stress disorder. the main objective of our study was to describe the availability of psychologists in french-speaking icus. patients and methods: internet survey conducted between march and may using surveymonkey (san mateo, usa). survey consisting of questions sent to subscribers of the srlf mailing list via mailchimp software (atlanta, usa). frequencies and percentages were determined for categorical variables and median and interquartile range for continuous variables. the icus with or without psychologist were compared using nonparametric fisher exact test. stata used (lakeway drive, te, usa). results: responses were obtained from unique icus in france (n = ), belgium (n = ), switzerland (n = ), algeria (n = ), morocco (n = ) and tunisia (n = ). ( %) icus were part of public hospitals, ( %) of private facilities. ( %) icus cared for adult patients, ( %) for children. the median number of beds was [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . ( %) icus were open to visitors / , ( %), to visitors > h/day and ( %) to visitors < h/day. psychological consults were established in ( %) wards ( icus did not answer). pediatric icus employed more psychologists than adult icus (p = . ). comparison of icus based on the presence or not of a psychologist appears in table . in icus where a consulting psychologist is available, their effective availability is . [ . - ] full time equivalent. consults are delivered to: patients ( %), families ( %) or healthcare workers ( %). out of the icus without a psychological consult, responders from ( . %) icus believe that a psychological consult is undesirable. out of the icus without psychological consult, ( %) responders cannot obtain a psychological consult, whatever the circumstances, ( %) can require an outside psychological consult when needed, while ( %) can require assistance from a psychologist working in another unit (several answers possible for each respondent). conclusion: psychologists consult in only half of adult icus but in almost all pediatric icus. % of icus are unable to provide a psychological consult. psychological consults are delivered in similar proportions to patients, their family and to a lesser extent to healthcare workers. responders from . % icus without an established psychological consult believe that the availability of a psychologist is undesirable. compliance with ethics regulations: na. rationale: comfort of patients in intensive care unit (icu) is now a real concern for the healthcare teams. perceived patient discomfort assessment is a daily practice for our staff. the primary objective of our study was to assess whether the overall discomfort score reported by patients hospitalized in a separate intermediate care unit differs from that reported by patients hospitalized in icu. a tailored multicomponent program consisting of assessment of icu-related self-perceived discomforts with a -item questionnaire, immediate and monthly feedback to healthcare teams and site-specific tailored interventions, was applied in our department, located in a general hospital, and comprising a -bed icu and a separate -bed intermediate care unit rationale: the transition period surrounding the discharge from icu to hospital ward is a critical period in the course of the patient. handoff of complex patients is at high risk for communication failures between providers, inaccurate cares and icu readmission. a transition program including a post icu follow-up has been proposed to improve handoff quality. post icu consults by icu team represent, also, an opportunity for improving feedback on the quality of icu cares. the goal of the present study is to assess the feasibility and the impact of a systematic early post-icu consult (epicuc) program on handoff quality in a bed mixed icu. patients and methods: before the development of the epicuc program, standardized handoffs were already applied including identified day and hour of discharge and both verbally communicate and written medical and nurse information for receiving team. from st march to th october , all patients who were discharged to the ward of our hospital were candidates for epicuc. epicuc were performed by icu staff (at least one icu physician) within the days following discharge. the epicuc consisted of a face-to-face discussion with the receiver team to assess the accuracy, completeness and understanding of passing information and of a patient visit. a standardized form was used for collecting data. the impact of epicuc on handoff quality was assessed by the number of communication failures and the number of patients in whom epicuc resulted in a management change. personal feeling of epicuc providers on its usefulness was assessed by a - rating scale. results: among the candidates for epicuc, were dead and already discharged alive from hospital at epicuc time. epicuc were performed in patients ( %) within ± days after icu discharge. epicuc ( %) were performed by both, nurse and icu physician. ( %) patients and receiver teams ( %) were available at epi-cuc time. epicuc duration was ± min. a communication failure was identified in epicuc ( %), either a rectification of passing information (n = ; %) and/or a change in patient management (n = ; %). the usefulness of the epicuc was rated at ± and ± by icu physicians and nurses, respectively. conclusion: the time spent for epicuc appears reasonable. epi-cuc identified a communication failure in one-third of handoffs and allowed care readjustment in one quarter of patients. factors associated with handoff failures will be presented during the congress. compliance with ethics regulations: yes. rationale: surviving a critical illness is a challenging condition for patients and relatives. the psychological aspects are directly affected by physical status and performance. patients can feel depressed or anxious facing difficulties during recovery time. the aim of this study was to correlate patients' perceptions of his health status and his clinical performance measured after icu discharge. patients and methods: this is a prospective pilot study of an icu follow-up clinic conducted in a single center from january to july . this clinic is multidisciplinary and includes two visits at and months after icu discharge. patients with more than days of icu los were eligible. all patients at and -m visit were evaluated with sf- , mwt, mrc and time-up-and-go test. we conducted an analysis comparing clinical performance data and qualitative data between and months after icu discharge. the investigation included patients who had at least days of icu length of stay. patients attended the consult at -m and patients attended the consult both times. the median age (iqr) was ( - ) and % were men. %, % and % of patients had medical, scheduled surgical and emergency surgical admission causes respectively, with median (iqr) saps iii score ( - ). %, % and % of patients had sepsis, delirium and mechanical ventilation as a support. the physical status was progressively increased overtime likewise the physical capacity assessed by sf- score with p-value . between and -m. however, no significant difference between the subjective dimension of sf- , which analyses the perception of the patient about his physical capacity, assessed at -m and at -m was demonstrated (p . ). in this pilot-phase of following a cohort of critically ill patients, the natural physical improvement does not seem to change the patient's perception of their performances. this paradigm rouses a different perspective that should take into account when setting up rehabilitation programs. compliance with ethics regulations: yes. post-traumatic stress disorder after discharge from an acute medical unit basma lahmer , naoufel madani , , jihane belayachi , , redouane abouqal rationale: post-traumatic stress disorder (ptsd) occurs after exposure to a traumatic event and comprises of symptoms of repeated re-experiencing of the said event, avoidance of reminders, emotional numbing and persistent hyperarousal. in individuals exposed to "medical stress", various studies found evidence of ptsd occurring after the onset, diagnosis, or treatment of physical illness. our study aims to determine ptsd's risk factors in patients of an acute medical unit (amu) after their discharge. patients and methods: it was a prospective, analytical study conducted over a period of months at an acute medical unit. we collected sociodemographic and clinical data, patients' medical history, and evaluated the symptoms of anxiety and depression during their stay using the hospital anxiety and depression scale (hads). the prevalence of severe ptsd symptoms was assessed with the impact of events scale-revised (ies-r) at weeks and months using a cutoff of . associations between ptsd as evaluated by ies-r at months and patients' characteristics, including hads scores at admission were investigated using unadjusted linear regression, for univariate and multivariate regression analysis. statistical analyses were carried out using spss for windows (spss, inc., chicago, il, usa). we included patients in our study with a mean age of . ± . . in our population, . % of patients scored higher than a ies-r cutoff at weeks compared to . % at months. the mean hads-anxiety score is . ± and that of the hads-depression score is . ± . . on one hand, higher hads-anxiety score during the stay in the amu was linked to higher ies-r scores at months β: rationale: objective of critical care includes restoration of functional capacities. prompt identification of muscle acquired weakness (icu-aw) is crucial to target efficient rehabilitation. in published literature, data of quadriceps strength (qs) cannot be compared because of insufficient standardization of measurement protocols. we recently validated a highly standardized protocol of qs measurement. in order to build basic and comparable knowledge and to identify the weakest patients, this study aimed to describe qs of critically ill (ci) patients during their short-term evolution, and to compare them to surgical (s) and healthy (h) subjects. patients and methods: this observational study included ci patients who spent at least days in icu, patients scheduled for elective colorectal surgery (s) and young healthy volunteers (h). maximal isometric qs was assessed using a handheld dynamometer (microfet ® ) and expressed in newton/kg (n/kg). dominant leg was tested in supine position using a highly standardized procedure. ci and s patients were tested at t (as soon as collaborative in icu) and month after discharge (m rationale: the post intensive care syndrome (pics) gathers various disabilities, associated with a substantial healthcare use. however, patients' comorbidities and active medical conditions prior to intensive care unit (icu) admission may partly drive healthcare use after icu discharge. to delineate the relative contribution of critical illness and pics per se to post-critical illness increased healthcare use, as opposed to pre-existing comorbidities, we conducted a population-based evaluation of patients' healthcare use trajectories. patients and methods: using discharge databases in a . -million-people region in france, we retrieved, over three years, all adult patients admitted in icu for septic shock or acute respiratory distress syndrome (ards), intubated at least days and discharged alive from hospital. healthcare use (days spent in healthcare facilities) was analyzed two years before and two years after icu admission. healthcare trajectories were next explored at individual level: patients were assembled according to their individual pre-icu healthcare use trajectory by clusterization with the k-means method. results: eight-hundred and eighty-two ( ) patients were included. median duration of mechanical ventilation was days (interquartile ranges [iqr] ; ), mean saps was , and median hospital length of stay was days (iqr ; ). prior to icu admission, we observed, at the scale of the whole study population, a progressive increase in healthcare use. however, clusterization of individual according to pre-icu healthcare trajectories identified patients with elevated and increasing healthcare use (n = ), and two main groups with low (n = ) or no (n = ) pre-icu healthcare use. patients with high healthcare use had significantly more comorbidities than those with low healthcare use. in icu, however, saps , duration of mechanical ventilation and length of stay were not different across the groups. interestingly, analysis of post-icu healthcare trajectories for each group revealed that patients with low or no pre-icu healthcare (which represented % of the population) switched to a persistent and elevated healthcare use during the two years post-icu. conclusion: for % of ards/septic shock survivors, critical illness appears to have a pivotal role in healthcare trajectories, with a switch from a low and stable healthcare use prior to icu, to a sustained higher healthcare recourse two-years after icu discharge. this underpins the hypothesis of long-term critical illness and pics-related quantifiable consequences in healthcare use, measurable at a population level. compliance with ethics regulations: yes. ( ) to describe the pre-hospital grading protocol developed by the northern french alps emergency network (trenau) for children, ( ) to evaluate its quality to detect the most severe trauma patients and ( ) to assess the accuracy of this procedure to perform an adequate triage. patients and methods: our regional trauma system included hospitals categorized as level i, ii or iii pediatric trauma centers. eachpatient was graded a, b or c by an emergency physician, according to the seriousness of their injuries at presentation on scene. the triage was performed according to this grading and the categorization of centers. this study is a registry analysis of an -year period ( to ). results: a total of children (mean age years, % were boys) with severe trauma were included in the cohort. fifty-seven, % and % of patients were admitted to a level i, ii and iii, respectively. road accident was the main mechanism of injury ( % of patients). thirtysix percent of patients had a severe trauma, defined as an injury severity score (iss) higher than . one quarter of patients had at least severe lesions and one-third of patients had a trauma brain injury. the pre-hospital gradation was closely related with injury severity score (iss) and intra-hospital mortality rate. the triage protocol had a sensitivity of % and a specificity of % to predict adequate admission of patients with iss more than . using a specific trauma score (including occurrence of death, an admission in intensive care unit and the need for urgent surgery), sensitivity and specificity reached and %, respectively. fourty-six percent of patients were not graded at the scene (non-graded group). undertriage rate was significantly reduced in the graded group compared with the non-graded group, ( % versus %), without significant modification of the overtriage rate ( % versus %). overall, mortality at discharge from hospital was %, but % in grade a patients. conclusion: implementation of a regional pediatric trauma system with a specific pre-hospital triage procedure was effective in detecting severe pediatric trauma patients and in lowering the rate of prehospital undertriage. compliance with ethics regulations: yes. rationale: critically ill children suffer from pathophysiological changes, leading to large between-subject variability in drug clearance. since piperacillin is eliminated mainly via the kidney, changes in renal function go along with a modified elimination, and possible subtherapeutic or toxic drug concentrations. we aimed to determine the most accurate glomerular filtration rate (gfr) estimation formula for assessing piperacillin clearance in critically-ill children. patients and methods: all children hospitalized in pediatric intensive care unit and receiving piperacillin were included. piperacillin was quantified by high performance liquid chromatography. pharmacokinetics were described using the non-linear mixed effect modeling software monolix. in the initial pharmacokinetics model, gfr was estimated according to the schwartz formula. in the study, gfr was estimated with additional formulas, developed with plasma creatinine and/or cystatin c. biases, precisions, spearman's rank correlation coefficient and normalized prediction distribution error (npde) were used to assess the models. results: we included children with a median (range) postnatal age of . ( . - ) years, body weight of . ( . - ) kg and estimated gfr according to the schwartz formula of . ( - ) ml min- . . m . piperacillin concentrations were best predicted with the model using the creatinine clearance. the correlations were most accurate: r = . between the population-predicted and the observed concentrations, r = . and r = . for the npde versus population-predicted concentrations and time, respectively. concerning the individual predicted concentrations, bias and precision were respectively − . mg l − and . mg l − . gfr estimations based on serum creatinine were higher than those based on cystatin c (p = . ). conclusion: in summary, the -h creatinine clearance is the best predictor of piperacillin clearance and this could be investigated for drugs with renal elimination. as a whole, literature and our findings strongly suggest using creatinine clearance to also estimate gfr in critically ill children. the gap between the gfr estimations is large depending on the formulas, with higher estimations with equations based on serum creatinine. compliance with ethics regulations: yes. rationale: acute pancreatitis (ap) incidence have increased dramatically over the past years. new guidelines in were recently published in order to standardize the definition and management of ap. the aim of this study is to describe the management of children that were diagnosed with ap from the pediatric intensive care unit (picu) in two french hospitals. patients and methods: this retrospective cohort study included children aged under years old, who were admitted to the picu of robert-debré hospital and trousseau from to with a discharge diagnosis of ap. data collected included management, severity and outcomes. we have also obtained data on clinical, biological and radiological presentation. results: sixty patients were included, the median age was years ( - ) and % had a co-morbidity mainly hematologic ( / ). most of the ap were moderate ( %) or severe ( %). hemodynamic failure was the main reason for picu admission requiring a median fluid resuscitation ml/kg complemented by a median intravenous fluid therapy of ml/kg/h ( - ) during the first h. twenty patients ( %) required mechanical ventilation. fasting has been instituted in patients ( %) for a median of days ( - ), whereas patients ( %) received parenteral nutrition, only patients ( %) received enteral nutrition. antibiotic therapy was given to patients ( %) including % for curative therapy. the median length of stay in picu was days ( ) ( ) ( ) ( ) ( ) . the mortality rate was %. conclusion: this is the first french study which precisely described the management of patients with ap in picu. it highlighted the differences withthe new international guidelines. this study could improve the management of pa in picu and open research perspectives. compliance with ethics regulations: yes. rationale: apheresis and therapeutic plasma exchange (tpe) for children diseases has been poorly investigated in mostly small-uncontrolled studies. the purpose of this study is to describe indications and safety of tpe in children. patients and methods: in this single center and retrospective study, we included patients who underwent tpe with an age < years old in the pediatric center of necker-enfants-malades hospital from january to december . data were retrospectively collected in an electronic case report form via a web-based data collection system. results: patients with a median age of . years [range . ; . ] were selected. they achieved a total number of procedures. indications were antibody-mediated rejection (n = ; %) or desensitization therapy (n = ; %) for solid organ or hematopoietic transplantations; microangiopathy (n = ; %); renal diseases (n = ; %) and pediatric inflammatory diseases (n = ; %); or hyperviscosity syndrome (n = ; %). each patient had an average of procedures for the first session [range ; ] with a median volume of ml [range ; ml] corresponding to a median (rang) total plasma volume (tpv) equivalent of . l/m [ . - . ]. within days since the beginning of sessions, patients ( %) present a total of adverse events (aes) potentially related to tpe. there was a median (range) of aes/patients [ - ]. there was no association between aes and diseases, severity of patients, venous access, plasma substitute and body weight. few of aes (n = for patients) were potentially life-threatening and concerned mostly critically ill children. allergic reactions represented only aes for patients (grade i n = ; grade ii n = ; grade iii n = ). at the months endpoint, ( %) patients died and ( %) patients had severe persistent disease. no death had been related to the tpe process. we describe one of the largest retrospective pediatric cohort updated to the last international recommendations. tpe in children is performed for specific and potentially refractory disease. it is feasible without a major risk of life threatening adverse events. compliance with ethics regulations: yes. yacine benhocine university hospital nedir mohamed, tizi-ouzou, algeria correspondence: yacine benhocine (yacine @yahoo.fr) ann. intensive care , (suppl ):f- rationale: although analysis of literature data shows that implantable chamber catheters (iccs) are less at risk of infectious complications than other central venous catheters, these complications can be serious, which may differ from ongoing treatments such as chemotherapy, and may lead to the removal of the implanted device. the literature on preventing these infections is quite disparate, as practices. purpose: to evaluate the incidence of infections, to identify responsible germs and to measure the impact of preventive measures. patients and methods: prospective, descriptive, mono-centric study, from january to january . all patients under the age of who have benefited from an implantable chamber catheter, whose insertion procedure is as follows: local anesthesia, surgical asepsis (polyvidone iodine) in an operating room, double disinfection, no antibiotic prophylaxis, routes used: subclavian ( %), internal jugular ( %) by anatomic registration. the main criteria of judgment are: the incidence of local and general infections, their time of onset, responsible microorganisms. statistical analysis used the statistical package for the social sciences software. results: patients were included, the average incidence density of early infection is . / day-catheters. the time of onset of infection is essentially between the nd and rd week post-exposure, of which % is general infection. ablation involved % of infected catheters. the causative organisms are mainly gram-positive cocci ( . %), gram-negative bacilli are less involved ( . %), with a significant number of candida infections ( %). discussion: higher incidence of data from the literature. to remedy this requires the implementation of additional hygiene measures: antiseptic showers preoperatively, chlorhexidine??, and practice changes: echo guidance, antibiotic prophylaxis or locks? second generation catheters? our practices are disparate especially since the recommendations specifically concerning the prevention of infectious risk associated with internationally published iccs are rare. conclusion: at the end of this work, our perspectives are to: update the procedure, highlight risk factors on which it is possible to act, the adhesion of the different staff to the protocols. compliance with ethics regulations: yes. rationale: the sepsis and septic shock pediatric guidelines advise to treat patients using care bundles. in the first hour, the «resuscitation bundle» contains an appropriate fluid resuscitation, a broad-spectrum antibiotics administration after blood cultures, and initiation of inotrope if needed. the objectives were to evaluate the resuscitation bundle compliance in a cohort of septic children with cardiovascular dysfunction, and to analyze the effect on severity and outcome in pediatric intensive care unit (picu). patients and methods: retrospective analysis of the diabact iii study. this study analyzed the care course of children with severe community-acquired bacterial infection, hospitalized in picus in france's west departments, between august and january . children with severe sepsis and cardiovascular dysfunction were retrospectively included. results: we included children of whom ( . %) had compliant bundled care. the severity scores at picu's admission were similar between groups (p = . for the prism score and . for the pelod ). there was the same proportion of fluid-refractory shock (p = . ), mechanical ventilation (p = . ), neurological dysfunction (p = . ) and cardiac arrest (p = . ). in the «resuscitation bundle compliant» group, . % died versus . % in the other group (p = . ). we highlighted a severity bias: the sickest patients were more likely to receive compliant bundled care. conclusion: in our cohort, the resuscitation bundle's compliance was low. we did not show some effect on morbidity nor mortality. however, this study helps understand the factors associated with resuscitation bundle's compliance. rationale: nosocomial infections with extended-spectrum β-lactamase (esbl) producing gram-negative bacilli (gnb) are an important cause of hospital morbidity and mortality. the objective of this study was to determine the incidence and risk factors of nosocomial esbl-producing gnb infections in a paediatric intensive care unit (picu). patients and methods: a prospective surveillance study was performed from january through march in a picu. all patients hospitalized for more than h were included. centers for disease control and prevention criteria were applied for the diagnosis of nosocomial infection. results: during the study period, patients (median age: ± days) were included. the average length of stay was ± days with a total of , days of hospitalization. newborns accounted for . % of patients. sixty-two per cent of patients were colonized with multi drug resistant gram-negative rods, on admission or during their stay in the picu. one hundred and nineteen bacterial infectious episodes were registered ( . / patient days). one hundred infectious episodes were caused by a gnb and ( . %) by esbls producing gnb with an incidence of . / patient days (bloodstream infections: episodes, ventilator acquired pneumonia: episodes). esbls producing gnb infection had a specific incidence of . per catheter-days, and . per mechanical ventilation-days. fifty-nine percent of patients infected with esbls producing gnb had a prior digestive colonization with a multidrug-resistant gnb. forty-one episodes ( %) occurred in patients with central venous catheters. klebsiella pneumoniae was the most frequently isolated bacteria ( . %). mortality in the esbls producing gnb group was high ( . %). associated factors of nosocomial esbls producing gnb infection were mechanical vrntilation (p < . ), central venous catheterization (p < . ) and colonization with multiple drug-resistant gram-negative bacteria (p < . ). conclusion: nosocomial esbl-producing gnb infection had an incidence of . per patient days in our unit and seems to increase the mortality rate. factors associated with this infection were identified. marie lemerle , aline schmidt , valérie thepot-seegers , achille kouatchet , valérie moal , mélina raimbault , corentin orvain , jean-francois augusto , julien demiselle chu angers, médecine intensive réanimation, angers, france; chu angers, maladie du sang, angers, france; chu angers-ico, angers, france; chu angers, pharmacie, angers, france; chu angers, labora-toire de biochimie, angers, france; chu angers, néphrologie dialyse transplantation, angers, france correspondence: marie lemerle (marielemerle@yahoo.fr) ann. intensive care , (suppl ):f- rationale: acute kidney injury (aki) is associated with high morbidity and mortality in the setting of tumor lysis syndrome (tls). thus, strategies aimed at preventing aki occurrence represent a major goal to improve prognosis of patients with tls. the role of hyperphosphatemia as a risk factor of tls has been poorly analyzed. the aim of this study was to study the association between hyperphosphatemia and aki, and to determine whether a cut-off value of phosphatemia or phosphatemia's variation was associated with aki development during tls. patients and methods: in this retrospective and monocentric study, we included all patients with tls and whithout aki at admission, admitted to hematology, nephrology and intensive care units of the university hospital of angers between / / and / / . results: one hundred and thirty tls episodes were identified in patients. aki developed during episodes of tls ( %). hospital mortality was much higher in aki patients ( . % versus . %, p = . ). phosphate maximal values ( . ± . versus . ± . ) and ldh maximal values ( . ± . versus . ± . ) were higher in tls with aki, before aki occurrence (p = . and p = . , respectively). we found no association between the other biological parameters of tls and aki (serum calcium, uric acid and potassium). after adjustment for cofounders, there was a strong association between a rise in phosphate level of . mmol/l (hr . ic % [ . - . ], p < . ), exposure to platinum salts (hr . ic % [ . - . ], p = . ) and increasing maximal ldh value (hr per ui/l increase . ic % [ . - . ], p = . ) with aki. conclusion: this study highlights the utmost importance of serum phosphate in the setting of tls: phosphate is an early relevant biomarker for the risk of aki development. further studies are needed to assess whether aggressive prophylactic treatment to control serum phosphate concentration, such as renal replacement therapy before aki onset, constitutes a valuable approach. compliance with ethics regulations: yes. retrospective cohort of patients admitted to the medical icu of university affiliated hospital after carts treatment between august and august . results: of the patients treated by carts in the haematology department, ( %) were subsequently admitted to icu. median age was [ . - . ] years, and ( . %) were female. carts were indicated for r/r lymphoma. the median time between carts injection and icu admission was [ . - . ] days. all patients had cytokine release syndrome (crs), and ( . %) developed car-related encephalopathy syndrome (cres). median sofa score and saps were [ - . ] and [ . - . ], respectively. four ( . %) patients had hypotension treated by fluid bolus (n = ) or vasopressors (n = ), and ( . %) had acuterespiratory failure requiring oxygen therapy (n = ) or mechanical ventilation (n = ). six ( . %) patients had neurological symptoms (impaired consciousness n = , confusion n = , transient aphasia n = ), of whom one developed refractory convulsive status epilepticus afterwards. all patients received broad spectrum antibiotics, of whom ( . %) had documented infections. six ( . %) patients received interleukin- inhibitor (single dose n = , multiple doses n = ), and ( . %) received intravenous dexamethasone. one patient died in the icu from septic shock. median icu and hospital length of stays were [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and [ . - . ] days, respectively. two ( . %) patients died from relapsing malignancy before hospital discharge. three months after icu admission, four ( . %) patients were alive in complete remission. conclusion: more than % of patients treated with carts required icu admission for the management of a crs or a cres. early icu admission, close collaboration between haematologists and intensivists, and prompt administration of appropriate therapy (il- inhibitor and/or dexamethasone) and supportive care resulted in a good prognosis. compliance with ethics regulations: yes. rationale: tisagenlecleucel (ctl ) is a chimeric antigen receptor t cell therapy that reprograms autologous t cells to target cd + leukemia cells, approved in the us since august and in the eu since august for children and young adult (< years old) with relapsed/refractory b-cell acute lymphoblastic leukemia (b-all). this study reports the experience of picu management of ctl toxicity in patients treated in robert-debré university hospitals. patients and methods: all patients (age < years old) treated by tisagenlecleucel infusions between march , and september , , included in sponsored-clinical trials or treated within the french compassionate program or with the commercial product, were retrospectively analyzed. results: twenty-four patients were infused and patients ( %) were managed in picu for stays. ( stays: n = and stays: n = ). median age at picu admission was . years old [ . ; . ] with a median delay after car-t cells infusions of days [ . ; ] . the median length of stay in picu was days [ . ; ] with a max at days. cytokine release syndrome (crs) was the main indication of picu hospitalization ( . %, n = ) with grade (n = ) and grade (n = ) according to american society for transplantation and cellular therapy (astct) consensus grading system and treated by corticosteroid (n = . ) and tocilizumab (n = , only one infusion). norepinephrine was the only vasopressor used. the median vaso-inotrope score (vis) for grade was [ . ; . ] with a maximum at . neurologic toxicity was observed in patients with a grade (status epilepticus) and grade (focal edema on neuroimaging with depressed level of consciousness) according to immune effector cell-associated neurotoxicity syndrome (icans) grading system from astct consensus. the status epilepticus was managed with anti-epileptic drugs without mechanical ventilation. the focal edema was related to hhv and toxoplasmosis encephalitis. evolution was positive with foscavir and ganciclovir and days of mechanical ventilation. one patient was hospitalized for septic shock secondary to gram-negative central line bloodstream infection in aplasia, with a vis score at . evolution was favorable with antibiotics and central line removal. no death in picu from severe tisagenlecleucel toxicity was observed since the beginning of the car-t cells program. conclusion: toxicity profile of tisagenlecleucel required frequent and early picu hospitalization after infusions for severe crs and icans management. compliance with ethics regulations: yes. rationale: car-t cell (chimeric antigen receptor t) therapy is a promising treatment in refractory acute lymphoid leukemia (all) and diffuse large b cell lymphoma (dlbcl). the main complication consists in a cytokine release syndrome (crs) leading to an inflammatory state that can be very severe with life-threatening organ failure. neurological toxicity is also reported. we aim to describe car-t cells-related complications in icu patients. patients and methods: this is a single-center prospective study conducted between july and august . all the patients who have received car-t cells and who required icu admission were included. crs grading was defined according to the most recent classification of the asbmt and neurological toxicity was assessed with the cartox scale. each admission is considered independent and therefore corresponds to one patient. results: admissions, representing patients ( men and women), were considered. the median age was years . twothirds of the patients have been diagnosed with dlbcl (n = , %) and one-third with all (n = , %), months [ - ] ago. they had received lines [ ] [ ] of chemotherapy and had a high tumor burden ( % of lymphomas classified stage iv). the majority of the patients was admitted because of hemodynamic failure (n = , %) or respiratory failure (n = , %), days [ ] [ ] [ ] [ ] [ ] after car-t cells infusion. sofa at admission was [ ] [ ] [ ] [ ] [ ] . all the patients presented at least one complication ( figure) , the most common being crs (n = , %) with a median grade of [ ] [ ] . neurological toxicity was reported in ( %) patients (worst grade at [ ] [ ] [ ] ). documented bacterial infection involved % of the patients and consisted in catheter-related infections for half of the cases. in the icu patients were managed with fluid resuscitation (n = , %) during the first day, vasopressors (n = , %) and broad spectrum antibiotics ( %). a single patient required mechanical ventilation and two patients underwent dialysis. tocilizumab (anti-il receptor) was given to patients ( % of crs) in a median time of . h [ . - . ] after icu admission. patients ( %) received corticosteroids. the median icu length of stay was . days [ ] [ ] [ ] [ ] . patients ( %) died in the icu and hospital mortality was %. the -fluorouracil ( -fu)-induced hyperammonemic encephalopathy is a rare but serious -fu adverse drug reaction, which could require the admission of patients in intensive care unit (icu). given the paucity of data regarding this -fu adverse drug reaction, we performed a retrospective national survey from the french pharmacovigilance database to better characterize -fu-induced hyperammonemic encephalopathy and its management. patients and methods: since the inception of the french pharmacovigilance database, we identified all patients that experienced -fu-induced encephalopathy. variables regarding epidemiology, characteristics, management and prognosis of these patients were collected and analyzed. results: from from to years-old, % of women) were included. overall mortality was % (n = ) and % (n = ) of patients were admitted in icu. the -fu-induced hyperammonemic encephalopathy started [ ] [ ] [ ] [ ] days after the onset of -fu infusion. the most common neurological disorders were consciousness impairment, confusion and seizures. abnormalities in ct scan, mri, electroencephalogram and lumbar puncture were found in %, %, % and % of the whole population respectively, similar in icu and non-icu patients. ammonemia was dosed in % of the whole population and in % of icu patients. hyperammonemia tended to be higher in icu than in non-icu patients ( [ - ] vs. [ - ] µmol/l, respectively, p = ns) and in patients with the lowest glasgow outcome scale, but was not different between survivors and non-survivors. among icu patients, % required mechanical ventilation and % anti-epileptic drugs administration. besides -fu discontinuation, lactulose intake, renal replacement therapy or ammonium chelators were used to decrease hyperammonemia in %, % and % of patients respectively. a complete neurological recovery was observed in up to % of icu and non-icu patients within a delay of [ - ] days. a dihydropyrimidine deshydrogenase (dpd) deficiency was found in % of tested patients. a -fu rechallenge was considered in % (n = ) of patients with complete neurological recovery, including a patient with a partial dpd deficiency, within a delay of [ - ] days after recovery. a -fu-induced hyperammonemic encephalopathy relapse was observed in % of patients with -fu rechallenge. no relapse was observed when -fu rechallenge was performed with a decreased -fu dosage. conclusion: we report the first national survey and the largest cohort of patients with -fu-induced hyperammonemic encephalopathy so far. this serious -fu adverse drug reaction must be known by intensivists, since more than half of patients are admitted in icu and specific treatments are available. compliance with ethics regulations: yes. immune related adverse events: a retrospective look into the future of oncology in the intensive care unit adrien joseph , annabelle stoclin , antoine vieillard-baron , guillaume geri , jean-marie michot rationale: immune checkpoint inhibitors (ici) represent a paradigmatic shift in oncology. with their new position as a mainstay in cancer treatment, new toxicities called immune related adverse events (iraes) have emerged. patients and methods: retrospective study including patients admitted in the icu within days after treatment with an ici in french hospitals. patients were classified into groups according to the reason for admission: irae, intercurrent adverse event (intae) or event related to tumor progression (tumprog). results: patients were admitted during the course of an ici treatment, including irae, intae and tumprog, with a significant increase between (n = ) and (n = patients, p for trend < . ). irae included pneumonitis, colitis, diabetes complications, hypophysitis, nephritis, myocarditis and cardiac disorders, hepatitis or allergic reaction and meningitis. the immune related nature of the complication was known before admission in only ( %) cases. mean age was (± ) years and % had a performance status of - . primary tumors were melanomas ( , %), non-small cell lung cancers ( , %) , urothelial carcinomas ( , %) and hodgkin lymphomas ( , %) . ici at the time of admission included anti-ctla ( , %), anti-pd /pdl ( , %) and anti-ctla /anti-pd combination in ( %) patients. mean duration of stay in the icu was . (± ) days. three patients required vasopressor therapy alone, with mechanical ventilation and one with extracorporeal membrane oxygenation. three patients required non-invasive ventilation and renal replacement therapy alone. six required only endocrine or electrolytic equilibration and others did not receive any form of organ support. icu mortality was %. compared with other admissions, anti-ctla or anti-ctla /anti-pd combination treatments were associated with irae diagnosis (or = . [ . - . ] , p = . for anti-ctla and . [ . - . ] for anti-ctla /anti-pd , p = . ) and so was the diagnosis of melanoma ( . [ . - . ] , p = . ). there was no difference in terms of icu and post-icu survival between irae (median post-icu survival months [ -na]), intae ( . [ . -na]) and ). six patients admitted for an irae were rechallenged with the same ici after icu discharge and achieved complete response. conclusion: we conducted the first study describing patients admitted in the icu for iraes. their specific and heterogeneous profile, along with the expected increase in the number of admissions, underlines the need for an in-depth knowledge for icu physicians in order to take part in the multidisciplinary care required by these patients. compliance with ethics regulations: yes. rationale: patients with advanced-stage non-small-cell lung cancer have high mortality rates in the intensive care unit (icu). in this context, acute respiratory failure due to cancer involvement is the worst situation. in the last two decades, targeted therapies have changed the prognostic of patients with lung cancer outside the icu. unlike cytotoxic chemotherapy, the fast efficacy of targeted therapies led some intensivists to use them as rescue therapy for icu patients. we sought to investigate the outcomes of patients with lung cancer involvement responsible for acute respiratory failure and who received tyrosine kinase inhibitor during icu stay. patients and methods: we performed a national multicentric retrospective study with the participation of the grrroh (groupe de recherche en réanimation respiratoire en onco-hématologie). all patients with non-small-cell lung cancer admitted to the icu for acute respiratory failure between and were included in the study if a tyrosine kinase inhibitor was initiated during icu stay. cases were identified using hospital-pharmacies records. we collected demographic and clinical data in icu charts. vital status was assessed at the time of study completion (august ). the primary outcome was overall survival days after icu admission. results: twenty-nine patients (age: ± years old) admitted to a total of icus throughout france were included. seventeen patients ( %) were nonsmoker. the most frequent histological type was adenocarcinoma (n = , %) and a majority had metastatic cancer (n = , %). epithelial growth factor receptor mutation was the most common oncologic driver identified (n = , %). during the icu stay, ( %) patients required invasive mechanical ventilation, ( %) catecholamine infusion, ( %) renal replacement therapy and one ( %) extracorporeal membrane oxygenation. in addition to tyrosine kinase inhibitor, ( %) patients received steroids (beyond . mg/kg/day) and ( %) cytotoxic chemotherapy during icu stay. seventeen patients ( %) were discharged alive from icu and ( %) were still alive after days (see kaplan-meier curve figure) . moreover, patients ( %) were alive one year after icu discharge. conclusion: despite a small sample size this study showed that, in the context of lung cancer involvement responsible for acute respiratory failure, the use of tyrosine kinase inhibitor should not be refrained in patients with severe condition in icu. compliance with ethics regulations: yes. rationale: acute respiratory failure is the leading reason for intensive care unit (icu) admission in immunocompromised patients and the need for invasive mechanical ventilation has become a major clinical end-point in randomized controlled trials (rct). however, data are lacking on whether intubation is an objective criteria that is used unbiasedly across centers. this study explores how this outcome varies across icus. patients and methods: hierarchical models and permutation procedures for testing multiple random effects were applied on both data from observational cohort (the trial-oh study: patients, icus) and randomized controlled trial (the high trial: patients, icus) to characterize icu variation in intubation risk across centers. results: the crude intubation rate varied across icus from % to % in the observational cohort and from to % in the rct. this center effect on the mean icu intubation rate was statistically significant, even after adjustment on individual patient characteristics (observational cohort: p-value = . , median or . [ . - . ]; rct: p-value: . , median or . [ . - . ]). two icu-level characteristics were associated with intubation risk (the annual rate of intubation procedure per center and the time from respiratory symptoms to icu admission) and could partly explain this center effect. in the rct that controlled for the use of high-flow oxygen therapy, we did not find significant variation in the effect of oxygenation strategy on intubation risk across centers, despite a significant variation in the need for invasive mechanical ventilation. conclusion: invasive mechanical ventilation has become an important endpoint in immunocompromised patients with acute respiratory failure. however, we found significant variation in intubation risk across icu in both an observational cohort and a randomized controlled trial. our results highlight the need to take into account center effect in analysis because it could be an important confounder. reasons for heterogeneity are various (case-mix differences, center practices). this gives opportunities to future improvement in care management and study design. compliance with ethics regulations: yes. rationale: influenza virus (iv) infection is a major cause of ards that has been the focus of attention since the pandemic h n (h n pdm ) iv. although iv-mediated damage of the airway has beenextensively studied emphasizing specificity compared to other causes of ards, the impact of iv infection on the prognosis of ards patients, compared to the other causes of ards, has been few assessed. patients and methods: systematic detection of iv in times of epidemic using rt-pcr in respiratory specimen is routine practice in our icu along with prospective data collection of patients admitted to our icu for ards with pao /fio ratio ≤ mmhg. all patients received lung-protective ventilation, the sequential organ failure assessment (sofa) score was calculated on the first days of mechanical ventilation. the primary endpoint compared the -day survival from the diagnosis of ards between patients with and without iv infection. results: from october, to may, , patients (pts) [median saps ii score = ( - ); age years ( - ); pao / fio ≤ mmhg, n = ( %)] were admitted to our icu for ards with pao /fio ratio ≤ mm/hg, including pts ( %) with iv infection (h n pdm iv a, n = ; h n a virus, n = ; b virus, n = ; associated bacteria, n = ). other main causes of ards were bacterial pneumonia without iv ( %), aspiration ( %), non-pulmonary sepsis ( %). ( %) received prone positioning, and ( %) extra-corporeal membrane oxygenation. the overall mortality rate at day- for the entire population was % ( pts ( %) with iv infection versus pts ( %) without iv infection, p = . ). kaplan-meier survival curves showed that survival was significantly higher in patients with iv infection than in those without iv infection. iv infection remained independently associated with a better prognosis at day- when entered as dichotomous variable (iv infection, yes/no) (adjusted hazard ratio (hr) = . , % ci . - . , p = . ) and when iv infection only was distinguished from other causes of ards including mixed infection iv plus bacteria (adjusted hr = . , % ci . - . , p = . ). of note, within the first days of mechanical ventilation, non-pulmonary sofa scores were significantly lower in iv patients although similar pulmonary sofa scores. conclusion: our results suggest that patients with iv related ards have less severe non-pulmonary organ dysfunctions than those with ards from other and a lower mortality at day- despite similar ards severity. compliance with ethics regulations: yes. rationale: acute respiratory distress syndrome (ards) remains frequent in intensive care unit (icu) with % to % mortality. according to joint theater trauma system, ards occurs among % of war casualties: direct lung trauma, blast lesions, burn, massive transfusion and systemic inflammatory response syndrome lead to ards development. however, there is no data reporting ards among french evacuated casualties from forward environment. our study's aim is to describe ards incidence and its severity concerning medical evacuations from war theater. patients and methods: this is an observational retrospective multicentric study analyzing all evacuated patient from war theater and admitted in icu. all patients developing ards according to berlin definition have been included. study has been approved by local ethic committee. primary study endpoint was ards developing. second study endpoints were ards severity, duration of invasive ventilation, ards treatments, icu length of stay and mortality. results: patients have been admitted in icu between and . have been excluded. a total of patients have been analyzed. % (n = ) were military aged ( - ) years. % (n = ) developed ards. we found % (n = ) war casualties, % (n = ) trauma not related to war and % (n = ) medical patients. among severe trauma, median iss was ( - ), ais thorax ( ) ( ) ( ) , and % benefited from surgery on forward environment and % (n = ) received massive transfusion. % (n = ) suffered from mild ards, % (n = ) moderate ards and % (n = ) severe ards. evacuation time was ( - ) h. at admission in icu, pao /fio ratio was ( - ) (fig. ). all patients were intubated. ards treatments used were curarization ( %, n = ), prone position ( %, n = ), inhaled nitric oxide (noi) ( %, n = ), almitrine ( %, n = ) and extracorporeal life support (ecls) ( %, n = ). invasive ventilation duration was ( - ) days, length of stay ( - ) days, and -month mortality % (n = ). conclusion: according to our study, ards among french evacuated patients from war theaters remains frequent: it occurs on % among icu admitted patients. % suffer from severe ards with % global mortality. those datas are consistent with us studies. also, we wonder if we must adapt our treatment capacities on forward environment for the most severe patients. in us army, a specialized team (acute lung rescue team) is trained to care the most hypoxemic war casualties with more treatment options as noi, ecls. compliance with ethics regulations: yes. rationale : we recently reported that septic shock patients with pneumonia exhibit a high risk of icu-acquired pneumonia, suggesting that a primary pulmonary insult may drive profound alterations in lung defence towards secondary infections ( ) . given their importance in lung immune surveillance, alveolar macrophages (am) are likely to play a pivotal role in this setting. the objective of this experimental study is to address the impact of primary pulmonary or non-pulmonary infectious insults on lung immunity. patients and methods: we established relevant double-hit experimental models that mimic common clinical situations. c bl/ j mice were first subjected either to polymicrobial peritonitis induced by caecal ligation and puncture (clp), or to bacterial pneumonia induced by intra-tracheal instillation of staphylococcus aureus or escherichia coli. respective control mice were subjected to sham laparotomy or intratracheal instillation of phosphate-buffered saline. seven days later, mice that survived the primary insult were subjected to intra-tracheal instillation of pseudomonas aeruginosa (pao strain). we assessed survival and pulmonary bacterial clearance of post-septic animals subjected to p. aeruginosa pneumonia, as well as the distribution and functional changes in alveolar macrophages. results: when compared to sham-operated mice, post-clp animals exhibited increased susceptibility to secondary p. aeruginosa pneumonia as demonstrated by defective lung bacterial clearance and increased mortality rate ( % vs. %, p < . ). in contrast, all postpneumonia mice survived and even exhibited improved bacterial clearance as compared to their control counterparts. when addressing whole-lung immune cell distribution at the time of second hit (day ), amounts of am were decreased in post-clp mice while preserved or even increased in post-pneumonia mice. antigen-presenting functions of am appeared similar in all conditions. percentages of apoptotic (annexinv + ) and necrotic ( -aad + ) am were comparable at day and day after the first hit. interestingly, both ly c high and ly c low monocytes were sustainably increased in the lungs of post-clp mice, while only transiently expanded following pneumonia, suggesting that differences in am counts could be related to modulated turnover from precursor monocytes. conclusion: using clinically relevant double-hit experimental models, a primary pulmonary infection conferred resistance to secondary bacterial pneumonia. ongoing investigations are aimed at addressing the antibacterial am functions, as well as the turnover-driving mechanisms.compliance with ethics regulations: yes. rationale: little is known on the role of exit-site signs in predicting intravascular catheter infections. the current study aimed to describe the association between local signs at the exit-site and catheter-related bloodstream infection (crbsi), which factors substantially influenced local signs and which clinical conditions may predict crb-sis if inflammation at insertion site is present. patients and methods: we used individual data from multicenter randomized-controlled trials in intensive care units (icus) that evaluated various prevention strategies regarding colonization and crbsi in central venous and arterial catheters. we used univariate and multivariate logistic regression stratifying by center in order to identify variables associated with redness, pain, non-purulent discharge, purulent discharge and ≥ local sign and subsequently evaluate the association between crbsi and local signs. moreover, weevaluated the role of thedifferent local signs for developing crbsi in subgroups of clinically relevant conditions. results: a total of patients, , catheters ( , catheterdays) and crbsi ( . %) from icus withdescribed local signs were included. redness, pain, non-purulent discharge, purulent discharge and ≥ local signs at removal were observed in ( . %), ( . %), ( . %), ( . %) and ( . %) episodes, respectively. the sensitivity of ≥ local sign for crbsi was by . %, whereas the highest specificities were observed for pain ( . %) and purulent discharge ( . %). positive predictive value (ppv) was low for redness ( %), pain ( %), non-purulent discharge ( %) and ≥ local sign ( %), but increased for purulent discharge ( . %). negative predictive values were high for all local signs. after adjusting on confounders, crbsi was associated with redness, non-purulent discharge, purulent discharge and ≥ local sign (fig. ). conditions independently associated with ≥ local sign were age ≤ years old (or . , % ci . - . , p < . ), sofa score (sofa < or . , % ci . - . , p < . ), non-immunosuppression (or . , % ci . - . , p < . ), catheter maintenance > days (or . , % ci . - . , p < . ) and insertion site (or for subclavian site . , % ci . - . , p < . ). however, the presence of ≥ local sign was more predictive for crbsi in the first days of catheter maintenance (or . , % ci . - . vs. > catheter-days or . , % ci . - . , p heterogeneity = . ). conclusion: this post hoc analysis showed that local signs were related to crbsis in the icu. local signs were independently associated with specific patient's and catheter's conditions. in the first days of catheter maintenance, local signs were predictive for crbsi. compliance with ethics regulations: yes. rationale: pneumococcal meningitis (pm) is the leading cause of bacterial meningitis in adult patients requiring icu admission and is associated with a high case fatality rate (cfr), ranging from to more than % ( ) ( ) ( ) . patients with pm may develop sepsis or septic shock that may impact management and outcomes. we aim to describe the epidemiology and outcomes of pm associated with sepsis in adult patients in france. we analysed the occurrence of pm with sepsis from to in adult patients, using the national french hospital database pmsi (programme de médicalisation des systèmes d'information). for all analyses, only the first hospital admission was considered. cases were identified using a combination of a diagnosis code for pm plus a diagnosis code for sepsis (either a code for organ failure or a procedure code for organ support). data recorded included comorbidities ( ), characteristics of the hospital stay, severity of the patients including major intracranial complications and characteristics of the infection. costs and endpoints were determined at the end of all the hospital stays related to the first admission for pm with sepsis. standardized incidence, hospital mortality, and cfr were estimated. temporal trends were assessed using cochran armitage tests of trends and linear trend analyses. results: a total of pm with sepsis aged ≥ years were hospitalized in france during - . the incidence of pm decreased from . to . per m inhabitants (p < . ) (fig. ) . most of them came from home ( %), were admitted in an academic institution ( %) and benefited from icu ( %). their median age was [ ; ] years. twothird of them had at least one comorbidity. the initial neurological presentations included coma ( %), focal signs ( %), seizures ( %) and brain stem involvements ( %). the saps ii score was [ ; ] points. the main neurological complications were cerebrovascular complications ( %), cerebral abscess ( %) and hydrocephaly ( %). pm was associated with pneumococcal septicaemia or pneumococcal pneumonia in % and % of cases respectively. the length of icu and hospital stays were [ ; ] and [ ; ] days respectively and only icu length of stay decreased over time (p < . ). the prognosis was poor since only . % of the patients were discharged to home. indeed, . % of them died and % were transferred to rehabilitation units. no temporal trends could be observed for these outcomes. the average hospital costs per case were , € [ . ; . ] . conclusion: pm with sepsis in adult in france remained a real burden associated with a high mortality rate, and disability. compliance with ethics regulations: na. rationale: mucormycosis is an emerging fungal infection, especially in patients with hematological malignancies. although this infection may lead to multi organ failure, no study has been dedicated to critically ill patients with hematological malignancy. the primary objective was to assess outcome in this setting. the secondary objective was to assess prognostic factors. patients and methods: this retrospective cohort study was performed in icus. critically ill adult patients with hematological malignancies and mucormycosis were included between and . mucormycosiswas classified as "probable"or "proven" regarding eortc criteria. variables are reported as median [iqr] or number (%). adjusted analysis was performed using cox model. results: twenty-six patients were included with a median age of years [iqr, . acute leukemia was the most frequent underlying disease (n = , %). nine patients ( %) were allogeneic stem cell transplantation (sct) recipients. nineteen patients ( %) had neutropenia and patients ( %) had received steroids. the main reason for admission was acute respiratory failure (n = , %) followed by shock (n = , %). the median sofa score at admission was [iqr, - ] points. only patients ( %) had received prior anti-fungal prophylaxis effective against mucorales. mucormycosis was "proven" in patients and "probable" in patients. diagnosis was made by histopathologic examination in patients, direct microscopy or culture in , and polymerase chain reaction in . rhizopus and mucor were the most frequent documented species. seven patients ( %) had concurrent aspergillus infection. mucormycosis was diagnosed day [− to + ] after icu admission. ten patients ( %) had pulmonary involvement whereas five patients ( %) had rhino-cerebral involvement. infection was disseminated in eight patients ( %). twenty-two patients ( %) were treated with liposomal amphotericin b. twelve patients ( %) received antifungal combination including posaconazole in . eight patients ( %) underwent curative surgery. multiple organ failure was frequent, patients ( %) requiring invasive mechanical ventilation (imv), ( %) vasopressors, and ( %) renal replacement therapy. icu and hospital mortality rates were % and %, respectively. only two patients were alive at day . three variables were associated with mortality in a cox model including allogeneic sct . ]; figure), sofa score (hr . [ % ic . - . ]) and dual therapy (hr . [ % ic . - . ]) (fig. ) . conclusion: mucormycosis is associated with a high mortality rate in patients with hematological malignancies, especially in allogeneic sct recipients. futility of icu management in these patients is to be considered and strategies aiming to improve these patients' outcome are urgently needed. compliance with ethics regulations: yes. rationale: sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. several mediators, alone or in combination, were proposed to characterize individual response, but none was proven to have good external validity. the aim of this work was to establish whether some combinations are linked to clinical phenotypes in patients with presumed sepsis, using the data collected in the captain multicenter cohort which methods and first results were previously published (parlato, icm ). patients and methods: patients were prospectively included at the time of sepsis criteria, ( %) of whom with a secondary confirmed infection. community acquired pneumonia was causal in % of infections. saps score = points [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , age = years , male sex = %. patients were followed for more than days, in whom usual icu clinical and biological parameters were collected, as well as plasma biomarkers and leucocyte associated rnas. patients were clinically classified according to their acute severity (sofa score, serum lactate), confirmed initial infection, outcome (secondary infection occurrence, icu survival). non-supervised principal component analysis of the maximal values of biomarkers assessed on first days of sepsis, and varimax rotation technique of the selected components using sas software. results: patients, med sofa day = pts, med serum lactates day = . meq/l, bacterial infection = ( %), enterobacteriaceae infection = ( %), vap and/or bacteremia after day = ( %), alive at icu d/c = ( %). five components explain % of the variance of the biomarkers. the first component ( % of the variance) was not linked to the clinical predetermined phenotypes. the second component ( % of the variance) was principally made of hla-dr rna, cd rna and cx cr rna, and linked to a lower initial severity (r = − . , p = . ), a less frequent confirmation of initial infection (p = . ), a lower occurrence of pneumonia or bacteremia (p = . ) or death (p = . ). conclusion: in our cohort, using non supervised analysis, we could separate a biomarker association linked to lower initial severity, lower rate of a bacterial cause to sepsis, and better outcome. the markers found are among those which are regularly considered as describers of the peripheral alteration of the immune system observed during sepsis (pachot, ccm ; friggeri, cc ; peronnet icm ) . compliance with ethics regulations: yes. ( ) compared a standard of care to a procalcitonin (pct) oriented use of antimicrobials for sepsis in icus. serial blood samples were biobanked in / icus ( / patients enrolled for pro-adrenomedullin (proadm) and pct concentrations). patients and methods: the aim of the study was to evaluate the respective impact of serial pct and proadm measurements in predicting relapse or superinfection and death on day *. relapse was defined as the growth of one or more of the initial causative bacterial strains (i.e., same genus, species) from a second sample taken from the same infection site at h or more after stopping of antibiotics, combined with clinical signs or symptoms of infection. superinfection was defined as the isolation from the same or another site of one or more pathogens different from that identified during the first infectious episode, together with clinical signs or symptoms of infection [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] points at amission, medical admission: ( %), immunocompromised: ( %), on mechanical ventilation ( %), pct and proadm at inclusion were [ . - . ] ng/ml and . [ . - . ] nm/l respectively. ( %) patients developed a first episode of recurrence or supereinfection after a median delay of days [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and ( %) died before d . the hr maximization process proposed an optimal cut point of ng/ml for pct and nm/l for pro adm to predict d death. in the multivariate cox model, both pct and proadm were associated with death but not with relapse or superinfection (table ) . conclusion: conclusion: both serial measurements of pct and proadm are independent predictors of death in patients treated for sepsis in icu. our study confirmed the use of nm/l as a good prognosis cut point for proadm. . compliance with ethics regulations: yes. rationale: the performance of serum ( - )-β-d-glucan (bdg) and its evolution to predict the occurrence of invasive fungal infection (ifi) in a high risk non immunocompromized population remains to be determined ( ). in a post hoc analysis of the empiricus randomized clinical trial ( ), we aimed to assess the prognostic value of repeated measures of bdg on the occurrence of invasive fungal infections. patients and methods: non-neutropenic, non-transplanted, critically ill patients with icu-acquired sepsis, multiple candida colonization, multiple organ failure, exposed to broad-spectrum antibacterial agents, and enrolled between july and february in french icus were included. bdg were collected in icu at day , , , and after inclusion. a value time of more than pg/ ml, pg/ml and an increase by more than % from the previous measurement (threshold of measurement error) were assessed at baseline and overtime. for that purpose, we conducted cause specific hazard models with death as a competing risk. we also planned subgroup analyses on the placebo and the micafungin groups. cumulative risk (cumrisk) of ifi at day were derived from models. [ . ; . ] ). neither a bdg > pg/ml, nor an increase by % of bdg over time were associated with the occurrence of ifi. similar results were found in the placebo subgroup. conclusion: among high risk patients, a first measurement of bdg over pg/ml was highly associated with the occurrence of ifi. neither a cut-off of pg/ml, nor repeated measurements of bdg over time seemed to be useful to predict the occurrence of ifi. the cumulative risk of ifi in the placebo group if bdg > pg/ml is . % questioning about the potential interest of empirical therapy in this subgroup. compliance with ethics regulations: yes. rationale: since the sepsis- conference, the distinction between sepsis and septic shock is based on blood lactate value. septic shock may be encountered in the pre-hospital setting. in order to reduce the mortality, the precocity of treatments implementation has been emphasized, particularly early antibiotic administration. prior antibiotic administration, and blood culture drawing must be performed. the aim of this survey was to clarify the capabilities of french prehospital emergency service (pems) to measure blood lactate and to draw blood culture prior to hospital admission for septic shock. patients and methods: we performed an electronic survey of auto-questions addressed to the deputy chair of the french pems in . results: sixty pems ( %) participated in the survey. twenty-five percent are able to measure blood lactate and % are able to draw blood culture in the prehospital setting. ninety-five percent declared lactate measurement is helpful in assessing severity. ninety percent claimed that the lactate value influences the hospital facility, emergency department vs. intensive care unit. twenty-eight percent believe that the impossibility to draw blood culture precludes prehospital antibiotic administration. sixty-three percent estimate that a protocol for septic shock management would be beneficial. conclusion: few french pems are able to measure lactate and draw blood culture in the prehospital setting. the impact of blood lactate measurement and blood culture drawing by pems on septic shock outcome requires further studies. compliance with ethics regulations: yes. rationale: head injury is a common cause of morbidity and mortality in the first four decades of life, accounting for approximately , annual hospital admissions in the united kingdom. the majority of patients recover without intervention, however some may develop a long-term disability or even die. the early detection of pathology is therefore absolutely critical in determining patients' prognosis, helping to provide appropriate timely management. the national institute for health and care excellence (nice) adult head injury guidelines, recommend that head injuries with specific risk factors should have a ct scan within h of risk factors being identified. furthermore the provisional report should be made available within h of the scan. this audit assessed the compliance of staff to the nice adult head injury guidelines. patients and methods: the previous adult ct head scans, requested due to head injury, from the emergency department (ed) at london north west healthcare nhs trust were analysed for compliance to the nice guidelines. the standards measured were: ( ) time from request of scan to completion of scan should be within h; ( ) time from completion of scan to publication of provisional report should be within h. the locally agreed target for both standards was %. results: on review of the ct scans, ( %) were completed within h of request. from the scans ( %) not completed within the hour, were due to porter unavailability, due to an uncooperative patient and the remaining reasons were not clear from documentation. following completion of the scan, scans ( %) were provisionally reported within h. conclusion: this study highlighted a good compliance by hospital staff in ensuring patients with head injuries are managed appropriately, following detection of risk factors indicating a ct head scan. having said that, the locally agreed targets were just short of being met. one factor resulting in delayed scans was porter availability. an intervention recently introduced is the use of the "e-portering" application, which will endeavour to save time for referrers requesting porters and allow patient tracking. it is also worth educating porters, via email bulletins, on the importance of priority scans, such as ct head following injury. furthermore, the findings of the audit were relayed to the radiology department to help improve reporting times and to the ed to re-emphasize prompt requesting of ct head scans when clinically indicated. compliance with ethics regulations: yes. rationale: continuous insufflation of oxygen (cio) performed with specific endotracheal tube during cardiopulmonary resuscitation (cpr) is as effective as intermittent ventilation on endotracheal tube. experimental data suggest that cio improves the efficacy of external cardiac massage and reduces gastric dilatation. as endotracheal intubation is a cause of cpr interruption and requires skilled staff, a specific device has been developed to perform cio without intubation. this device has been implemented progressively in our fire department since . we evaluated this practice. patients and methods: longitudinal study comparing the patients with out-of-hospital cardiac arrest managed by our fire department with cio or bag-valve ventilation between january and april . patients who received mechanical chest compression were excluded. the main outcome was hospital survival. secondary outcomes were the return of spontaneous circulation (rosc) and cpr quality. univariate and multivariate analysis was performed in the whole cohort and in the sub-groups of patient with shockable and non-shockable rhythms to take into account factors associated with survival (shockable rhythm, witness, age). results: among the patients included, have been ventilated with cio and with valve-bag. the mortality was similar in the two groups (cio: . % valve-bag: . % p . ). mortality and rosc were not associated with cio in the multivariate analysis (odds ratio or . %-confidence interval ci [ . - . ] and . [ . - . ], respectively). cpr quality was better with cio than with valve-bag regarding cpr fraction (ratio of duration of chest compressions on total duration of cpr, versus % p < . ) and adequacy to the guidelines of the rhythm and depth of chest compressions ( % vs % p < . and % vs % p < . , respectively). in both subgroups of patients, cpr quality was still better with cio than with valve-bag. in the subgroup of patients with shockable rhythm, univariate analysis showed a lower mortality among the patients with cio than among the patients with valve-bag ( . % vs . % p < . ) but this difference was not confirmed by the multivariate analysis (or . ci [ . - . ], p . ). conclusion: cio without intubation is associated with an improvement of cpr quality but neither with mortality nor return of spontaneous circulation in case of out-of-hospital cardiac arrest. compliance with ethics regulations: yes. rationale: cardiovascular accidents are a leading cause of death. a cardiopulmonary resuscitation (cpr) of quality has well shown that can reduce the mortality; despite this, survival rate has not changed significantly during last years. the aim of this study is to test a new wearable glove to provide lay people with instructions during out-ofhospital cpr. patients and methods: we performed a blinded, controlled trial on an electronic mannequin ambuman to test the performance of adult volunteers, non-healthcare professionals performing a simulated cpr both, without and with glove, following the glove instructions. the group without glove, also called "no-glove" is intended as control group. each compression performed on the electronic mannequin ambuman was recorded by a connected laptop computer, drawing a depth frequency curve over the time. primary outcome was to compare the accuracy of the two simulated cpr sessions in terms of depth and frequency of chest compressions performed by the same lay volunteers. secondary outcome was to compare the decay of performance and percentage of time in which the candidate performed accurate cpr. finally, the participants were asked if the glove was useful for cpr maneuvers. the difference between the two groups in regard to change in chest compression depth over time due to fatigue, defined as decay were also analyzed. results: chest compressions were included: in control group, in glove group (table ) . mean depth of compression in the control group was . mm versus . mm in the glove-group (p = . ). compressions with an appropriate depth were not statistically different ( . % vs . %, p = . ). mean frequency of compressions in the group with glove was . rpm vs . rpm in the control group (p < . ). the percentage of compression cycles with an appropriate rate (> rpm) was . % in the group with the glove versus % in the control group, with an observed difference of . % between the two groups, which was statistically significant (p < . ,ci = %). a mean reduction over time of compressions depth of . mm (sd . ) was observed in the control group versus a mean reduction of . mm in the group wearing the glove (sd . ), but this mean difference in the decay of compressions delivery was not statistically significant (f-ratio = . , ss = . , df = , ms = . , p = . ). conclusion: the visual and acoustic feedbacks provided by the device were useful in dictating the correct rhythm for non-healthcare professionals, translating in a significantly more accurate cpr. compliance with ethics regulations: yes. rationale: neuroprognostication after cardiac arrest (ca) is a crucial issue and current guidelines recommend delayed multimodal approach. we aimed to describe reasons for death in a prospective cohort of ca patients and evaluate the diagnostic accuracy of early combined neurological prognostication tools such as automated pupillometry (ap), continuous amplitude electroencephalography (aeeg) and cardiac arrest hospital prognosis (cahp) score performed h after return of spontaneous circulation (rosc). we set up a monocentric prospective cohort of adult ca patients admitted in icu after sustained rosc and collected data according to utstein style recommendations. reasons for death were described under recently proposed classification: withdrawal of life-sustaining therapies (wlst) for neurological reasons, wlst due to comorbidities, refractory shock or recurrence of sudden ca or respiratory failure. for patients who kept abnormal neurologic state after rosc with glasgow coma scale < , we analysed accuracy of early neuroprognostication tools (ap, aeeg and cahp score) to predict poor neurological outcome, i.e. cerebral performance category (cpc) > at hospital discharge. results: patients were admitted after sustained rosc from ca during the period ( . . to . . ). in-hospital mortality was %. neurological wlst was the first reason for death ( %). exhaustive early neuroprognostication with ap, aeeg and cahp score was available for patients. among them, poor neurological outcome at hospital discharge (cpc > ) was observed for patients ( % (fig. ) . this strategy would falsely misclassificate % of patients in a good neurologic outcome category. other survivors ( %) should then be investigated with further classical delayed neuroprognostication tools. compliance with ethics regulations: yes. rationale: management delay is one of the determining factors in the assessment of emergency department quality of care. asking for a specialized advice seems to increase the time of delay. our study aimed at measuring the delays in obtaining specialized advice and identify their major causes. patients and methods: we conducted a prospective study over the period of month. we included all adult patients presenting to the emergency department who required specialized advice. data of all patients was collected. waiting times and influencing factors were studied. results: a total of patients were included. the main reason for calling for a specialized advice was to ask for a department transfer in % of cases. the time of the day when specialized advice was solicited (n (%)): in the morning ( ); in the afternoon ( ); in the evening ( ). the main solicited specialties were (n (%)): visceral surgery ( ), trauma medicine ( ), cardiology ( ), urology ( ), and pulmonology ( ). the average waiting time between calling for and getting the specialized advice was ± min. seventy-five percent of the specialized advice was obtained within h. the causes of the delay were (n (%)): physician busy in the operating room ( ), unreachable physician ( ), physician in the outpatient clinics ( ). the impact of the waiting time was (n (%)): conflict ( ), worsening patient state ( ). the average time between calling for the specialized advice and reaching a management decision was ± min. conclusion: the increasing length of stay of patients in the ed is strongly correlated to the delay in obtaining specialized advice. the implementation of a strategy to reduce the waiting time is necessary to avoid overcrowding the emergency departments and provide optimal care. compliance with ethics regulations: yes. rationale: hypnoanalgesia has been used since few years to reduce icu-patients physical and psychological discomfort during invasive procedures. however, feasibility of overall well-being management of intubated patients with hypnosis has not been described. patients and methods: we report here the hypnotic accompaniment of a -year old patient without significant medical history hospitalized in our icu for a severe gbs during months. the gbs was diagnosed by electrophysiological study and immunologic markers. patient had nearly complete paralysis of all extremities, but no facial or bulbar muscles. he received mechanical ventilation during days, including weaning time. tracheotomy was performed at day . sedative drugs were stopped days after intubation. hypnosis sessions were startedvery early after intubation by one of our trained intensivist. eight hypnotic sessions of hypnoanalgesia or hypnotherapy were performed after approval of the patient and his parents. time distribution is reported in fig. . first and second sessions were performed in order to induce relaxation and reduce anxiety. following sessions were dedicated to: ) decrease pain intensity (initially neuropathic, then induced by physiotherapy), ) attenuate the negative perception of paralysis, ) reduce the discomfort of tracheotomy ) promote the belief in healing ) facilitate swallowing exercises. furthermore the patient was quickly trained to use self-hypnosis in order to dissociate him from pain, anxiety and icu pollutions. results: feasibility of hypnosis was judged satisfactory by the operating physician, despite mechanical ventilation. after extubation, final debriefing with the patient indicates that the most efficient sessions were those focused on anxiety disorders (using the suggestion of a safe place) and suggestions of mobility (using a mangas metaphor). the patient reported very positive perception of hypnosis use. he explained that self-hypnosis was effective to reduce many discomfort. he used it frequently (generally twice a day) for a puff of anxiety or before enoxaparin injection. our observation suggests that hypnosis seems feasible in icu-awake patients and may be an interesting way to improve their icu lived experience in combination with validated measures. further investigations are needed to evaluate its effects on post-traumaticstress disorder. compliance with ethics regulations: yes. rationale: there is little medical reference for hypnosis in the intensive care field. closed specialties such as anesthesia, emergency medicine can help and refer to hypnosis for certain technical procedures. objective: to propose landmarks for a successful implementation of hypnosis by intensivists within the intensive care unit. patients and methods: this monocentric prospective observational study was performed from february to june in the -bed medical icu of brest university hospital. collected data were: characteristics of patients and hypnosis sessions performed, demographic data, physiological parameters (heart and respiratory rates) and objective and subjective evaluation of hypnosis sessions quality. results: patients were included (mean age . ± years, saps ii . ± points). hypnosis sessions were performed, of which / under mechanical ventilation. patterns of hypnosis sessions were: anxiety/comfort ( %), during a technical procedure ( %): toe, cvc placement, thoracic drainage, upper digestive or bronchial endoscopy), initiation of noninvasive ventilation or before intubation. most of time, the hypnotic trance was permitted by formal hypnosis techniques with travel and nature themes suggestion. efficacy was qualitatively assessed and rated as "total effectiveness" for % of sessions. qualitative evaluation by hypnotherapist, technical operator and observers was respectively . ± . , . ± . and ± / . heart rate decreased from ± to ± bpm and respiratory rate/min decreased from ± to . ± rpm during sessions. discussion: after a meeting, the healthcare team carried out a brainstorming to propose hypnosis in our unit. several difficulties were observed to explain implementation failures such as: finding competent patient, respiratory assistance, difficult communication, noisy environment, many nursing care, unexpected emergencies, etc.…). this experience allowed writing a vademecum to perform hypnosis in intensive care. our aims are to get more trained caregivers and to integrate hypnosis during our postresuscitation consultation, especially for post-traumatic stress. conclusion: hypnotic tools can facilitate technical procedures and improve patients' and caregivers' quality of life within the icu. compliance with ethics regulations: yes. effect of a musical intervention during central venous catheterization in an intensive care unit: the music cat prospective randomized pilot study sophie jacquier, brice sauvage, gregoire muller, thierry boulain, mai-anh nay chr, orléans, france correspondence: sophie jacquier (sophie.jacquier@chr-orleans.fr) ann. intensive care , (suppl ):f- rationale: evaluate the effect of a musical intervention on patient anxiety during a central venous access or a dialysis catheter implantation in an intensive care unit. patients and methods: the music cat study was a prospective, single-centre, controlled, open-label, two-arm randomized trial, conducted from february to february . central venous catheterization with musical intervention was compared to standard care, i.e., the usual procedure of central venous catheterization without listening to music. eligible patients had to be able to hear, understand explanations and consent. randomisation was stratified according to ventilation type (mechanical ventilation or not) and catheter site (superior vena cava or femoral vein). the music care ® (paris, france) application was used to make the patients listen to music through headphones. each patient chose his/her musical topic on a digital tablet, just before the catheterization. the primary outcome was the change in anxiety visual analogic scale (vas) between the beginning and the end of the catheterization procedure (t -tf anxiety vas). secondary outcomes included the patient's pain vas at the end of the procedure (tf pain vas). results: patients were included in the standard care group versus in the musical intervention group. main reasons for admission were the need of central catheter for chemotherapy ( , %), and sepsis and/or shock in both groups ( , %). catheters were inserted in the internal jugular vein in most cases ( , %) and about one-third were tunnelled in both groups. there was no between-group difference regarding median t -tf anxiety vas: [iqr:− to ] in the standard care group versus − [− to ] in the music intervention group (p = . ) (fig. ) , with no significant interaction between the variables of stratification or the operator experience and the intervention. the median tf pain vas was not statistically different between groups: [ to . ] in standard care group and [ to ] in music intervention group (p = . ), with no significant interaction between the variables of stratification or the operator experience and the intervention. conclusion: in this first randomized pilot study of musical intervention for central venous catheterization in awake patients in the intensive care unit, the musical intervention did not reduce patients' anxiety as compared to usual care. as the study may have been underpowered, larger size trials are needed. compliance with ethics regulations: yes. rationale: sleep is markedly altered in icu-patients under mechanical ventilation and may be due to noise, light, patient-care activities, patient-ventilator asynchronies, or the result of acute brain dysfunction induced by sedative drugs. to our knowledge, sleep has never been studied at icu admission before any sedation. our study aimed at assessing sleep quality of non-intubated sedation-free patients admitted to icu for acute respiratory failure. patients and methods: observational study performed in a single centre of a teaching hospital. patients admitted to icu for acute respiratory failure (respiratory rate ≥ breaths/min and pao / fio < mm hg under high-flow nasal oxygen) could be enrolled. patients with hypercapnia, central nervous disease, intubated early after admission and those with a do-not-intubate order were excluded. sleep was evaluated by complete polysomnography (psg) that started in the afternoon following admission and was continuously performed until the next morning. results: over a -year period patients were screened and patients were included. among them, patients were excluded for the following reasons: patient was intubated shortly after psg initiation, psg was lost, and eeg recordings ( %) were stopped before midnight (electrodes turned off or loss of signal). therefore, patients in whom psg was complete during the nocturnal period were retained in the analysis ( rationale: convulsive status epilepticus (cse) is a common neurological emergency associated with high mortality and morbidity rates. there are strong experimental data suggesting a potential impact of secondary brain insults (sbi) on outcome after cse. however, there is no clinical proof to support this hypothesis. our objective was to evaluate the association between sbi (mean arterial blood pressure, arterial partial pressure of carbon dioxide, arterial partial pressure of oxygen, temperature, natremia, and glycemia) at day and neurological outcomes days after cse. patients and methods: this was a post hoc analysis of the hyber-natus multicenter open-label clinical trial randomized critically ill patients with cse requiring mechanical ventilation to either therapeutic hypothermia ( - °c for h) plus standard care or standard care alone. patients still alive at day after inclusion were enrolled from march to january in french medico-surgical icus. the primary outcome was favourable outcome days after cse defined as a glasgow outcome scale score of . results: median age was of years . a previous history of epilepsy was noted in ( %) patients. most episodes ( / , %) occurred out-of-hospital, and ( %) were witnessed from their onset. cse was refractory in ( %) patients and total seizure duration was min ( - ). a favorable -day outcome occurred in ( %) patients. maximal glycemia value and hyperglycemia > . mmol/l at day were the only sbi variables associated with outcome in univariate analysis. by multivariate analysis, age > years (or, . ; % ic, . - . ; p = . ), refractory cse (or, . ; % ic, . - . ; p = . ), and primary brain insult (or, . ; % ic, . - . ; p = . ) were associated with an increased risk of poor outcome, and a bystander-witnessed onset of cse (or, . ; % ic, . - . ; p = . ) was associated with a decreased risk of poor outcome. conclusion: in our population, secondary brain insults were not associated with outcome in critically ill patients with convulsive status epilepticus; whereas age, bystander-witnessed onset of status epilepticus, refractory status epilepticus and primary brain insult were identified as strong predictors of -day functional impairment. further studies are warranted to confirm our findings. compliance with ethics regulations: yes. rationale: acute stroke (as) is a leading cause of morbidity and mortality worldwide. however, data on the prognosis andfunctional outcome of patients with as requiring icu management is limited. our purpose was to identify factors associated with good outcome (defined by a modified rankin score (mrs) of - ) months after icu admission. patients and methods: retrospective cohort of patients admitted to the medical icu of a university-affiliated hospital between january and december and coded for acute stroke using the icd- criteria. patients with traumatic stroke and isolated subarachnoid hemorrhage were excluded. results: we identified patients. median age was [ . - ] years and ( . %) were males. main reasons for icu admission were coma ( %), hemodynamic instability ( . %), acute respiratory failure ( %), and cardiac arrest ( . %). glasgow coma score at icu admission was [ ] [ ] [ ] [ ] [ ] [ ] [ ] and points. types of stroke were hemorrhagic in ( . %) patients and ischemic in ( . %). mechanical ventilation was required in patients ( . %). seizures occurred in . % of the patients and convulsive status epilepticus in . %. pneumonia was diagnosed in ( . %) patients (aspiration pneumonia n = , ventilator associated pneumonia n = ). thrombolysis or thromboaspiration were performed in ( %) patients with ischemic stroke. surgical evacuation of expanding hematoma was performed in ( . %) patients, ( . %) had craniectomy, and ( . %) had external shunt for hydrocephalus. icu and hospital mortality were . % and %, respectively. six months after icu admission, ( . %) patients had a good outcome (mrs - ), ( . %) had significant disability (mrs - ), and ( . %) were deceased (lost follow-up n = , . %). on multivariable analysis, age (or . per year ( . - . ), p = . ), saps (or . per point ( . - . ), p = . ), and hemorrhagic stroke (or . ( . - . ), p = . ) reduced the likelihood of good outcome (mrs - ) months after icu admission. conclusion: in our study, prognosis of acute stroke requiring icu admission was poor and a good functional outcome occurred in less than % of the patients at months. age, severity at icu admission, and type of stroke predicted outcome. compliance with ethics regulations: yes. rationale: in intensive care units, severe spontaneous hemorrhagic brain injuries have a poor prognosis for mortality and functional outcomes. affected patients face particular ethical issues regarding the difficulty of anticipating their eventual recovery. in this context, prognostic scores can help clinicians in patients/relatives counseling and therapeutic decisions. the previous reviews pointed out many prognostic tools for intracranial hemorrhage and subarachnoid hemorrhage but did not focus on injuries explicitly severe nor assessed the methodological limitations of the models. our systematic review aimed to assess methodologically prognostic tools for functional outcomes in severe spontaneous haemorrhagic brain, with particular attention to their clinical utilities. patients and methods: following prisma recommendations, we queried medline, embase, web of science, and the cochrane by february , . we included multivariate prognostic models explicitly developed or validated on adults with severe intracranial or subarachnoid haemorrhage. we evaluated the articles following the charms recommendations (checklist for critical appraisal and data extraction for systematic reviews of prediction modelling studies) and the tri-pod statements (transparent reporting of a multivariable prediction model for individual prognosis. results: our review confirmed the multiple publications of prognostic scores, as we found articles aiming to develop or validate prognostic tools. relying on guidelines, we discarded articles due to the lack of prognostic capacities, validation, or predictor selection. articles developed and validated a prognostic tool and externally validated existing models (fig. ) . no score was of good methodological quality in intracranial hemorrhage. we highlighted two prognostic scores in subarachnoid hemorrhages: the sahit predicting unfavorable outcome or mortality at months and the fresh predicting unfavorable outcome at months. conclusion: although prognostic studies on haemorrhagic brain injuries abound in the literature, they generally lack of methodological robustness or show incomplete reporting. with the numerous published scores, we believe that it is time to stop developing new scores. ongoing validation, recalibration, and impact studies would keep improving existing good tools. the use of "patient-centered" approaches could also enhance them, and be more appropriate to inform patients and families about their long-term potential recovery. these considerations should drive future research in the modern era of neurocritical care prognosis. compliance with ethics regulations: na. rationale: respiratory pattern analysis by a visual examination is an important part of clinical assessment but is dependent on caregiver expertise and is subjective. furthermore, there is no easy medical device used in picu to measure tidal volume (vt) and minute ventilation (mv) in spontaneous breathing patients. the clinical research unit in critical care of chusj and ets have developed a non-invasive computerized d video analyzing system (retract system) to detect and perform a video analysis of respiratory status in children. the aim of this study is to test the reliability of the retract system to monitor respiratory distress in critically ill children. the retract system is detailed in reference . in summary, cameras reproduce in d the thorax and abdomen of a subject. the respiratory status (respiratory rate (rr), tidal volume (vt), minute ventilation (mv)) assessed by the retract system was compared on a bench test (high-fidelity mannequin) and in critically ill children, to the ventilator measurements and clinician expert evaluation (gold standard). bland-altman plots were used for comparison. results: we observed a significant agreement, on mannequin, between retract system and gold standard method in estimating vt, rr and mv, i.e. % of the paired differences were within the limits of agreement in bland-altman plots, as illustrated in fig. . in critically ill children (n = ), the correlation between the pairs of measures was also high (r > . , p < . ) and thecoefficient of determination with a high fit ( . < r < . , p < . ). for good correlation, the retract system needed to have a visual access to thorax and abdomen in a quiet subject. the retract system measurements of vt, rr and mv for respiratory distress monitoring in patients seems reliable. more testing are required to validate this method in usual practice and to develop the retractions signs video analysis. compliance with ethics regulations: yes. rationale: severe bronchiolitis requires hospitalization in paediatric intensive care unit (picu). non-invasive ventilation (niv) has been demonstrated to treat them since twenty years, its use is well defined but there is no consensus for the weaning. this study evaluated the application of a nurse-driven niv weaning protocol in hospitalized infants with severe bronchiolitis and verified its safety. this was a retrospective monocentric study in a picu of robert debré hospital-paris, france. in the epidemic period of bronchiolitis between and , all patients under one year old with severe bronchiolitis and requiring niv were included. two groups were compared: one group using the nurse-driven niv weaning protocol and one group without using this protocol. occurrences of complications, duration of ventilatory support and length of stay (los) in picu and total los were compared. results: patients were included in the study, in the no-protocol group, and in the protocol group. the nurse-driven protocol was using at the rate of % (n = / in the protocol group (p = . ). picu los were . days [ ] [ ] [ ] in the no-protocol group versus days [ - . ] in the protocol group (p = . ), hospital los was days [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in the no-protocol group versus days [ ] [ ] [ ] [ ] [ ] [ ] in the protocol group (p = . ) (fig. ) . the use of this first nurse-driven niv weaning protocol was feasible and simple with a very good application rate. its utilization was safe. the occurrence of complications did not increase by the use of this protocol. it would allow an optimal niv weaning without prolonging the ventilatory support duration nor picu los or hospital los. the professional practices appeared to be coordinated and the nurses appeared to be more autonomous. compliance with ethics regulations: yes. no-protocol and protocol groups comparison: cpap duration ( ), ventilatory support duration ( ), picu los ( ), hospital los ( ) rationale: first-line management of severe acute bronchiolitis in infants is mainly based on non-invasive ventilation (niv) and high-flow nasal cannula (hfnc) therapy. however, pediatric data regarding weaning from niv/hfnc are lacking. this study aims to identify the weaning practices from niv/hfnc in children with severe bronchiolitis. the weaniv-survey is a cross-sectional survey. a questionnaire was sent to french-speaking physicians with key roles in pediatric intensive care units. results: a total of % ( / ) of french university hospital were represented in the study. only % of pediatric centers used a protocol for weaning from niv/hfnc and nurses were considered as key-actors of the weaning process for half of participants. continuous positive airway pressure (cpap) was the mode of ventilation mainly used as the first-line therapy in clinical practice. the main criteriaconsidered toinitiate weaning process were: noor slight respiratory distress, a fio < %, a respiratory rate < /min and no significant apnea. three strategies to discontinue niv/hfnc were identified: /gradual decrease of ventilatory parameters (pressure or flow), /abrupt discontinuation and /gradual increase in off-ventilation time. abrupt weaning strategy was the most commonly used, no matter the mode of ventilation. a significant level of respiratory distress, the presence of apneas, an increase in oxygen requirement, and a respiratory rate > / min were identified as weaning failure criteria by most pediatric intensive care physicians. conclusion: in most centers, the weaning process does not follow any protocol. abrupt weaning seems to be commonly used as weaning strategy in children with severe bronchiolitis supported by niv/hfnc. based on the study findings, we suggest that criteria for weaning initiation and for weaning failure must be defined and weaning protocols generated. compliance with ethics regulations: yes. complications secondary to prone positioning occured for patients ( . %). conclusion: this first study, which evaluate prone positioning efficacy in severe p-ards shows evidence that prone positioning improves oxygenation parameters and survival rate. these results highlight the necessity to develop a multicentric prospective randomized study to confirm these conclusions. compliance with ethics regulations: yes. ( vs ) and vasoactive-inotropic score (vis) ( vs ) were significantly higher in the non-survivor group. cannulation was veno-venous ( %) or veno-arterial ( %) and patients ( %) were finally not initiated on ecmo. we observed an increase of patients cannulated in our picu over time (fig. ). there was no significant difference in mortality between patients transported on ecmo after cannulation in our picu and those who were transported to be cannulated in a referral ecmo center. the median time between the decision and the cannulation was . h and the median time taken in charge by picu transport team was approximately h. these periods were not significantly different between cannulation on site or in an ecmo center and between survivors and not-survivors. conclusion: in our study, multiple organ dysfunction, particularly hematologic and acuterenal failures, seems to be a risk factor of mortality. the delay between decision and management is similar whatever the cannulation site. specific ecmo mobile team and picu transport team seem to be essential, fast and trained to transfer these patients. it would be interesting to compare our cohort with children requiring ecmo already hospitalized in a referral ecmo center. compliance with ethics regulations: yes. rationale: life expectancy in patients with metastatic breast cancer (mbc) has substantially improved over the last decade. life threatening complications result from advanced diseases, infection and treatment-related toxicity. only few studies have assessed outcomes in this setting. we performed a hospital-wide study to investigate how icu resources are needed in patients with mbc. patients and methods: all patients with mbc managed at our hospital between and were retrospectively included. the primary outcome was overall survival (os). factors associated with icu mortality were identified using a multivariable cox proportional hazard model with sensitivity analysis. results are expressed as median [interquartile ranges] unless stated otherwise. results: among the patients managed at our hospital, ( %, including male) were admitted to the icu ( [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] patients per year). age was [ - ] years. patients were receiving their nd [ st- rd] line of treatment and had [ ] [ ] metastatic sites. sofa score at admission was [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . main reason for icu admission was sepsis (n = , %), acute respiratory failure (n = , %), coma (n = , %) and metabolic disorder (n = , %). invasive mechanical ventilation was required for patients ( %) and renal replacement therapy for ( %). sixteen ( %) patients died in icu. following icu discharge, median os was . months ( % ci [ . - . ]) and / ( . %) patients died within months. an antineoplastic treatment was resumed for / ( %) patients alive after icu discharge. factors independently associated with mortality were performance status ≥ (hr . , % ic [ . - . ] ) and sofa score at day (hr . per point, % ic [ . - . ] ). after sensitivity analysis, the number of treatment lines at icu admission was not associated with mortality. conclusion: icu admission is required in the course of the mbc disease for % of the patients. determinants of short term outcomes rely on performance status and disease severity but not on the characteristics of the underlying disease. ongoing analyses will assess whether icu survivors reach life expectancy of patients never admitted to the icu. compliance with ethics regulations: yes. hubert gheerbrant , jean-françois timsit , nicolas terzi , stephane ruckly , mathieu laramas , matteo giaj levra , emmanuelle jacquet , loic falque , denis moro-sibilot , anne-claire toffart chu grenoble alpes, grenoble, france; aphp, paris, france; outcom-erea, bobigny, france correspondence: hubert gheerbrant (hgheerbrant@chu-grenoble.fr) ann. intensive care , (suppl ):p- rationale: the prognosis of critically ill cancer patients admitted in intensive care unit (icu), remains an issue. our objective was to assess the factors associated with -and -month survival of icu cancer survivors. patients and methods: based on the french outcomerea ™ database, we included solid cancer patients discharged alive, between december and november , from the medical icu of the university hospital in grenoble, france. patient characteristics and outcome at and months following icu discharge were extracted from available database. results: of the cancer patients with unscheduled admissions, ( %) were discharged alive from icu. the main primary cancer sites were digestive ( %) and thoracic ( %). the -and -month mortality rates were % and %, respectively. factors independently associated with -month mortality included ecog performance status (ecog-ps) of [ ] [ ] . . - . ). interestingly, cancer chemotherapy prior to icu admission was independently associated with lower -month mortality (or, . ; % ic: . - . ). among patients with an ecog-ps - at admission, % (n = ) and % (n = ) displayed an ecog-ps - at and months, respectively. at months, ( %) patients received anticancer treatment, ( %) were given exclusive palliative care. discussion: factors associated with -month mortality are almost the same as those known to be associated with icu mortality. we highlighted that most patients recovered an ecog-ps of - at and months, in particular those with a good ecog-ps at icu admission, and could benefit from an anticancer treatment following icu discharge. conclusion: these results should be taken into account when deciding upon icu admission. it is of paramount importance to have an evaluation of both patient's general condition and anticancer treatment opportunities following icu discharge. compliance with ethics regulations: yes. rationale: the decision to urgently initiate medical anti-cancer treatment in cancer patients admitted to intensive care unit for cancerrelated organ failure is an issue. we currently lack criteria to select patients who may benefit from the treatment initiation. the purpose of our exploratory study was therefore to evaluate the characteristics of patients whose medical anti-cancer treatment is initiated in icu and to identify prognostic factors for in-hospital mortality. in these patients. patients and methods: we analyzed retrospectively, over a period of years ( / / to / / ), cancer patients over -year old admitted to our icu bordet and in whose anti-cancer medicaltreatment was initiated during in-icu stay. to identify prognostic factors for in-hospital mortality, we carried out a multivariate analysis of the factors influencing this mortality, considered as a binary. we also analyzed the long term survival of patients alive after their hospital stay (from the day of going out of hospital). results: overall, patients were included, men ( %) and women ( %), with a median age of years ( - ). of these, patients ( %) had a solid tumor and ( %) had a hematological tumor. in-icu mortality is % ( % ci - %) and in-hospital mortality % ( % ci - %). the prognostic factors for in-hospital mortality were age (mean vs in those who survived), the sofa score (median vs ), the saps ii score (mean vs ), the charlson score (mean vs. . ), the number of organ failure (mean . vs . ) and the presence of a therapeutic limitation (ntbr stated within h: % vs %). survival at year of patients who survived the hospital stay was % and median survival time was estimated to be . year ( % ci . - . ). in patients with a solid tumor, -year survival was % and % in those with a hematological tumor (p < . ). conclusion: we observed, in selected cancer patients admitted to the icu for a cancer-related complication, that the initiation of an anti-cancer medical treatment is feasible and can lead to interesting results, particularly in patients with a hematological tumor. compliance with ethics regulations: yes. rationale: considerable progress in the management of onco-hematology (oh) malignancies led to an increase in the number of patients proposed for intensive care unit (icu) admission. several guidelines offer decision models for icu transfer of these patients. we aimed to describe prognosis, adequacy of icu admission and denial in oncohematology patients. we included all oh patients proposed for icu admission in a tunisian medical icu, between january and july . from an admission proposal registry, were collected patient underlying condition, functional status, malignancy and predicted prognosis, acute critical illness and its reversibility, adequacy of icu rationale: cancer patients frequently need intensive care support for a life-threatening condition due to the underlying neoplasm or an adverse therapy-related event. however, there are poor data on their characteristics and outcomes in the intensive care setting. the aim of the present study was to describe clinical characteristics and to identify factors associated with in-icu mortality in critically ill cancer patients. patients and methods: it is a retrospective study conducted in the medical icu of farhat hached teaching hospital between january and december . all cancer patients with complete records were included. baseline characteristics, clinical parameters, severity of illness, primary tumor location and outcomes were collected. univariate and multivariate regression analyses were carried out to identify factors independently associated to poor prognosis. rationale: prognostic impact of underlying malignancy seems limited in most studies assessing outcome of critically ill cancer patients [ ] . however, only limited number of characteristics, namely disease progression status and preexisting stem cell transplantation, were usually assessed [ ] . primary objective of this study was to assess influence of hematological malignancy aggressiveness on hospital outcome. secondary objective was to assess influence hematological malignancy aggressiveness on type of infection. patients and methods: post-hoc analysis of prospective multicenter cohort performed in hospitals in france and belgium and including critically ill adults with underlying hematological malignancy admitted in icu from jan to may . a cox model was used to adjust for confounding variables then a propensity score matching on characteristics associated with underlying malignancy aggressiveness was performed. results: of the included patients, ( . %) had low grade malignancy (lg), the most frequent being myeloma (n = ), chronic lymphocytic leukemia (n = ), and myelodysplasia (n = ). patients with lg malignancy were older, underwent more frequently autologous stem cell transplantation (sct) and had less frequently altered performans status. they had more severe organ failure at icu admission (sofa score [ ] [ ] [ ] [ ] [ ] [ ] vs. [ ] [ ] [ ] [ ] [ ] [ ] , p = . ). before adjustment, mortality was % (n = ) and . % (n = ) respectively in patients with and without lg malignancy (p = . ). after adjustment for confounder using a cox model, a higher mortality was associated with nonlow grade malignancy (or . ; % ic . - . ). a propensity score then allowed a : matching upon variable associated with malignancy aggressiveness. after matching unadjusted mortality was % (n = ) in patients with lg malignancy and . % (n = ) in patients with high grade malignancy (p = . ) (figure) . in the matched cohort and after adjustment for confounder, high grade malignancies were associated with lower mortality (or . ; % ic . - . ). risk of fungal infection was unchanged by underlying malignancy before adjustment ( % vs. . % of patients with and without lg malignancy; p = . ) or after adjustment (hr . ; % ic . - . ). conclusion: despite anti-cancer advances, aggressiveness of hematological malignancies is associated with overall icu outcome. lowgrade malignancies displaying a better prognosis than non-low grade. aggressiveness of the underlying malignancy is not associated with risk of fungal infection. compliance with ethics regulations: yes. rationale: guillain-barré syndrome is the most common cause of acute flaccid paralysis and is associated with pulmonary embolism due to the mobility limitation. the aim of this study is to describe the incidence, the severity of pulmonory embolism in patients admitted to an intensive care unit (icu) for guillain-barre syndrome (gbs). patients and methods: twenty-eight adults patients with confirmed diagnosis of gbs were admitted to the icu in our university hospital center over a -year period and they were all included. prevalence, risk factors and course of vte were analyzed in icu patients with various forms and severity of gbs. results: during the study period, adult gbs patients were included. five ( . %) developped pulmonary embolism. the mean age was . ± . years and the sex ratio was . . the comparaison betewen the groups with and without pe showed that factors associated with the development of this complication were: respiratory failure requiring mecanical ventilation (p = . ), infectious complications (p < . ), blood pressure lability (p = . ), the delay of icu admission (p = . ), the delay to treatment initiation (p = . ), the sofa score (p = . ) and the presence of quadriplegia (p = . ). conclusion: pulmonary embolism is a frequent complication in patients with gbs. factors associated with this complication were: respiratory failure requiring mecanical ventilation, infectious complications, the delay of icu admission, the delay to treatment initiation, a high sofa score and the presence of quadriplegia. preventive measures in this category of patients have to be improved. rationale: acute respiratory distress syndrome (ards) is a life-threatening pathology associated with very high morbidity and mortality ( - %) in intensive care units (icu) and with even higher mortality among the severly burned patients worldwide ( à %). the aim of our study was to describe in tunisia burn patients with ards and to identify prognosis factors. patients and methods: we conducted a descriptive retrospective study between - - to - - , in burns icu, in ben arous, in tunisia. all burns who presented an ards, according to the berlin definition, during their stay in the icu, were included. when clinical or gasometric data was uncomplete, these patients were excluded. results: during the study period, patients were admitted to our burn unit including ventilated patients. fifty patients presented an ards: fifteen patients were excluded for lack of information, and patients were retained. the sex ratio was . . patients had a mean age of ± years, an average burned area of % ± %, an average unit of burn skin score (ubs score) of ± and an average sequential organ failure assessment score (sofa score) of . none of the patients had a history of cardiovascular or pulmonary diseases. the average time of onset of ards was ± days. ards was mild in case, moderate in and severe in . the etiology of ards was pulmonary in cases ( %) and extra-pulmonary in ( %). the pulmonary ards had as cause pneumonia isolated in patients, an isolated pulmonary burn in patients and a combination of pneumonia and lung burns in patients. extra-pulmonary ards were all due to sepsis and mainly to bacteremia. septic shock was associated with ards in patients ( %). the treatment was a conventional treatment based on protective ventilation, curarization and prone positioning in addition to the etiological treatment. the average length of stay in icu was days and mortality was % in these patients. conclusion: mortality from ards in burns in tunisia, is important especially in those with pulmonary burns as well as those with sepsis. the introduction of new treatments, such as extracorporeal membrane oxygenation, remains essential to improve the prognosis of burn patients. compliance with ethics regulations: yes. rationale: aspiration pneumonia (ap) is common in intensive care unit (icu). the incidence of ap among adults hospitalized with pneumonia ranges between and . %. usually one or more risk factors are identified to be involved in ap. the aim of this study was to determine the risk factors and predictors of mortality on patients with ap. patients and methods: we retrospectively included patients aged more than years and who were hospitalized in our icu for ap. patients were excluded if they had history of tuberculosis, if they have bronchiectasis or metastatic brain tumor. results: a total of patients were included. history of diabetes, hypertension, epilepsy and ischemic stroke were found respectively in . %, . %, . %, and . % of cases. the reason of icu admission were coma ( %), acute respiratory failure ( %), poisoning ( %) and cardiac arrest ( %). the incidence of acute respiratory distress syndrome (ards) was %. the most common organism isolated was staphylococcus aureus ( cases). risk factors for ap were epilepsy ( %), swallowing disorders ( %), ischemic stroke ( %), copd ( %) and degenerative neurological disease ( %). the mortality rate was . %. the median duration of mechanical ventilation was days [iqr - ]. in multivariate logistic regression analysis; saps ii score (or = . , % ic [ . - . ], p = . ) and ards (or = . , % ic [ . - . ], p = . ) were independently associated with mortality. conclusion: risk factors for aspiration pneumonia were epilepsy, swallowing disorders and ischemic stroke. ards and saps ii score were independent predictive factors of mortality. compliance with ethics regulations: yes. undetermined. the aim of this study was to evaluate the impact of hyperoxia on morbidity and mortality. patients and methods: this was a prospective study performed in the icu of abderrahmen mami hospital during a -month period. all patients admitted in icu during the study-period were included. those who didn't need oxygen therapy or in end of life stage were excluded. arterial blood gases were analyzed daily and each day with at least one value of oxygen arterial saturation (sao ) > % was considered as a day with hyperoxia. for each patient included, the number of times and days spent in hyperoxia was recorded as well as complications during the icu stay and the outcome. results: during the study-period, patients were included but only were eligible. mean age was ± years. acute on chronic respiratory failure was the most frequent reason of admission ( %). non-invasive ventilation was required for % of patients and invasive mechanical ventilation was necessary in % of cases. overall mortality was %. hyperoxia was observed in % of cases, with an average of ± times during the icu stay and ± days. a statistically significant association was observed between a long duration of hyperoxia and the occurrence of ventilator acquired pneumonia (p < - ), ventilator acquired bronchitis (p = . ), acute respiratory distress syndrome (p < - ), atelectasis (p < - ), septic shock (p < - ), rythm disorders (p = . ), reintubation (p < - ) and tracheostomy (p = . ). on multivariate analysis, independent factors of mortality were: simplified acute physiology score ii, cardiac failure, need for invasive mechanical ventilation and septic shock. hyperoxia was not independently associated with mortality. conclusion: hyperoxia is frequent in icu. it is significantly associated with icu complications but not independently associated with mortality. compliance with ethics regulations: yes. experience of the practice of prone position in patientswith acute respiratory distress syndrome in intensive care (chu oran) nabil ghomari, soumia benbernou, djebli houria faculté de medecine d'oran, oran, algeria correspondence: nabil ghomari (nabilghomari@hotmail.fr) ann. intensive care , (suppl ):p- rationale: mechanical ventilation (mv) in the prone position (pp) and low tidal volume have become recommendations with a high level of scientific evidence in recent years. the pp has been practiced for years in the chu oran emergency resuscitation service. we wanted to report the service experience in the practice of pp in patients with ards. patients and methods: retrospective study performed in patients with severe hypoxia ards with spo < % under fio > % or pao /fio < during the period march to december . results: patients received ventilation in pp. ards was secondary to thoracic trauma in % of patients, septic shock in % and aspiration pneumonitis in %. analysis of the success factors and improvement of oxygenation found that lobar ards, the delay < h and a duration of pp ≥ h were statistically significant. conclusion: the pp must be integrated into the arsenal of care of the patients in ards especially in our country where we do not have all the therapeutic options. compliance with ethics regulations: yes. julien goutay, nicolas cousin, thibault duburcq, erika parmentier-decrucq chu de lille, pôle de réanimation, hôpital salengro, lille, france correspondence: julien goutay (julien.goutay@gmail.com) ann. intensive care , (suppl ):p- rationale: in veno-venous extracorporeal membrane oxygenation (vv-ecmo) therapy, blood flow is the main determinant of arterial oxygenation and should be - ml/kg/min in adults. this flow rate is determined by several factors including the size of the inflow cannula. the impact on clinical outcomes of arterial cannula's size in veno-arterial ecmo (va-ecmo) has already been studied, and showed no difference for survival to discharge, weaning success rate and initial flow rate between a small cannula group and a larger one. our first objective was to describe the impact of inlet cannula size on the assistance flow rate in patients treated with vv-ecmo. secondary objectives were to analyze its impact on ecmo weaning, mechanical ventilation characteristics and mortality. patients and methods: we retrospectively reviewed all cases of respiratory failure treated with vv-ecmo admitted in the medical intensive care unit (icu) of lille's teaching hospital from january st, through march st, . inlet cannula size was collected and divided into two groups: the "small cannula" group had inlet cannula less than or equal to fr, while "large cannula" were larger than fr. primary endpoint was the initial flow rate according to the inlet cannula size, and its changes during the first h of assistance. secondary endpoints were the analysis of predictive factors associated with the choice of a larger inlet cannula, and the impact of its size on clinical outcomes such as successful ecmo weaning. results: patients treated with vv-ecmo were admitted in our hospital. eleven ( %) were cannulated with a large inlet device. mean initial ecmo flow rate was statistically higher in the "large cannula" group than in the "small cannula" one: . l/min (± . ) versus . (± . ) respectively, p < . . the difference was also significant during the first h of assistance. we found no difference between the two groups on clinical outcomes such as ecmo weaning time. in univariate analysis, weight was heavier in the "large cannula" group [ (± ) kg] than "small cannula" [ (± )], p < . . conclusion: ecmo initial flow rate was higher in a "large inlet cannula" group (internal diameter more than fr) compared with a "small cannula" group. we found no correlation with cannula-related haemorrhagic or thrombotic complications. inlet cannula size did not influence ecmo weaning, and duration time, but this may be a lack of statistical power. further prospective studies should confirm this results. compliance with ethics regulations: yes. rationale: burn patients are at risk of multidrug-resistant (mdr) bacterial infections with high mortality rate. therefore, monitoring the emergence of mdr pathogens in these vulnerable patients is important. this study aimed to assess digestive colonization with carbapenemase-producing gram-negative bacilli (cp-gnb) in patients admitted to the burn intensive care unit. patients and methods: our study was prospective and conducted over a one-year period (january to december ). every admitted patient was subjected to the screening. a double swab set was used to collect rectal swab specimens. one swab was used for mdr screening by disk diffusion method on selective media; the other for multiplex real-time pcr (cepheid's genexpert ® ) allowing detection of the most common carbapenemase-encoding genes (ceg) (blaoxa- , blakpc, blandm, blavim and blaimp). results: among the studied patients, ( . %) were detected positive at admission for cp-gnb by the genexpert ® carba-r assay. eleven patients, initially not colonized, acquired positive faecal carriage subsequently during their hospital stay. forty-two colonized patients ( . %) developed cp-gnb infection during their hospitalization. the ceg blandm quantitatively dominated by far with detections; either alone ( cases) or associated with other ceg ( cases). the second most frequent gene was blaoxa- . it was detected alone eight times and in association with other ceg times. forty-three patients carried blavim gene, usually in association with other ceg ( %). however, only one patient carried blakpc gene. the parallel screening by classical microbiology methods (disk diffusion on selective media) detected the presence of cp-gnb in all molecular positive samples. conclusion: our study describes the characterization of carbapenemase in burn patients and highlights their alarming spread. this emphasizes the importance of an active surveillance program by early detection of cp-gnb carriers and an isolation policy to limit the mdr infections expansion. compliance with ethics regulations: yes. rationale: invasive fungal infections are increasingly observed in the icus especially in burn units. inthe absence of simple and accessible techniques for early microbiological diagnosis, the use of antifungal treatment is increasing. little is known about the extent of the problem of antifungal prescription in burn icus. we aimed to evaluate the antifungal prescription in major burn patients. patients and methods: during the study period ( - ), all prescriptions of antifungals were analysed. analysis concerned demographics, clinical circumstances, as well as the basis of antifungal prescribing (targeted vs. empiric). among the patients admitted in this period, patients were treated with antifungals (sex ratio: . ; mean age: ± years, with low associated comorbidity). the tbsa was . % [ . - . ], ubs was [ . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . most of the patients ( . %) were transferred from another hospital structure within ± h. antifungal treatment was started at the average of the seventh day post wound injury, more often on an empiric basis. sofa score at the beginning of the treatment was ± . . lymphopenia was present in % and thrombopenia was present in %. index of colonisation was positif only in cases. the average candida score was . ± . . microbiological results were tardily collected, within weeks, in %. fungal urine infections were found in cases. candidemia and catheter-related infections were considered only in cases. the risk factors of fungal infection as described in literature were found in most of the patients including mechanical ventilation ( . %), length period of stay ( days [ . - . ]), central venous line ( %), severe sepsis or septic shock ( %), large-spectrum antibiotherapy for more than days ( %). conclusion: the management of antifungal infections in major burn patients is still challenging. antifungal prescription is based on clinical presumption. the empirical prescription reflects the lack of efficient laboratory support and late microbiological results prompting physicians to rely on clinical informations. the management of fungal infections is based on the improvement of mycological investigations. compliance with ethics regulations: na. rationale: invasive candidiasis is a widespread and alarming infection in intensive care units (icu) patients. its diagnosis is often difficult because of the lack of specificity of clinical signs and the low sensitivity of blood cultures. while the candida albicans species remain the most common cause of bloodstream infections, non-albicans are emerging. these infections are serious, associated with high mortality rate and requiring early diagnosis and appropriate treatment. in tunisia, few data are available. we aimed to determine the epidemiological profile of a series of candidemia in icu, the risk factors associated with the occurrence of candidemia and to describe the modalities of the mycological diagnosis of candidemia and their etiological profile. patients and methods: a retrospective longitudinal descriptive study conducted in the parasitology-mycology laboratory with the collaboration of the medical icu of la rabta hospital-tunis over a -year period from january , to december , . all hospitalized icu patients with at least one candida-positive blood culture were included. results: forty-three patients among hospitalized patients during the study period had at least one candidemia infection. the main risk factors for development of candidemia infection include invasive procedures, a prior use of antibiotics and parenteral nutrition. c. albicans was the most common species, detected in . % of patients. nonalbicans candida species were prominent ( . %), represented by c parapsilosis, followed by c. tropicalis and c. krusei then c. glabrata and finally c. lusitaniae. all the isolates tested were sensitive to the common antifungal agents. the mortality rate of our patients was high ( . %), and the detection of the albicans species in blood cultures was the only prognostic factor identified (or = . [ . - . ], p = . ). conclusion: candidemia in the medical icu patients is common and is associated with high mortality rate. despite the progress of biological tools, the diagnosis is difficult and needs to take into account the risk factors of the patients as well as scores based on clinical and microbiological parameters. a better identification of risk patients may help to early initiate empirical antifungal treatment. compliance with ethics regulations: yes. necrotizing soft-tissue infections in the intensive care unit: a retrospective hospital-based study kais regaieg, sabrine nakaa, arnaud mailloux, madjid boukari, johana cohen, dany goldgran-toledano groupe hospitalier intercommunal le raincy-montfermeil, montfermeil, france correspondence: kais regaieg (kais.regaieg@gmail.com) ann. intensive care , (suppl ):p- rationale: the objective of our study is to describe the epidemiological and clinical characteristics of necrotizing soft-tissue infections (nsti) and to improve therapeutic management. we conducted a retrospective observational study that included patients admitted in the intensive care unit (icu) of general hospital between september and aout with a primary or secondary diagnosis of nsti. we collected demographic and clinical data, cultured pathogens, lengths of stay, and in-icu mortality. results: during the study period, a total of patients admitted to the icu were diagnosed with nsti ( . % of the total number of patients). the mean of age was years. the sex ratio (m/w) was . . ten patients ( %) were directly admitted to the icu, others were transferred from medical or surgical wards. the mean of saps ii was . ( . ). the main indication to admission in icu was shock ( %). the most common comorbidity was diabetes ( %). the other co-morbidities associated with nsti were cardiovascular diseases ( %), obesity ( %) and carelessness ( %). the sites most commonly affected were extremities in patients ( %) and abdomen/ano-genital in patients ( %). in icu, a total of patients ( %) were mechanically ventilated [ (median duration: . days ( . )], patients ( %) were given vasopressors, and patients ( %) underwent renal-remplacement. all patients underwent one or more chirurgical intervention. patients ( %) underwent radical necrosectomy. in cases, an amputation was necessary. polymicrobian infection was seen in patients ( %). in patients ( %), we used vacuum assited closure therapy, which in patients was followed by definitive reconstruction by split skin grafts. the mortality in icu was %. the mean stay in icu was days . the mean duration of hospitalization of the patients who survived was days ( - ). on the basis of a univariate analysis, higher saps ii score and lactate levels were associated with increased mortality (p < . ). conclusion: ntsi is rare in icu but it's a life-threatening and disabling disease with a high mortality requiring a multidisciplinary management. early diagnosis and adequate treatment are necessary to improve clinical outcome and must be known by everyone. more studies are needed to estimate the interest and delay of new strategies such as negative pressure therapy. compliance with ethics regulations: yes. rationale: nosocomial infections remain a major cause of mortality and morbidity in burn patients. providing information about the main causative bacterial agents and determination of their susceptibility to antibiotics may improve empiric therapy and early detection of emerging antimicrobial resistance. the aim of our study was to investigate the species distribution and antibiotic susceptibility of isolated strains from a burn intensive care unit (icu). patients and methods: this study was performed retrospectively on all bacteriological samples taken from the burn icu at the trauma and burn center in tunisia during a seven year period (from january to december ). all isolated microorganisms were identified on the basis of standard microbiological techniques. antibiotic susceptibility testing was carried out by the agar disk diffusion method, and susceptibility results were interpreted using clinical breakpoints according to ca-sfm guidelines. minimum inhibitory concentration of colistin was determined using the e-test ® method (biomérieux), then using the eucast broth micro-dilution method (umic, biocentric ® ) since may . results: during the study period, the most frequent identified species were pseudomonas aeruginosa ( . %), staphylococcus aureus ( %), klebsiella pneumoniae ( . %) and acinetobacter baumannii ( %). these strains have been mainly isolated from blood cultures ( %) and skin samples ( . %). pseudomonas aeruginosa resistance to ceftazidime increased from . % in to . % in and resistance to imipenem and ciprofloxacin was . % and . %, respectively. four strains were resistant to colistin. rationale: community-acquired peritonitis is a heterogeneous condition characterized by peritoneum inflammation in response to a bacteria injury. the aim of our study is to describe the epidemiological, clinical, bacteriological, etiological, therapeutic characteristics of community peritonitis, and to evaluate the prognostic factors. patients and methods: this is a retrospective descriptive and analytical study spanning three years (between january and december ) involving cases of community peritonitis, hospitalized in the surgical emergency resuscitation department p ibn rochd casablanca university hospital. our study included adult patients with community-acquired peritonitis who underwent medical and surgical management. the studied parameters are the demographic data, the clinical and paraclinical signs, the care taken and the evolution of the patients. the study showed that the mean age was . ± . years, with a sex ratio of . . patients medical history included tobacco ( . %), extra-abdominal signs [hemodynamic failure ( %), renal failure (n = , %), hematological disorders (n = , %) and respiratory disorders (n = , %)]. therapeutic management was based on perioperative resuscitation, treatment of organ failure, probabilistic antibiotic therapy and median laparotomy surgery. the main etiologies of community peritonitis were: digestive perforation ( . %), purulent effusion ( %), intestinal necrosis ( . %), cholecystitis ( . %). intraoperative bacteriological specimens yielded the following bacteriological profile: predominance of ngb ( . %) dominated by e. coli ( . %) followed by klebsiella pneumoniae and enterobacter cloacae ( . %) the mean hospital stay was . ± . days. the mortality rate was . %. conclusion: improvement in the prognosis of community-acquired peritonitis can only be achieved by constant assessment of very early diagnosis and initiation of appropriate resuscitation and antibiotic therapy associated with a complete surgery carefully codified according to guidelines. compliance with ethics regulations: yes. rationale: klebsiella pneumoniae carbapenemase (kpc)-producing bacteria are a group of emerging highly drug-resistant gram-negative bacilli causing infections associated with significant morbidity and mortality. the aim of our study is to point out the incidence of bloodstream infections (bsi) caused by kpc in icu patients, its clinical presentation and course. patients and methods: we conducted a retrospective descriptive study. all patients hospitalized in the icu of our hospital who developed bsi caused by kpc from january , to december , were included. results: during the study period, patients were included. the mean age was . ± . years ranging from to years. sex ratio (m/f) was . trauma was the major cause of hospitalization in cases ( %). the most common past medical diseases were arterial hypertension in patients ( %). length of hospital stay prior to icu admission was ± . days. at infection onset, mean saps ii was ± . , mean sofa was . ± . and mean apache ii was . ± . . during icu hospitalization, all patients required invasive mechanical ventilation during . ± . days, had a central venous catheter (cvc) and an indwelling urinary catheter in place, patients ( . %) had tracheotomy, ( %) underwent surgery, ( %) presented acute kidney failure and ( %) needed hemodialysis. before the isolation of kpc, all patients presented infections. antibiotics prescript were: colistin in patients ( %), carbapenems in patients ( %), amoxicillin/clavulanic acid in patients ( %), cephalosporins in patients ( %), fluoroquinolones in patients ( %), tigecycline in patients ( %), aminosids in patients ( %), rifampicin in patients ( %), fosfomycin in patients ( %), glycopeptides in patients ( %). the delay for kpc-bsi onset was . ± . days. the most common infection sources responsible of kpc-bsi were: cvc in patients ( %) and pneumonia in patients ( %). kpc infection was responsible of septic shock in patients ( %). resistance rates were: gentamycin ( %), amikacin ( %), colistin ( %), fosfomycin ( %) and tigecycline ( %). antibiotics used to treat kpc bloodstream infection were resumed in table . the mean length of icu stay was . ± . days. out of the included patients, patients died (the mortality rate was %). death was related to kpc infection in patients. conclusion: the high prevalence of kpc-bsi in icu patients dictates the importance of implementation of infection control measures and strict antibiotic policies. compliance with ethics regulations: not applicable. we identified episodes of nosocomial infections in patients, representing a cumulative incidence rate of . per exposed patients. the incidence density was . infections per days of hospitalization. the prevalence of pneumonia was . %, followed by urinary tract infections . %, central venous catheterization infections . %, bacteriemia . %, meningitis . % and surgical site infections . %. the incidence rate of intubation-related pneumonia was . / day of exposure. the incidence rate of bladder-related urinary tract infection was . / day of exposure. the incidence rate of positive culture of the central venous catheter was . / day of exposure. the incidence rate of bacteremia related to stay was . / day of exposure. the mortality rate was . % with a significant difference between infected and uninfected patients (p = . ). microorganisms were gram negative bacteria in % of cases. conclusion: epidemiological surveillance of healthcare-associated infections is needed to establish prevention plans. compliance with ethics regulations: not applicable. in the prehospital setting, early identification of septic shock (ss) with high risk of mortality is essential to guide hospital orientation (emergency department (ed) or intensive care unit (icu)) prior to early treatment initiation. in this context, the severity assessment is most of the time restricted to clinical tools. in this study, we describe the association between prehospital shock index (si) and mortality at day of patients with ss initially cared for in the prehospital setting by a mobile intensive care unit (micu in this study, we reported an association between prehospital si and mortality of patients with prehospital ss. a si > . is a simple tool to assess severity and to optimize prehospital triage between ed and icu of patients with ss initially cared for in the prehospital setting by a micu. the association of si with biomarkers may be helpful to improve the screening for ss and decision making of ss in the prehospital setting. compliance with ethics regulations: yes. the failure rate and complications were comparable between the groups, but the ultrasound-guided internal jugular catheter appears to be faster to insert and requires fewer punctures, so it could be an alternative to the femoral one in emergency situations. rationale: neuromyelitis optica (nmo) is a rare but severe disease. the prognosis of treated nmo attacks remains unclear. we evaluated our practice, the early evolution and the prognosis of nmo patients. patients and methods: an observational study was performed on patients with nmo attacks presenting with visual or medullar symptoms admitted for plasma exchange (pe) therapy from january to august . treatment efficiency was defined as a negative shift of the visual or motor disability score (edss). nonparametric mann-whitney and fisher exact tests were used for statistical analysis as required. results: twenty-four patients had pe sessions. characteristics of the cohort are described in table . ( . %) died from complications of nmo attacks. treatment had an effect in ( . %) patients. the shift in the ambulatory and visual edss was respectively − . + . and − . + . . the non-survivor patients had all aqp antibodies (p < . ). residual edss was higher in the non-survivor group ( . + . vs . + . , p < . ). pulse steroids were administered in ( %) patient in the non-survivor group vs ( %) patients in the survivor group (p < . ). twelve ( %) patients previously given pulse steroid therapy responded to pe. discussion: we assessed the handling of nmo attacks and identified our flaws. we concluded that pulse steroid therapy should not be withheld or replaced by lower dosage. we also need to find a way to make attacks identified by physicians earlier to shorten the delay between its onset and patient's admission in a specialized care unit. we observed that the mean improvement is modest during the early phase of our treatment. but a modest improvement in the edss can have a great impact in the patient's quality of life and even survival. conclusion: nmo attacks remain a threatening disease despite aggressive treatment. shortening the delay of treatment and ensure adequate pulse steroid therapy coupled to pe could be a way to improve the prognosis. compliance with ethics regulations: yes. rationale: acute kidney injury in trauma patients is a problem that has been little studied in the intensive care unit (icu). its occurrence has been shown to be associated with high morbidity and mortality. we aim to determine the outcome of icu trauma patients with acute kidney injury (aki), including the incidence of death in the icu, of nonreversible renal impairment and icu complications. patients and methods: this is a prospective study, conducted in the department of emergencies and icu, including trauma patients with a minimum icu stay of days. renal failure was defined based on the new kdigo classification. predictors of mortality and poor outcome were identified using univariate and then multivariate analysis. results: one hundred and fifty patients were admitted during the study period for the management of post-traumatic injuries, among which patients were included. the incidence of aki in the studied population was % ( cases) with ( %) diagnosed with stage one, ten ( %) with stage two and ten ( %) with stage three. the overall mortality of patients with post-traumatic aki was . % ( patients) with a mean icu lengh of stay (los) at ± days and of days on ventilator at ± . eight patients ( . %) needed renal replacement therapy and thirty-four had non-reversible renal impairement ( %). during icu stay, eight patients ( %) were diagnosed with pulmonary embolism. on univariate analysis, the following variables were associated to mortality in patients with post-tramatic aki including; age, hemodynamic instability on the day of diagnosis and bilirubin levels on the day of aki diagnosis. besides, according to our analysis, the use of renal replacement therapy and the non-reversibility of renal impairment during icu stay were also associated to icu mortality. among these factors, the non-reversibility of renal impairment in the icu was a predictor of mortality on multivariate analysis (p = . , or = , . in this cohort, the following variables were predictive of non-reversible renal impairment during icu stay; including age (with a best cut-off of years old), medical history of hypertension, higher iss and diuretics' administration. on multivariate analysis, the age (p = . , or = . , ci . - . ) and use of diuretics (p = . , or = , ci . - ) were associated to non-reversible aki in the icu. conclusion: our study confirms that post-traumatic aki in the icu is associated to high morbidity and mortality. the identification of outcome predictors could be valuable to guide the management of aki. compliance with ethics regulations: yes. rationale: the occurrence of acute kidney injury (aki) in trauma patients is a problem that has been little studied to date. its presence has been shown to be associated with an increased risk of morbidity and mortality in affected individuals. to determine the incidence of post-traumatic aki and identify its predictive risk factors that could be eventually prevented. patients and methods: this is a -month long prospective cohortstudy, conducted in the department of emergencies and intensive care unit (icu) of a university hospital, including trauma patients with a minimum icu stay of days. renal failure was defined based on the new kdigo classification. predictors of aki were identified using univariate and then multivariate analysis. results: one hundred thirty patients were admitted during the study period for the management of post-traumatic injuries, among which patients were included. the incidence of aki in the studied population was % ( cases) with ( %) diagnosed with stage one, ten ( %) with stage two and ten ( %) with stage three. on univariate analysis, older age and medical history of diabetes or hypertension were predictors of aki. injury assessment found traumatic brain injury (ais > ), glasgow (gcs) on admission, and the diagnosis of fat embolism to be associated to post-traumatic aki. moreover, hemodynamic instability on admission and during icu stay, shock-index on admission, the amount of fluid administered the use of vasoactive drugs, sepsis, hyperbilirubinemia, p/f ratio and acute respiratory distress syndrome (ards) were also associated to post-traumatic aki. among these factors, ards (p = . , or = , ci - ), fat embolism (p = . , or = , ci . ) without preload-dependence, and were unclassified. multivariate analysis (using variables collected prior to hypotension) identified the following variables as risk factors for the occurrence of hypotension associated with preload-dependence: preload-dependence before hypotension (odds ratio = . , p < . ), fluid removal rate by crrt (or = . per increase in sd, p < . ), and lactate levels (or = . per increase in sd, p < . ). in this single center study, preload dependence-associated hypotension was slightly more frequent than hypotension without preload dependence in icu patients undergoing crrt. testing for preload dependence to adjust fluid removal could help prevent hypotension incidence during crrt. rationale: few studies report the relation between functionnal brain alterations during and after icu stay and abnormalities of cbf displayed on tcd. using vti as hemodynamic parameter is unusual for evaluation of cbf. the purpose of this preliminary study was to compare the values of vti of healthy controls (c) versus icu (p) with usual parameters (i.e. diastolic (vd) and mean velocities (vm), resistance (ir) and pulsatility index (ip)). rationale: accurate diagnosis of the level of consciousness is a challenge and different states such as coma, vegetative state (vs) or minimally conscious state (mcs) are often confused while they convey meaningful prognostic information. this distinction rely on the coma recovery scale-revised (crs-r) gold-standard. however, this clinical scale is imperfect since unresponsive patients can exhibit genuine signs of consciousness using advance neuroimaging techniques. expanding the range of behaviors indexing consciousness at bedside is thus of decisive importance. patients and methods: we designed and proposed a new clinical sign of mcs, the habituation to auditory startle reflex (asr), based on the blink response to repeated sounds: either inhibition of the automatic asr response (extinguishable) or nohabituation (inextinguishable response). we prospectively tested this new sing in patients suffering from disorders of consciousness after severe brain injury and first compared its diagnostic performances with the current gold-standard (crs-r) using standard discrimination metrics (auc, sensitivity, specificity, likelihood ratios) and their % confidence interval. we then investigated the correlates of this new sign on two validated neuroimaging diagnostic procedures (multivariate eeg-based classification of the state of consciousness and fdg-pet metabolic index of the best preserved hemisphere) using an anova with the state of consciousness and the asr response as independent variable. rationale: although continuous electroencephalography (ceeg) is commonly recommended in neurocritical care patients, implementation of this monitoring in routine is facing the need for a specific training of professionals. we evaluated the effectiveness of a training program for the basic interpretation of ceeg to critical care staffs in a prospective multicentre study. patients and methods: after completion of a pre-test, participants (physicians and nurses) recruited in french intensive care units (icu) received a face-to-face eeg learning course, followed by additional e-learning sessions at day- (post-course), day- , day- and day- , based on training tests followed by illustrated and commented answers. each test was designed in order to evaluate knowledge and skills through correct recognition of predefined eeg sequences covering the most common normal and abnormal patterns. the primary objective was to achieve a success rate of more than % of correct answers at day- in at least % of participants. results: among participants, ( . %) completed the full training program and of these ( . %) full-training participants achieved at least % of correct answers at day- . paired comparisons between scores obtained at each evaluation demonstrated a statistically significant increase over time. at day , rates of correct answers were greater than % for all predefined usual eeg sequences, excepted for the recognition of periodic and burst-suppression patterns and reactivity, which were identified in only . % ( % ci . - . ) and . % ( . - . ) and . ( . - . ) tests, respectively. discussion: this multicentric prospective study, which evaluated a training program for the basics of electroencephalography offered to critical care teams, provides interesting information about the training process and its impact on learners according to their different characteristics. we believe that participants reflect the heterogeneity of the various use of ceeg in the critical care setting. participants came from university and non-university icus, and whereas some of them used to monitor patients with ceeg, others were in an implementation process when the last monitored neurocritical care patients with intermittent eeg. in accordance with previous studies, we focused to the entire medical and nursing icu staffs. conclusion: a -months training program aiming to teach the basic interpretation of continuous eeg in the intensive care units was associated with a significant attrition in participation over time. however, participants who received the full training program were capable to accurately recognize the vast majority of eeg patterns that are encountered in critically ill patients. compliance with ethics regulations: yes. mourad goulmane oran hospital and university center, oran, algeria correspondence: mourad goulmane (goulmane.mourad@univ-oran . dz) ann. intensive care , (suppl ):p- rationale: cerebral venous thrombosis (cvt) is a rare but very serious disease with various clinical and etiological aspects. unlike ischemic arterial accidents, epidemiological studies are limited. the aim of our work was to study the clinical, etiological and evolutionary features of cvt in the algerian population from a sample of patients. patients and methods: this is a retrospective observational study conducted in the neurology department of the chu d'oran between january and december . in a clinical context suggestive of cvt, the diagnosis of certainty was provided by brain mri coupled with mra. all subjects benefited from a complete etiological assessment. the anticoagulant treatment was based on the low molecular weight heparin relayed by the anti-vitamin k. the duration of the follow-up was months. results: the mean age was . ± . years, the sex ratio was ( f/ h), the onset was subacute in % of cases. the main early signs were headache ( . %), visual disturbances ( %), epileptic seizures ( . %) and motor deficit ( . %). thrombosis predominated in the upper sagittal sinus and lateral sinuses; parenchymal lesions were associated in / of the cases. gynecologic obstetric causes were by far the most frequent. the evolution was favorable in . % of the cases. discussion: cvt is characterized by its clinical polymorphism, its predominance in young women, and its most often favorable evolution. the causes are multiple and often intricate requiring the realization of a systematic etiological assessment even if the cause seems obvious. the treatment of choice remains early anticoagulation, based on heparinotherapy even in case of hemorrhagic softening. the characteristics of cvt in the algerian population are distinguished by a high frequency of gynecological obstetric causes. awareness campaigns for women of childbearing age are useful. compliance with ethics regulations: not applicable. rationale: the ct-dragon score was developed to predict longterm functional outcome after acute stroke in the anterior circulation treated by thrombolysis. its implementation in clinical practice is hampered by the plethora of variables included. in addition, the score has not been validated in important subgroups such as stroke patients undergoing thrombectomy. given these limitations, the current study was designed to evaluate the use of a simplified score based on machine learning, as a possible alternative. this single-centre retrospective study included patients treated for stroke, in the anterior and posterior cerebral circulation, between - and - . at days, favourable (modified rankin scale (mrs): - ) and miserable outcome (mrs: - ) were predicted by ct-dragon. machine learning selected the aim was to describe the adherence rates to gold guidelines in critically ill copd patients and to identify predictors of low adherence. patients and methods: a prospective cohort study conducted from december to april in a -bed medical intensive care unit of farhat hached hospital. all adult patients admitted for aecopd during the period of the study were included. demographic and clinical data were recorded. adherence to gold was evaluated. univariate and multivariate regression analyses were carried out to identify factors independently associated to non-adherence to gold guidelines. results: seventy-seven patients were recruited. patients' characteristics were : mean age, . ± years; male ( . %); median duration of the disease, [ - ] years; mmrc scale ≥ , ( . %); health insurance coverage rate, ( %); pulmonologist follow up, ( , %); frequent exacerbator (≥ exacerbations in the last year), ( . %); median exacerbations episodes, [ ] [ ] [ ] . long-term oxygen use and home mechanical ventilation were respectively used in ( . %) and ( . %). eight ( . %), ( . %) and ( . %) belonged to copd groups b, c and d, respectively. pharmacological treatment included: saba-ics combination, ( . %), laba-ics, ( . %), laba-lama, ( . %) and lama-laba-ics, ( . %). overall adherence to gold guidelines treatment recommendations for the different stages of copd was ( . %). two patients ( . %) were over treated and ( . %) were undertreated. inappropriate treatment rate was ( %) in gold b, ( . %) in gold c and ( . %) in gold d. univariate analysis identified two factors associated with non-adherence to gold : the absence of pulmonologist follow-up ( % vs. . %; p = . ) and the low income ( . % vs. . %; p = . ). in multivariate analysis only the lack of pulmonologist follow-up was identified as an independent risk factor associated with gold guidelines discrepancies (or, ; % ci [ . - . ]; p = . ). there is a lack of adherence to gold guideline treatment recommendations in tunisian copd patients. this may lead to severe exacerbations. discrepancies were due to the poor access of severe copd patients to an appropriate pulmonologist follow-up. compliance with ethics regulations: yes. the operating theaters concerned were: the otolaryngology block, ophthalmology, vascular and thoracic surgery, and gynecological surgery. all patients over years of age were enrolled using the clinical parameters of difficult intubation (arne score > ), which will benefit from orotracheal intubation. the main judgment criteria were: first-pass success rate, intubation time, which is defined as the time between inserting the slide into the patient's mouth and obtaining the capnography curve, the cormack-lehane score and the pogo score (percentage of opening of the glottis). statistical analysis used spss software. results: a total of patients were included. no cases of failure with this device were observed, the duration of intubation was on average . s (only cases required more than min). the cormack-lehane score and involved patients ( . %), and the pogo score greater than % involved patients ( . %). one case required the features of the simplified score. discrimination, calibration and misclassification of both models were tested. results: % had proximal anterior stroke, % proximal posterior stroke and % lacunar infarcts in either circulation. in % no thrombus was objectivated. % of patients were treated with thrombectomy, % received thrombolysis and % underwent both thrombolysis and thrombectomy. % only received anti-platelet therapy. the area under the receiver-operating-characteristic curve (auc-roc) for ct-dragon was . ( % ci . - . ) for favourable and . ( % ci . - . ) for miserable outcome. r ofct-dragon was . and . for favourable (lack of fit, p = . ) and miserable (lack of fit, p = . ) outcome respectively. misclassification rate was % for favourable and % for miserable outcome with ct-dragon. selection of predictors from the ct-dragon was done by logistic regression, bootstrap forest and decision tree analysis. nih stroke scale, pre-stroke mrs and age were identified as the strongest contributors to favourable and miserable outcome, and included in the simplified score. auc-roc was . ( ci% . - . ) and . ( ci% . - . ) for the prediction of favourable and miserable outcome respectively. r was . and . for the prediction of favourable (lack of fit p = . ) and miserable (lack of fit p = . ) outcome respectively. misclassification rate was % for favourable and % for miserable outcome with the simplified score. the simplified score had better discriminative power than ct-dragon for both outcomes (both p < . ). the ct-dragon score revealed acceptable discrimination in our cohort of both anterior and posterior circulation strokes, receiving a variety of treatment modalities. the simplified score had a better discrimination, while maintaining comparable and good specificity and misclassification rate for miserable outcome. the simplified score needs further validation in a prospective, multi-centre study. compliance with ethics regulations: yes. rationale: the gold report represents a major revision to gold strategy guidelines. it brings new recommendations regarding diagnosis, severity assessment, and both pharmacologic and non-pharmacologic treatment of copd. however, adherence to evidence-based therapeutic guidelines is often poor in low-income developing countries and represents a significant barrier to optimal management. the setting up of an lma-fastrach (desaturation). a case of glottic edema has been noted. discussion: this study shows a very high success rate with this technique ( . % in the first trial and . % in the second trial), in the context of a predictable difficult intubation. the video-airtraq allows a very good visualization of laryngeal structures, a shortening of the duration of intubation, and is rarely responsible for immediate or secondary complications. all the data in the literature go in the same direction. conclusion: at the end of this work, our perspectives are to update the difficult intubation procedure, integrating the video-airtraq into our algorithm, as well as into our difficult intubation trolley. to take into consideration the cost of this device to eventually generalize it to all our structures. compliance with ethics regulations: yes. ) and beds of continuous monitoring. the activity of the cp is organized in a medical visit in the morning and in conducting projects in the afternoon. the activity is presented using a -years balance sheet results: the activity of pharmaceutical interventions (pi) or answers to requests from teams is shown in table . the solicitations doubled the second year. the cp is involved in the conduct of internal or polar projects (set up of cooperative sedation, nutrition…), the good use of health products (relay iv/po, infusion, crushed tablets and compatibility with gastric probe, drug incompatibilities, proton pump inhibitors…), the efficiency of the drug circuit (link with the pharmacy, reflection on the improvement of the circuit, regular meetings with nurses), medico-economic analysis of health products spending and the formalization of actions by protocolisation. he is also very involved in clinical research: patient screening, clinical study setup: blipic study (beta-lactam's dosing in pneumonia in icu in patients treated by continuous renal replacement therapy; clinicaltrials nct ) or in candiarea project (invasive infections to candida and preemptive treatment guided by biomarkers; in progress). a satisfaction survey submitted at months to nurses ( answers/ ) or to doctors/ residents ( / ) reported cp competence in the accompaniment of teams (> %) [in medico-economical, contribution of knowledge, vigilance reflex…], relevance of information transmitted (> %) [administration of drugs, dosage adjustments, …] and his relationship adapted to the units (> %) [communication, availability] . the development of clinical pharmacy in icu involves mastery of the specificities of icu by the cp, requiring a learning period and relationships adapted to clinical situations and teams. many health products projects specific to critical care are coordinated by the cp and made possible by medical and paramedical involvement. the cp appears as a vector of good use both in medical (reasoned prescription) and paramedical (good practices) with increasing solicitation of teams since his arrival. this reception has been facilitated by an innovative approach of clinical pharmacy deployment in our icu on an impulse of the clinical pole compliance with ethics regulations: yes. predicting models such as the news has been developed in the emergency department, but it has only been fewly evaluated in the icu. heart rate variability (hrv) reflects the autonomic nervous system response in various pathological situations and may vary according to patients' physiological status. the rox index, which reflects the acute respiratory failure severity, seems to be a good predictor of high-flow nasal canula failure. the aim of this study was to evaluate the potential value of news, hrv and irox (inversed rox) as poor outcome predictors, using artificial intelligence and machine learning. a retrospective analysis of a prospective datawarehousing project (reastoc clinicaltrials identifier nct ) on icu patients who did not require invasive ventilation. physiological parameters were collected on admission, within a -h delay. news, hrv (in time, frequency, and non-linear domains), and irox were computed and integrated into the prediction model. analysis was performed using medcalc and matlab machine-learning work-package. results: one hundred and twelve patients were included. patients who died in the icu (n = ) had highest news as compared with icu survivors ( . [ . - . ] vs. . [ . - . ] respectively; p = . ). the irox was higher ( . [ . - . ] vs. . [ . - . ], p = . ) and most hrv parameters also depicted higher values for icu survivors. considering a composite icu prognostic outcome parameter (mortality and/or need for any form of respiratory assistance and/or an icu los > median los), there was also a difference for news, hrv and irox (p < . ). the best value to predict icu mortality for news was (auc = . , p = . ), irox > . (auc = . , p = . ) and hrv (shannon entropy) > . (auc = . , p = . ). the best model to predict the need fo respiratory assistance combines irox and hrv (sd /sd ; auc = . , p = . ). adding shannon entropy on this model predicts either the need for respiratory assistance and icu survival (respectively auc . , p = . and auc . , p = . ). in icu spontaneously breathing patients, news, irox and hrv are different in between survivors and patients who died. the best model to predict the need for respiratory assistance combines irox and hrv (sd /sd ). compliance with ethics regulations: yes. rationale: sepsis is known for its important mortality in critically ill patients. the last guidelines defined sepsis as life threatening organ dysfunction. it rejected the concept of systemic inflammatory response syndrome (sirs) associated to suspected or confirmed infection, and considered the concept of dysregulated response to infection. actual guidelines recommend the quick sequential organ failure assessment score (qsofa) to identify patients with sepsis especially when outside intensive care unit. thus, outcomes have mainly to judge the value of sirs in the sepsis- era. the purpose of our study was to compare whereas qsofa score or the sirs criterion are superior to predict in-hospital mortality, shock and mechanical ventilation use in sepsis. our study includes patients in whom the sepsis- definition is met. therefore, this inclusion was retrospectively performed throughout emergency department (ed) admission cases for clinically suspected infection. we collected patients admitted to ed for sepsis. mean age was years ± with bornes of and . men were % of the patients. death occurs in . % of patients, sepstic shock in % and the use of mechanical ventilation in . %. qsofa ≥ has a significant association with in-hospital mortality (p < . ) but not sirs ≥ ( . ). neither qsofa ≥ nor sirs ≥ has association with the use of mechanical ventilation (p = . vs. p = ). whereas, both have a significant association for prediction of septic shock. the absolute sensitivity and negative predictive value in our study can be explained by the small size of our sample. this needs confirmation with literature data about the fact that sirs criterion are superior in term of sensitivity and npv than qsofa to predict septic shock. despite the weak odds ratio (or) of sirs before that of qsofa and the poor specificity and positive predictive value (ppv), we can conclude that sirs according to its sensitivity and npv, seems to persist useful in the sepsis- era as a reliable prognostic tool in the ed. this may need more large studies for confirmation. conclusion: despite sirs has no significant association with mortality in sepsis, it has largely higher sensitivity and superior npv to predict septic shock than qsofa in ed. compliance with ethics regulations: yes. our study aimed to determine the predictive factors of mortality in our patients. retrospective study over years in the intensive care unit of the hospital august. all patients with septic shock were included. a p value < . was considered significant. results: patients were collected. the age ranged from to years old. the average duration of hospitalization in pre-intensive care was days. the reasons for admission: (febrile respiratory distress: % of cases, polytrauma: % and % for sepsis), the most frequent infections: pulmonary ( %) and blood ( %). % received prior antibiotic therapy and % were immunocompromised. the overall mortality was %. the analytical study of the data shows that the age, the length of stay before admission in intensive care and that in intensive care, fever, hypothermia, slimming, hypotension, collapse, failures (respiratory, hematological, renal, hepatic and neurological) and the use of catecholamines are correlated with mortality, whereas sex, chest pain, tachycardia or bradycardia and mottling are not predictive of mortality. conclusion: despite improved techniques for the diagnosis and treatment of patients with septic shock, mortality remains high, especially in the presence of certain risk factors, hence the value of prevention in immunocompromised patients and the reduction in their length of stay in a hospital setting. compliance with ethics regulations: yes. conclusion: p. mirabilis is among the leading bacteria responsible for nosocomial infections in icu. they are emerging highly drug resistant pathogens whose incidence is rapidly increasing in icu. so that, it early identification with in vitro testing is of paramount importance to the success of infectioncontrol efforts. compliance with ethics regulations: not applicable. rationale: influenza is a potential lethal disease causing dozens of thousands excess deaths per year both in europe and in the united states. besides hygiene procedures, vaccination is a cornerstone of influenza prevention and guidelines recommend for vaccination among health workers (hw), especially if they are in close contact with frail people. despite these recommendations, the vaccination coverage is low among health workers both in europe and in the us. the relevance of a mandatory vaccination for health workers is currently a hot topic but data are scarce regarding intensive care unit health workers' opinion. patients and methods: health workers from medical, surgical and polyvalent icus received a link to the electronic record of the survey. results: among the icus, icu health workers (hw) (medical: and paramedical: ) were questioned. three hundred and forty-one icu ( %) answered, ( %) medical health workers (mhw) and ( %) paramedical health workers (phw) (p < . ). among mhw / ( %) were vaccinated vs only / ( %) phw (p < . ). discrepancies exist between medical and paramedical icu health workers' opinions and beliefs about vaccination for influenza and its acceptance. medical health workers were more prone to consider influenza as a potentially lethal disease occurring not only among frail people but also in healthy people, to consider the vaccine efficient and safe. to agree with "vaccination for influenza is mostly related with gain for pharmaceutical industry" (or: [ . - ] ) and to disagree with "the risk of guillain-barré syndrome is higher after an episode of influenza than after vaccination for influenza" (or: . [ . - ] ) were independently associated to the disagreement with a mandatory vaccination for icu hw. conclusion: vaccination for influenza should be strongly recommended as a tool of individual protection for icu health workers as for general population. as confidence in vaccine efficacy and concerns about vaccine side-effects impact the vaccination rate, objective information should be provided to icu health workers about the efficacy and the side effects of vaccination for influenza. compliance with ethics regulations: yes. rationale: intra-abdominal infections are a major cause of morbidity and mortality. sfar recommendations on this topic were published in february . the purpose of this work was to evaluate whether our antibiotic therapy was adequate for these recommendations and whether they were adapted to our unit. the secondary objectives were to look for different risk factors for mortality, to evaluate the impact of inappropriate antibiotic therapy, to evaluate the relevance of carbapenem prescription. this is a single-center retrospective observational study of secondary peritonitis in the tourcoing intensive care unit. for each peritonitis, the epidemiological data and the co-morbidities of the patients were collected. bacteriology and anti-infectious therapies were described to determine the rates of adaptation of our antibiotic therapy and that recommended by sfar. the adequacy of our treatments to the recommendations was also quantifiable. the description of the stay, the occurrence of a death was specified. results: peritonitis were included. the rate of adaptation of the sfar antibiotic therapy was %. the rate of adaptation of our antibiotic therapy was % and its adequacy rate of %. the main differences in prescriptions concerned over-prescription of antifungals, molecule against gram positive bacillus and a sub-prescription of aminoglycosides and beta-lactams, in particular carbapenems. the different mortality risk factors found were sofa score > (or . % ci . - . ), the charlson score > (or . % ci . - . ), the hollow organ perforation (or . % ci . - . ). a comparison of the appropriate or not antibiotic groups did not reveal a significant difference in mortality, number of surgical revision and length of stay. in % of nosocomial peritonitis, antibiotic therapy with carbapenem was recommended. after recovery of microbiological data, it was only necessary for . % of cases. conclusion: our work showed a low rate of compliance with sfar recommendations. these recommendations are applicable to our service by providing a particular reflection for fungal infections. our study does not show a correlation between mortality and inadequate antibiotic therapy, surgery remaining the major treatment. compliance with ethics regulations:yes. rationale: acinetobacter baumannii is a gram-negative opportunistic bacteria that has gained several drug resistance mechanisms over the last decades. analysis of a. baumanii's resistance profile helps to establish a prompt control and a prevention program. the aim of this study was to evaluate the epidemiology and antimicrobial resistance of a. baumannii isolates in a trauma and burn center in tunisia. patients and methods: retrospectively, we studied all strains of acinetobacter baumannii isolated over a -year period (from january to december ). conventional methods were used for identification. antimicrobial susceptibility testing was performed with the disk diffusion method, and susceptibility results were interpreted using clinical breakpoints according to ca-sfm guidelines. data were analyzed using the sir-system. minimum inhibitory concentration (mic) of colistin was determined using the e-test ® method (biomérieux), then using the eucast broth micro-dilution method (umic, biocentric ® ) since may . results: during the study period, non-repetitive strains of acinetobacter baumannii were isolated representing . % of all isolates, % of gram-negative bacilli (gnb) and . % of non-fermenting gnb. in our center, infections due to a. baumannii were endemic with epidemic peaks. a. baumannii was mainly isolated from burn intensive care unit ( %) and anesthesiology department ( . %). the most frequent sites of isolation were blood cultures ( . %), catheters ( %), respiratory specimens ( . %) and skin samples ( % sampling duration is also reduced, improving workflow. evaluators consider that bronchosampler rationalizes the cumbersome sampling process and that the closed system design reduces the risk of losing sample or sample contamination. the set-up, the suction capacity, the sampling quality and quantity have all been evaluated better or far better than that usually observed with usual sampling techniques and devices. finally, ( %) of users prefer bronchosampler to commonly used method. conclusion: this satisfaction survey shows that with its simple but revolutionary design, bronchosampler brings a real effective benefit in sampling procedure enabling the clinician to perform it alone, and ( %) of the survey evaluators consider that bronchosampler should replace their current practice. compliance with ethics regulations: yes. rationale: the possibility of having a sensitive, specific and prognostic biological marker for bacterial infections is a considerable challenge. a step was taken with the discovery of pracalcitonin. patients and methods: this is a prospective observational cohort study of patients in the medical resuscitation department of the university hospital of casablanca during the -month period, including patients in whom the pct was dosed. the data collected allowed us to form two groups according to the pct value: pct+ group with pct > ng/ml and pct− group with pct < ng/ml. the statistical analysis of these different data was carried out using epi info software version . . . results: % of our patients had a bacterial infection and % did not have one. we also distinguished community infections ( % of i+ patients) and nosocomial infections ( % of i+ patients). we found that the highest rates of pct were in nosocomial infections and the lowest pct rates were found in community-acquired infections. then, in each type of organ involvement we tried to vary the pct thresholds to . - and ng/ml in order to find the best threshold for which pct allowed to diagnose bacterial infection, justifying our choice of departure. we concluded that the best pct cut-off value in general was ng/ml, because it gave us the best sensitivity/specificity ratio ( % and % respectively) with a positive predictive value of % and a negative predictive value of %. the link between pct and bacterial infection was moderate (yule q-factor at . ). by analyzing the different therapeutic aspects, we showed that % of our patients had been treated with atb before the pct assay and that the broadest spectrum antibiotics available to our service were used in patients with pct levels the highest. finally, concerning the evolution, the higher the rate of pct, the higher the death rate, especially since % of patients with pct > ng/ml died. conclusion: procalcitonin is considered to be one of the best markers of systemic bacterial infection. indeed, its elevation is earlier than that of crp and its specificity is better compared to il- and il- . the rate of procalcitonin remains low in the presence of viral infection. procalcitonin is also a prognostic marker, its elevation is correlated with the severity of the infection, and its decrease is a good indicator of the effectiveness of antibiotic therapy. compliance with ethics regulations: not applicable. rationale: due to induction immunosuppression infection is the most common cause of mortality within the first year after lung transplantation (ltx). the management of perioperative antibiotic therapy is a major issue, but little is known about worldwide practices. we sent by email a survey to ltx centers around the world dealing with daily clinical vignettes concerning perioperative antibiotic therapy. we considered perioperative period as the period of the transplant surgery (per operative) and the postsurgery time before any infection occurrence (postoperative). after general questions on local practices, we asked each center for colonization definition and their diagnostic methods for microbial screening in recipients and donors. the clinical cases were related to specific issues concerning the management of antibiotic therapy in different clinical situations, including no prior colonization, prior colonization with sensitive or multi-drug resistant (mdr) microorganisms including prior colonization with mdr bacteria not sensitive to beta-lactams. the invitation and a weekly reminder were sent to lung transplant specialists for a single consensus answer per center between june and september . we received a total of responses from countries, mostly from western europe (n = ) and the usa (n = ), (fig. ) . systematic screening for bronchial colonization before ltx was mostly performed with sputum samples ( %), regardless of the underlying lung disease. definition of colonization was very heterogeneous and the delay between the last bacterial isolation in pre-transplant and the ltx to consider if the therapy should target these bacteria varied between week and more than year. in recipients without colonization, antibiotics with activity against gram-negative bacteria resistant strains (piperacillin/tazobactam, cefepime, ceftazidime, carbapenems) were reported in % of the centers, and antibiotics with activity against methicillin-resistant staphylococcus aureus (mainly vancomycin) were reported in % of the centers. for these recipients, the duration of antibiotics reported was days ( %) or less ( %) or stopped when cultures of donor and recipients were reported negatives ( %). in recipients with pre-transplant colonization, antibiotics were adapted to the susceptibility of the most resistant strain isolated in pre-transplant samples and given for at least days ( %). conclusion: practices vary widely around the world, but resistant bacterial strains are mostly targeted even if no colonization occurs. the antibiotic duration reported was longer for colonized recipients. compliance with ethics regulations: not applicable. the vancomycin was therefore considered as justified or not and appropriate or not. occurrence of nephrotoxicity and supratherapeutic exposure in this study group was compared to critically ill children control group. results: thirty one children receiving vancomycin lines of treatment whose ( %) observed a risk of acute kidney injury (aki) (n = ) and an aki (n = ) during the vancomycin treatment period were included. there was a trend to inversed relationship between plasmatic concentrations of vancomycin and estimated creatinine clearance (r = . ). seven patients observed a nephrotoxicity related to vancomycin, they had a higher plasmatic concentration of vancomycin (p = . ). seven patients ( %) had a supratherapeutic exposure to vancomycin. nephrotoxicity and supratherapeutic exposure were higher in children with or combined liver-kidney transplantation than in comparative critically ill children group. we found blood stream infection due to the central catheter and blood stream infections probably due to the central catheter. one hundred thirtyfive bacteria were identified of which ( %) were staphylococcus coagulase negative. nineteen ( %) lines of vancomycin were appropriate and ( %) were justified. conclusion: vancomycin could have been avoided in one third of children with liver or combined liver-kidney transplantation during the early phase of postoperative stage. vancomycin is associated with a risk of both nephrotoxicity and supratherapeuric exposure. vancomycin should be used with caution, appropriate indications and dosing in this vulnerable population. compliance with ethics regulations: yes. rationale: early bacterial infection is a major and severe complication occurring within the first month after pediatric liver transplantation (lt). the rise of antimicrobial resistance, especially extended-spectrum beta lactamase producing enterobacteriaceae (esbl-pe), is henceforth a concern for these patients. this study aimed to assess the epidemiology of early bacterial infections, including those caused by multidrugresistant (mdr) pathogens, and to identify the risk factors for infection. rationale: the number of cancer patients admitted to emergencies is clearly increasing and digestive oncology is the leading cause of consultation. the aim of this work is to identify the epidemiological factors, the therapeutic modalities as well as the predictive factors of mortality and to compare them with the data of the literature. patients and methods: patients admitted to visceral emergencies for an urgent syndrome revealing or complicating a primary or secondary digestive cancer, and who required immediatemedical and/or surgical intervention and who had stayed at the surgical resuscitation level in our hospital center for a duration of years. several data were entered on excel and analyzed using the spss version software.-epidemiological, concerning age and sex; -clinics including risk factors, history, general condition of the patient and clinical examination data; -para-clinical, interesting biological assessments, and morphological examinations-medical and surgical therapeutics; -postoperative follow-up-treatment results. the three most frequent sites were rated in order of increasing frequency: colo-rectum ( %), pancreas ( %), and stomach ( %). the age group most found was age over years with % of cases, % of patients had under years. this series includes men and women with a sex ratio of , . the installation method was mostly gradual with % of cases. our patients have consulted for urgent clinical presentations mainly occlusive syndrome noted in % of patients. abdominal ct was the first examination performed, followed by abdominal ultrasonography in % and %, respectively. the therapeutic management was medico-surgical. the surgery done in % of patients, % for palliative indication: % were operated for an ostomy discharge, % for a digestive bypass, % for a palliative resection and % for a stoma feeding. postoperative outcomes were % morbidity and % mortality. the main cause of death was septic shock in % of cases, thanks to multivariate statistical analysis three factors were deduced significantly related to mortality: the asa score: p = . ; or = . ; ic: [ . ; . icu and hospital mortality rates were % (n = ) and . % (n = ), respectively. ten patients were alive months after with a median rankin score at [ - ]. more than half of the patients without stupor had a favorable neurological outcome (fig. ) . in univariate analysis, mechanical ventilation and stupor were correlated with mortality, whereas dic and apl were not. by multivariate analysis stupor was the only factor significantly associated with a higher mortality (hr: . [ . - . ] ). conclusion: intracranial hemorrhage is associated with a high mortality rate in al patients, stupor at the onset of intracranial bleeding being independently associated with poor outcome. up to one third of patients will nevertheless survive and experience a favorable neurological outcome. compliance with ethics regulations: yes. neurological outcome assessing by modified rankin scale according to stupor or coma at intracranial hemorrhage diagnosis (blank reflect missing data) rationale: sinusoidal obstruction syndrome (sos, previously known as veno-occlusive disease) is a complication of high dose chemotherapy, frequently occurring during bone marrow transplantation (bmt). severe sos is associated with a high mortality rate, related to multi-organ failure (mof). defibrotide being the only available option for prevention and treatment. prognosis of patients with sos requiring intensive care unit (icu) admission remains unknown. the primary objective was to assess the outcome of these patients. secondary objective was to assess risk factors associated with hospital mortality. patients and methods: retrospective study conducted between january and july in french icus. critically ill adult patients with sos (according to ebmt classification) who received defibrotide were included. results are reported as median [iqr] or number (%). adjusted analysis was performed using cox model. results: seventy-one patients were included with a median age of years . underlying hematologic diseases were acute myeloid leukemia ( %), lymphoma ( %),myelodysplasia/myeloproliferative neoplasm ( %) or acute lymphoid leukemia ( %). sos occurred during myeloablative allogeneic bmt ( %), reduced conditioning allogeneic bmt ( %), autologous bmt ( %) or chemotherapy ( %, including gemtuzumab ozogamycin in patients). median sofa score at icu admission was ]. ebmt prognostic score was often "very severe" ( %). main reasons for icu admission were respiratory failure (n = ), acute renal injury (n = ), shock (n = ), liver failure (n = ), coma (n = ) and monitoring (n = ). median bilirubin level at icu admission was µmol/l [iqr - ] and platelets count g/l . mechanical ventilation (mv), vasopressors, and renal replacement therapy (rrt) were required in % (n = ), % (n = ) and % (n = ) of patients, respectively. sixteen patients receiving defibrotide experienced bleeding events. icu and hospital mortality rates were % and % respectively, mainly related to organ dysfunction. in univariate analysis, delayed defibrotide initiation, bilirubin level, organ supports, sofa, and ebmt scores were associated with hospital mortality. cox model identified older age (hr . , % ci . - . ), mv (hr . , % ci . - . ), rrt (hr . , % ci . - . ), as associated with mortality. prophylactic defibrotide was correlated with a better outcome (hr . , % ci . - . ). similar results were observed after adjustment for center effect. conclusion: when organ support is required, icu management is associated with high mortality. organ support (namely rrt and mv) and older age were associated with poor outcome. prophylactic defibrotide was associated with survival either due to selection process or to efficacy in this setting. additional studies are needed to confirm these results. compliance with ethics regulations: yes. rationale: prognosis of critically ill immunocompromised patients (ciip) has improved over time. neutropenia is common and is found in one third of these patients. prognostic impact of neutropenia remains controversial and little data focus on ciip admitted in a context of acute respiratory failure (arf). primary objective was to assess prognostic impact of neutropenia on outcome of these patients. secondary objective was to assess etiology of arf according to neutropenia. patients and methods: retrospective analysis of prospective multicenter multinational dataset. adults immunocompromized patients with arf were included. adjusted analyses included ( ) a hierarchical model with center as random effect; ( ) propensity score (ps) matched cohort; and ( ) adjusted analysis in the matched cohort. results: overall, patients were included in this study. median age was [iqr - ] and patients ( . %) were of female gender. median sofa score was [ ] [ ] [ ] [ ] [ ] [ ] [ ] and ps was [ ] [ ] [ ] [ ] . main immune defect were hematological malignancy in patients ( %), solid tumor in ( %), systemic disease in ( . %), and other immunosuppressive drugs in ( %). neutropenia at admission was observed in patients ( %). initial oxygenation strategy was oxygen in patients ( %), high flow nasal oxygen in ( %), non-invasive ventilation in ( %) and invasive mechanical ventilation in ( %). before adjustment, hospital mortality was significantly higher in neutropenic patients ( % vs. % in non-neutropenic patients; p = . ). after adjustment for confounder in a mixed model, neutropenia was no longer associated with outcome (or . , % ci . - . ). after ps matching, neutropenic and non-neutropenic patients were compared. hospital mortality was similar in both groups ( % vs. % respectively; p = . ). after adjustment for variables associated with mortality, neutropenia was not associated with hospital mortality (or . , % ci . - . ). arf etiologies were distributed similarly in both neutropenic and non-neutropenic patients (fig. ) , main etiologies being bacterial pneumonia ( % vs. %), invasive fungal infection ( % vs. %), pneumocystis jiroveci pneumonia ( % vs. . %), and undetermined etiology ( % vs. %) (p = . ). conclusion: neutropenia at icu admission is not associated with hospital mortality in this cohort of ciip admitted for arf. surprisingly, arf etiology did not differ despite the multiplicity of observed immune defects. compliance with ethics regulations: yes. rationale: hepatic dysfunction (hd) is commonly observed in patients with hematologic malignancies and associated with an increased mortality in allogeneic hematopoietic stem cell transplantation patients. we aimed to assess incidence, risk factors and prognostic impact of hd in a large multicenter cohort study of critically ill patients with hematologic malignancies. patients and methods: this research was a post hoc analysis of a franco-belgian multicenter prospective study assessing the prognosis of patients with hematologic malignancies admitted to intensive care unit (icu) between january and may . hd was defined as serum total bilirubin ≥ µmol/l at icu admission. for patients with hd, a review of medical hospital records was performed by an expert panel to assess management of hd by attending physicians. results: among the patients with hematologic malignancies admitted to icu, were included in the study, mainly patients with non-hodgkin lymphoma ( . %) or acute myeloid leukemia ( . %). hd at icu admission occurred in patients ( . %). factors independently associated with hd were the use of cyclosporine (or = . , % ci . - . , p < . ) and antimicrobial treatment (or = . , % ci . - . , p = . ) before icu admission, abdominal symptoms at icu admission (or = . , % ci . - . , p < . ), ascites (or = . , % ci . - . , p = . ), hepatic charlson comorbidity (or = . , % ci . - . , p = . ), increased creatinine at icu admission (or = . , % ci - . , p = . ), neutropenia (or = . , % ci . - . , p = . ) and myeloma (or = . , % ci . - . , p = . ). hospital mortality was . % and . % in patients with hd and patients with no hd respectively (p < . ). hd appeared as an independent factor of hospital mortality after adjustment with other organ failure (oradj = . , % ci . - . , p = . ). factors independently associated with hospital mortality among patients with hd at icu admission are reported in table . etiologic diagnoses for hd by physicians were undetermined for patients ( . %) including ( . %) for whom the existence of hd has not even been mentioned in the medical record. investigations were performed in % and only % of patients received a specific treatment for hd. conclusion: hd at icu admission is common, underestimated, poorly investigated, and impairs outcome in critically ill patients with hematologic malignancies. hd should be considered and managed as other organ dysfunctions. it raises the importance of an early severity assessment of hd and a development of diagnosis strategies to get therapeutic options, in close collaboration between hematologists and intensivists. compliance with ethics regulations: yes. rationale: acute respiratory failure (arf) is the main cause for admission to the icu for patients with hematological malignancies (hm). viral pneumonia is poorly described in this population. respiratory viruses pcr is a rapid and sensitive diagnostic tool. thoracic ct allows to guide the diagnosis but is also poorly described. the primary objective was to describe ct features suggesting viral pathogenicity. secondaryobjectives were to assess risk factors associated with the use of invasive mechanical ventilation (imv) and icu mortality. rationale: high-dose methotrexate (hd-mtx) is commonly used in the treatment of solid tumours and hematological malignancies. severe toxicities are frequent, leading to organ dysfunction, multiple organ failure and death. outcome of these patients when critical illness occurs is poorly studied. this study aims to describe mtx-induced toxicities and to assess outcome in critically ill patients. in this retrospective study conducted in the icu of one university hospital between january and december , all the patients who were given hd-mtx (single dose greater than mg/m ) in the icu were included. results are presented as median [interquartile range] and number (percent). results: patients ( men and women) aged years [ - ], were included. b-cell lymphoma had been diagnosed in patients (burkitt, n = ; diffuse large b cell lymphoma with cns (central nervous system) involvement, n = ; primary cns lymphoma, n = ) and t-cell lymphoma in two patients. patients were mainly admitted for coma (n = ; %) or acute kidney injury (n = ; %). mtx was administered at a median dose of . g [ - ] . fourteen patients had concomitant medication interacting with mtx. median mtx clearance was days [ ] [ ] . frequent mtx-related complication were mucositis (n = , %), diarrhea (n = , %) or hepatic failure (n = , %). during icu stay, patients experienced acute kidney injury (kdigo stage . [ ] [ ] ). two patients received carboxypeptidase and three underwent dialysis. overall, patients ( %) required mechanical ventilation, ( %) vasopressors. hospital mortality was % (n = ). cox model identified mtx concentration h after administration higher than . µmol/l as associated with hospital mortality (hr . , % ci . - . ) (fig. ) . conclusion: to our knowledge this is the first study assessing characteristics and outcome of critically ill patients receiving hd-mtx. mtx concentration at h was associated with hospital mortality. despite underlying malignancy, icu support of these patients was associated with a meaningful survival. compliance with ethics regulations: yes. rationale: high-dose methotrexate ( g/m ; hdmtx) is the cornerstone of chemotherapy in acute lymphoblastic leukemia (all) and several high-grade non-hodgkin lymphoma (hnhl). despite standardized prevention, acute kidney injury (aki) and other life-threatening complications still occur. given the cost of glucarpidase, an enzyme that metabolizes mtx in few minutes, and the complexity of hematological patients admitted to the icu, a better comprehensive view of the factors that predict hdmtx toxicity, as well as the role of glucarpidase as rescue therapy in patients with organ failure, is mandatory. patients and methods: retrospective monocenter study including all the adult patients referred for all or hnhl in a french university hospital, and who received hdmtx. aki was defined according to the kdigo classification. univariate analysis (fischer exact or mann-withney tests) followed by multivariate analysis (stepwise logistic regression) were used to identify before hdmtx the clinical and biological predictive factors of aki. outcomes following glucarpidase were also addressed. results: from dec- to sept- , patients received hdmtx (median dose g/m ; all n = , hnhl n = ), totalizing hdmtx pulses. sixty-nine patients ( . %) developed aki after a median time of days (stage n = , stage n = , stage n = including one requiring dialysis in the first week). by multivariate analysis, only age, body mass index and a diagnosis of all were significantly and independently associated with the risk to develop aki. mtx exposure (maximal serum concentration at h - ) was also associated with aki (auc . , p < . ). glucarpidase was used in patients ( %) that differed by a higher age and bmi, and a lower basal egfr. glucarpidase was followed by a rapid renal improvement but serum creatinine did not return to baseline ( vs. micromol/l). thirty patients with aki or delayed mtx elimination did not receive glucarpidase but none required renal replacement therapy and egfr was only slightly but not significantly reduced at the end of follow-up. extra-renal adverse-events (rbc and platelets transfusions, neutropenia, hepatitis, severe diarrhea, mucitis) were more frequent in patients that developed aki. eighteen patients were admitted to the icu, including and that required mechanical ventilation or vasopressor drugs, respectively. conclusion: few actionable factors predict the development of aki after hdmtx, suggesting additional genetic factors. aki was reversed by glucarpidase but progression toward ckd was the rule. further studies will have to identify patients that will actually beneficiate from glucarpidase. compliance with ethics regulations: yes. khaoula ben ismail, sana khedher, ameni khaled, nassereddine foudhaili, mohamed salem usi digestif-service de gastroenterologie-eps charles nicolles.tunis-tunisie., tunisia, tunisia correspondence: khaoula ben ismail (khaoula @hotmail.fr) ann. intensive care , (suppl ):p- rationale: infection is common and accounts for major morbidity and mortality in cirrhosis. patients with cirrhosis are immunocompromised and have increased susceptibility to develop spontaneous bacterial infections, hospital-acquired infections, and a variety of infections from uncommon pathogens. we aimed to evaluate the impact of infection on hepatic encephalopathy. patients and methods: this is a prospective study, conducted over a period of years from january to december . consecutive patients with approved decompensated cirrhosis admitted to our department are included. all clinical and biological data were collected from the medical records. univariate and multivariate analysis were used to identify the impact of infection on hepatic encephalopathy. results: a total of patients diagnosed with decompensated cirrhosis were enrolled in this study. mean of age was years ( - ). sex ratio was . . hcv ( %) was the main etiology of cirrhosis. the reasons of hospitalization were: oedema with ascitic syndrome ( % of cases), digestive bleeding ( % of cases), fever ( % of cases), and encephalopathy ( % of cases). patients with infection seemed to have a high incidence of hepatic encephalopathy with % versus % when the patients are none infections. the results also showed that in those with hepatic encephalopathy, an effective antibiotic treatment accelerates significantly wakefulness under h with a rate of % vs. % (p < . ) . in addition, the infection does not influence mortality or length of stay compared to other complications such as digestive bleeding. conclusion: we found that infection caused more episodic hepatic encephalopathy than other complication and an effective antibiotherapy accelerate wakefulness. compliance with ethics regulations: yes. rationale: hepatic encephalopathy (he) is a common cause of hospitalization in patients with cirrhosis. pharmacologic treatment for acute (overt) he has remained the same for decades. to compare polyethylene glycol electrolyte solution (peg) and lactulose treatments in patients with cirrhosis admitted to the hospital for he. we hypothesized that rapid catharsis of the gut using peg may resolve he more effectively than lactulose. patients and methods: this is a prospective study, conducted over a period of years. from janury to december , we have been interested in cirrhotic patients with hepatic encephalopathy. all clinical and biological data were collected from the medical records. univariate and multivariate analysis were used to identify the difference beteween peg and lactulose in the treatement of hepatic encephalopathy. results: a total of patients diagnosed with decompation of cirrhosis were enrolled in this study. mean of age was years ( - ). sex ratio was . . hcv ( %) was the main etiology of cirrhosis. the hospitalization reasons were: edematous-ascitic syndrome in %, gastro-intestinal bleeding %, fever in %, and encephalopathy was present in % of cases. a total of patients were randomized to each treatment arm. baseline clinical features at admission were similar in the groups. twelve of patients in the standard therapy arm ( %) had an improvement of or more in hesa score, thus meeting the primary outcome measure, compared with of evaluated patients receiving peg ( %) (p < . ). the mean ± sd hesa score at h for patients receiving standard therapy changed from . ± . to . ± . compared with a change from . ± . to . ± . for the peg-treated groups (p = . ). the median time for he resolution was days for standard therapy and day for peg (p = . ). adverse events were uncommon, and none wasdefinitely study related. conclusion: we found that peg led to more rapid he resolution than standard therapy, suggesting that peg may be superior to standard lactulose therapy in patients with cirrhosis hospitalized for acute he. compliance with ethics regulations: yes. acute pancreatitis and pregnancy janati adnane, lina berrada obstetric intensive care unit, casablanca, morocco correspondence: janati adnane (adnanejanati@gmail.com) ann. intensive care , (suppl ):p- rationale: the association of acute pancreatitis and pregnancy is rare but not negligible, it often cause a diagnostic problem given the gravidal context that can lead to serious repercussions. the objective of our study is to assess the particularities in the diagnosis, management and prognosis of acute pancreatitis during pregnancy patients and methods: this is a retrospective study about cases of acute pancreatitis occurred during pregnancy over a -year period ( - ) at the obstetric intensive care unit of the meriem maternity hospital in the chu ibn rochd casablanca. a retrospective analysis of the medical files of these patients was carried out, considering epidemiological and etiological criteria, the treatments administered and maternal/fetal fate. we found cases during this period, with an incidence of / . the average age of onset was years, % of cases occurred in the rd trimester. epigastric pain and vomiting were the common symptomatology. ultrasound showed biliary lithiasis in % of cases with increased pancreas size in % of cases. maternal mortality was zero. uncomplicated benign forms are the most common ( %). severe hypokalemia was found in % of patients. neonatal morbidity was marked by six premature deliveries. among them, a newborn died at day- of life discussion: the association of acute pancreatitis and pregnancy is rare, more frequent during the rd trimester, it mainly affects the young woman. lithiasic biliary pathology remains by far the most frequent etiology. the diagnosis is clinical most often represented by epigastralgia with vomiting and biological via lipasemia and amylasemia dosage. uncomplicated benign forms are the most common. hydroelectrolytic disorders are often found. abdominal ultrasound allows the etiological diagnosis. the treatment is above all symptomatic whose objective is the digestive rest, the correction of the hydroelectrolyte disorders but first of all relieve the pain. conclusion: acute pancreatitis is a rare event in pregnant women, but can have a maternal and fetal prognosis. it must be systematically evoked in front of the acute abdominal pains of the pregnant woman because the confirmation of the diagnosis is easy and the maternal results depend mainly on therapeutic management. prematurity remains the predominant factor in neonatal morbidity. compliance with ethics regulations: not applicable. rationale: aclf is a clinical concept defined in patients with chronic liver disease who presented organ failure(s) secondary to an acute decompensated event. liver transplantation in this indication showed good results in selected patients. the aim of this prospective study was to evaluate the outcome and the factors associated with a favorable selection to liver transplantation in this population. patients and methods: all consecutive patients admitted to the icu with cirrhosis and aclf, were recruited. patient with age < years or with fulminant hepatitis were excluded. results: between july and february , cirrhotic patients were admitted to icu. mean age was . ± . years ( . % male). cirrhosis was due to alcohol in . % of the patients. aclf grading at admission was: . % aclf (n = ), . % aclf (n = ), . % aclf (n = ), and . % aclf (n = ). of the patients, . % (n = ) were considered to be eligible for a transplant project and were assessed for liver transplantation. the main reasons were alcohol abuse ( . %, n = ), death within days after admission ( . %, n = ) and rapid improvement of the liver disease. of the eligible patients, % were transplanted with a mean time between admission to icu and liver transplantation of . ± . days. twelve patients died on the waiting list ( % of the listed patients), mainly of septic shock. among those who were assessed for liver transplantation but not listed (n = ), . % died before the listing (n = ) and . % were not listed because of severe comorbidities (n = ). the global mortality rate was . % (n = ). the and days rate mortality were respectively . % and . %. the overall -month patient survival was respectively % and % in the transplant and non-transplant group (p < . ) for the entire cohort. among eligible patients, factors associated with the absence of liver transplantation, in the multivariate analyses, were mechanical ventilation (hr . , % ci rationale: body composition is known to be a prognostic factor in cirrhotic patients. however, the link between this and the prognosis of patients in intensive care unit (icu) is unknown. the computed tomography offer accurate estimations of muscle mass by analysing a cross-section usually going through the third lumbar vertebrae. this retrospective study aimed to assess the feasibility of body composition (bc) analysis in cirrhotic patients with septic shock, using computed tomography (ct) and evaluate the impact of bc (muscle mass, subcutaneous and visceral fat) on outcome. patients and methods: this retrospective study included cirrhotic patients with septic shock hospitalized in icu who underwent an abdomino pelvic ct scan within h of admission. we collected the surface areas of muscle mass and adipose tissue on the ct scans. we compared bc data with mortality and with the number of organ failures. the average age was years . the average child and meld scores were respectively . [ - ] and . . the prevalence of sarcopenia was %. it was not associated with a higher mortality rate at day (p = . ) or with a higher number of organ failures at day (p = . ). we observed a higher subcutaneous adiposity index in patients who died at day (p = . ) and in patients with renal insufficiency at admission (p = . ). there was a trend (p = . ) towards more visceral fat in patients who died in icu. the assessment by ct of body composition reveal evaluation of bc using ct is feasible and reproducible and may constitute a promising tool to evaluate in cirrhosis critically ill patients. visceral fat mass seems associated with poor outcome in cirrhotic patients with septic shock compliance with ethics regulations: yes. rachid jabi, mohammed bouziane chu mohammed vi, oujda, morocco correspondence: rachid jabi (jabirachid@gmail.com) ann. intensive care , (suppl ):p- rationale: the infection of the necrosis constitutes a pejorative element in the management of the necrotico-haemorrhagic pancreatitis, in the absence of the drainage the mortality approaches %. the morbidity and mortality of surgery can be avoided with minimally invasive treatments. purpose: to compare the morbidity and mortality of the two groups of post-ercp pancreatitis and the other etiologies. patients and methods: a retrospective study over years between and and a comparison between pancreatitis secondary to post-ercp and other etiologies of pancreatitis. a p value of . is considered significant. the surgical treatment used in cases of superinfection post ercp against seven cases of other etiologies of pancreatitis. high mortality in post-ercp pancreatic arm % vs. % (p = . ). high morbidity in the operated group % vs. % (p = . ) represented mainly digestive haemorrhages. duration of stay was significantly longer in the operated group vs. days (p = . ). thrombocytopenia and beta-lactamase-producing enterobacteria have further complicated management in the post-ercp infected pancreatitis arm. the antibiotic resistance of infected pancreatitis in post-ercp patients is . % for ciprofloxacin, . % for imipenem and % for amikacin. conclusion: pancreatitis the most common adverse effect of ercp with significant morbidity and mortality. the collaboration between the intensive care unit gastroenterologist and the surgeon improves management since the risk factors are mainly related to the patient and can not be modified. compliance with ethics regulations: yes. gautier nitel, aghiles hamroun, anne bignon, gilles lebuffe chru lille, lille, france correspondence: gautier nitel (gautier.nitel@gmail.com) ann. intensive care , (suppl ):p- rationale: liver transplantation (lt) has been recently experiencing an expansion of its indications, allowing patients with potentially more co-morbidities to access to transplantation. in our era of graft shortage, we should focus on the identification of the best lt candidates. the aim of our work is to study the determinants of early morbidity and mortality after lt from three angles: occurrence of a major cardiovascular event (mace) or acute renal failure (kdigo stage - aki) in the first days postoperative, and death in the year following lt. retrospective study investigating the occurrence of mace or aki (kdigo - ) within days post-operative and mortality at year after lt, including patients who received a first lt between january and december in our center. analysis of risk factors by a multivariate step-by-step analysis. statistical significance for p < . . data presented in odds ratio (or) rationale: infectious complications are frequently reported in critically ill patients supported by veno-arterial extracorporeal membrane oxygenation (va-ecmo) for refractory cardiogenic shock, but their diagnosis is challenging. no study has specifically studied bloodstream infection (bsi) in this population and some recommendations suggest performing systematic blood culture (bc). in our unit, systematic bc are daily sampled. we investigated the interest of systematic bc to detect bsi under va-ecmo. patients and methods: in a retrospective analysis ( - ), and after exclusion of patients dying within h, all adult patients from cardio-vascular intensive care unit supported by va-ecmo were included. systematic daily and "on demand" bc (at the physician's discretion) performed from va-ecmo implantation to days after withdrawal were analyzed. bsi was defined as at least one bc positive to a pathogen (except for contaminants bsi which required at least two positive bc with the same bacteria in h). multivariable logistic regression was performed to identify risk factors for positivity of systematic bc. rationale: fungal infections are constantly increasing in hospitals. indeed, the increase in these infections and especially candida yeast infections is almost parallel to the increase in the widespread use of a wide range of implanted medical devices such as catheters. for this reason, we have been investigating, isolating and identifying candida yeast colonizing vascular catheters and studying the epidemiological and clinical characteristics of patients with colonized catheters. patients and methods: it is a prospective, transversal study conducted at the intensive care and neurosurgery services of the sétif university hospital, evaluating the fungal colonization of vascular catheters. these are collected from hospitalized patients for a period of months. a culture of the distal end of the catheter is performed directly after its ablation. the results obtained showed that among the samples taken, six are colonized by the yeasts, the incidence is %. six yeast of candida spp were isolated, % of them were candida albicans species, . % candida parapsilosis and . % were candida glabrata. conclusion: it appears that colonization of catheters occurs most frequently in patients with the following characteristics: extreme ages of life, male sex, antibiotic therapy and length of hospitalization or prolonged catheterization. compliance with ethics regulations: yes. rationale: the threat of emergent extensively drug-resistant bacteria (exdr) dissemination worldwide is real. it has become a global public health issue. in fact, glycopeptides-resistant enterococcus faecium (gre) and carbapenemase-producing enterobacteriaceae (cpe) are the lead microorganisms in the high resistant bacteria category. the aim of our study was to characterize the molecular mechanisms and to determinate the antimicrobial susceptibility profiles of gre and cpe isolated from burn patients. patients and methods: prospectively, we studied all cpe and gre strains isolated from burn patients between january and december . all isolated microorganisms were identified on the basis of conventional microbiological techniques. antibiotic susceptibility testing was carried out by the agar disc diffusion method, and susceptibility results were interpreted using clinical breakpoints according to ca-sfm guidelines. molecular characterization was performed by multiplex real-time pcr (cepheid, genexpert ® ) allowing detection of the most prevalent carbapenemase encoding genes (blavim, blandm, blaimp, (blaoxa- and blakpc) as well as the genes vana and vanb of gre. results: during the study period, exdr were isolated from burn patients. the most frequent sites of isolation were blood cultures ( %) and skin samples ( . %). cpe represented . % of isolated exdr ( strains). among them, the most frequently identified species was klebsiella pneumoniae ( . %) followed by enterobacter cloacae ( %). twenty-four cpe ( . %) expressed the blandm gene. the blaoxa- gene was found in strains ( . %) and ten strains ( . %) carried both genes. of the cpe, . % revealed ertapenem mic > mg/l whereas most strains were susceptible to imipinem and meropenem with . % and . % of susceptibility, respectively. the antibiotics showing the highest resistance rates were cefotaxime ( . %), piperacillin-tazobactam ( . %), ciprofloxacin ( . %) and amikacin ( . %). the most active agents were colistin and fosfomycin with . % of resistance for each. seven strains of gre were isolated ( . % of exdr). all of them expressed the vana gene, with vancomycin mic > mg/l. however, teicoplanin mics ranged from to mg/l. all gre strains were beta-lactam resistant and highly resistant to aminosides. linezolid and tigecycline were the only active antibiotics. the dissemination of these extensively drug-resistant bacteria must be contained by implementation of strict isolation methods and better hygienic procedures in order to limit their economical and health consequences. compliance with ethics regulations: yes. rationale: stenotrophomonas maltophilia has emerged as an important pathogen that induces nosocomial infections. it is a non-fermentative, gram-negative bacillus that causes severe infectious diseases, particularly bacteremia in the hospital setting. morbidity and mortality due to stenotrophomonas maltophilia seems to be high, particularly in critically ill patient. the aim of this study was to describe the clinical features, management and outcome of patients with stenotrophomonas maltophilia infections. patients and methods: this was a retrospective analysis of prospectively collected data of patients hospitalized in intensive care unit (icu) between january and december . collected data were: age, gender, comorbidities, severity scores on admission, prior infections, use of antibiotics, use of invasive devices (urinary tract catheter, or mechanical ventilation), microbiological data, and antimicrobial therapy and outcome. results: during the study period, patients with stenotrophomonas maltophilia infection were included, with a mean age of ± years. the simplified acute physiology score ii and acute physiology and chronic health evaluation ii on admission were respectively ± and ± . bacteremia caused by stenotrophomonas maltophilia was observed in patients ( %) and ventilator acquired pneumonia in two patients ( %). twenty four episodes were classified as primary bacteraemia and only one as secondary bacteraemia due to urinary infection. four patients ( %) developed septic shock. mean sofa on the day of stenotrophomonas maltophilia infection was ± . prior antibiotic use was observed in % including an antipseudomonal agent in % of cases. infection due to stenotrophomonas maltophilia was considered in cases. empiric antibiotic therapy was administered to patients ( %) and had included an appropriate agent in only five cases ( %). after adapting antibiotics, monotherapy was the choice for six ( %) patients while a combination of two antibiotics was indicated in the others ( %). the most used antibiotic was the colistin in episodes ( %). intensive care mortality was %. univariate comparison between dead and survivors showed a significant difference in prior nosocomial infection and respiratory comorbidities. no independent risk factor of mortality was found in multivariate analysis. rationale: thrombocytopenia is a frequent disorder in critically ill patients, and several studies have reported its correlation with poor prognosis. considering the major role of platelets in hemostasis, a significant drop in platelet count is an alarming sign in septic patients. the aim of this study was to show the relationship between thrombocytopenia and platelet level changes and mortality in septic patients. patients with criteria for septic shock (based on the third international consensus definitions for sepsis and septic shock) at admission or at any time during hospitalization were included in a prospective study conducted for a period of months (january -august , ) in a medical surgical intensive care unit. patients hospitalized for less than h were excluded. thrombocytopenia was defined as a platelet count less than . /mm , and recovery was defined as returning to levels more than . /mm after presenting thrombocytopenia. we assessed the platelet count during the hospitalization and its outcomes. we included patients. the mean ± sd age was . ± . years. sex ratio was . . thrombocytopenia during sepsis (group ) was found in patients ( %) with a mortality rate at %. the mortality rate among patients not showing thrombocytopenia (group ) was significantly lower % (p = . ). the receiver operating characteristic showed that in (group ), a drop in the platelet count (from admission to septic shock day) more than % was associated with poor outcome (sensibility = %, specificity = %, auc = . ). among the (group ), % showed recovered platelet counts. the mortality was significantly higher in the patients with uncovered thrombocytopenia ( % vs. %, p = . ). conclusion: thrombocytopenia was shown to be an indicatorof poor prognosis in our study. in addition, drops of > % and failure to recover the platelet counts were further determinants of unfavorable outcomes. compliance with ethics regulations: yes. mehdi gaddas , sarra dhraief , karim mechri , imen jami , amenallah messaadi , lamia thabet rationale: pseudomonas aeruginosa is known as an opportunistic pathogen frequently causing serious infections. multidrug resistance in this bacterium is increasing worldwide and poses a major problem in the treatment of infections due to this microorganism. analysis of resistance profile to antibiotics of p. aeruginosa helps to establish a prompt control and prevention program. the aim of this study was to evaluate epidemiological profile and antimicrobial resistance of p. aeruginosa isolates in a trauma and burn center. patients and methods: retrospectively, we studied all p. aeruginosa isolates over a -year period (from january to december ). conventional methods were used for identification. antimicrobial susceptibility testing was performed with disk diffusion method and susceptibility results were interpreted using clinical breakpoints according to ca-sfm guidelines. data were analyzed using the sirsystem. minimum inhibitory concentration of colistin was determined using the e-test ® method (biomérieux), then using the eucast broth micro-dilution method (umic, biocentric ® ) since may . results: during study period, non-repetitive strains of p. aeruginosa were isolated, representing % of all isolates. in our center, infections due to p. aeruginosa were endemic with epidemic peaks. p. aeruginosa was mainly isolated from burn intensive care unit ( . %) and anesthesiology department ( . %). the most frequent sites of isolation were skin samples ( . %), blood cultures ( . %), catheters ( . %) and urines ( . %). the survey of antibiotic susceptibility showed high percentage of resistance to the different antibiotics: . % of strains were resistant to ceftazidime, % to ticarcillin, . % to pipercaillin-tazobactam, % to imipenem, . % to ciprofloxacin and % to gentamicin. resistance to colistin was rare. it concerned only four strains, isolated from burn patients. the survey of antibiotic susceptibility evolution have shown a global increase of resistance to commonly prescribed antibiotics between and : from % to . % to imipenem, from . to . % to ticarcillin-clavulanate, from . % to % to ceftazidime and from . to % to gentamicin. whereas ciprofloxacin resistance rate have decreased from . to %. antibiotic resistant strains were mainly isolated from burn intensive care unit, with % of resistance to imipenem and . % to ceftazidime. the dissemination of multidrug-resistant strains of p. aeruginosa in our center must be contained by the implementation of strict isolation methods and better hygienic procedures. compliance with ethics regulations: yes. acinetobacter baumanii: therapeutic impasse sabah benhamza, mohamed lazraq, abdelhak bensaid, youssef miloudi, najib el harrar réanimation de l'hôpital du août, casablanca, morocco correspondence: sabah benhamza (benhamzasabah @gmail.com) ann. intensive care , (suppl ):p- rationale: acinetobacter baumanii (ab) is frequently responsible for nosocomial infection in the intensive care units, and its resistance to antibiotics continues to increase. the objective of our study is to determine the epidemiological profile and antibiotic sensitivity of isolated bacteria in the intensive care unit august , in order to optimize the probabilistic antibiotherapy of bacteremia in intensive care. patients and methods: this is a retrospective study performed in the intensive care unit of the hospital august , , spread over a period of years from january to january . results: the incidence of ab infection in our department was . % for all patients admitted to intensive care. the average age was years ± , male predominance (sex ratio . ). the average time to onset of infection was days. during the study period, ab strains were isolated, % of which were pulmonary, % blood, and % urinary. resistance to c g reached % in , % in and % in . for imipenem resistance was % in , % in , % in . for amikacin, resistance was % in , % in , and % in . for fluoroquinolones resistance was % in , % in and % in . cotrimoxazole resistance was around % in the last years conclusion: the resistance of ab to antibiotics has reached very alarming levels, especially for carbapenems. this requires resuscitators to change their antibiotic prescription behavior and to invest in the prevention of nosocomial infections. compliance with ethics regulations: yes. this is a prospective observational study conducted at the ed during the period of year. data of all patients admitted with suspected infection of any cause were collected. poor outcomes were defined as death and transfer to an icu within h. results: during the study period, a total of patients with a mean age of ± were included. % were male. within h of management in the ed, % of patients were transferred to the icu and % died. independent predictors of icu-transfer and death included low systolic blood pressure, fever and tachycardia. a prediction model containing these independent predictors had a good predictive accuracy with an area under the curve of . ( % ci . - . ). sensitivity was %, specificity %, positive predictive value % and negative predictive value %. conclusion: assessing readily available clinical variables at arrival to the ed can aid in predicting poor outcomes. [ ] [ ] [ ] [ ] [ ] [ ] . the most common co-morbidities were chronic respiratory failure (crf, n = ) and hypertension (n = ). respiratory distress (n = ) and coma (n = ) were the major indications for iv. us diaphragmatic exploration was performed at a median delay of iv at days [ ] [ ] [ ] [ ] [ ] [ ] . % of patients received sedation and . % received neuromuscular blockers. the ventilator mode was control volume in patients via endotracheal tube (n = ) and tracheostomy cannula (n = ). no major incident was detected during the turning of patients. both tid and ted decreased from the sp to the pp (fig. ) : tid (mm) ( in sp vs. . in pp, p = . ), ted (mm) ( . in sp vs. in pp, p = . ). the observed dtf was lower in the pp but without significance ( . vs. . %, p = . ). no difference was showed when the comparison between sp-dtf and pp-dtf was adjusted on the ventilator mode, obesity, neuromuscular blockers and crf. the positioning in pp in ventilated patients reduces both tele-inspiratory and tele-expiratory diameters of the diaphragm but not altered its contractile function. compliance with ethics regulations: yes. significance was considered at p < . . results: results are presented in the table below. discussion: nebuliser type influences the efficiency of aerosol delivery, with the vmn delivering a significantly higher % aerosol dose than the jn at the two circuit positions (p = . on inspiratory limb; p = . at the dry side of humidifier). in agreement with previous reports using bias flow, for both nebulisers, the location within the circuit has a significant effect, with the nebuliser on the dry side of the humidifier delivering more aerosol than on the inspiratory limb (p = . for vmn; p = . for jn). conclusion: for a mechanically ventilated adult tracheotomy patient, the type of nebuliser and the location of the nebuliser within the circuit influences aerosol delivery. rationale: automatic tube compensation (atc) is a mode available in most icu ventilators. it compensates for the resistive pressure into endotracheal tube/tracheostomy canula by continuously providing a pressure assistance based on internal diameter of a new endotracheal tube/tracheostomy tube. its use in icu is unclear. we designed a survey to further explore this. patients and methods: the survey was endorsed by the acute respiratory failure section and the clinicaltrials group of the european society of intensive care medicine (esicm). the pool was sent out via an email on june to the esicm members worldwide. the following closed questions were: country, years in icu, kind of icu, kind of hospitals, kind of respirators, atc use (never, always or in some patients), reasons to or not to use atc, ventilatory mode in which atc was used. the database was frozen on august st after two reminders. we used the gross national income per capita (usd) provided by the world bank to transform the respondent's country into a geographical-economical variable with levels: high-europe, high-noneurope and middle ( ) . atc use was coded as yes or no. the primary end-point was atc rate of use and the hypothesis was that less than % of the respondents do use it. variables were expressed as counts. groups were compared by chi square test. a logistic regression analysis was performed to explore the contributing factors to atc use. we received responses without any doublons, of which six were empty, from countries. four-hundred and nine respondents used atc always or in some patients ( % atc rate of use). this rate was not different between economical-geographical regions, icu, hospitals and years in icu. for those respondents who did not use atc the reasons were: atc mode not available in icu ventilators ( . %), atc not helpful mode ( . %), atc not known ( . %) and atc provides too much pressure assistance ( . %). for those respondents who used atc the reasons were: helpful in weaning ( . %), set by default ( . %) and physiological benefit ( . %). they used atc during spontaneous breathing trial ( . %), with any assisted mode ( . %) and with specific modes ( . %). we found no risk factor for atc use in the logistic regression model (fig. ) . the atc rate of use was unexpectedly high in this survey. this may result from respondents selection bias or from an a priori underestimation of its use. compliance with ethics regulations: yes. rationale: during pressure support ventilation (psv), adjusting the level of assistance mainly aims at maintaining the patient's respiratory effort within a normal range. however, respiratory effort measurement is impeded in clinical routine by the need of esophageal pressure recording. in this study, we evaluated the accuracy of assessing the respiratory effort from the flow and airway pressure signals using several machine learning algorithms based on the equation of motion of the respiratory system. patients and methods: using the asl simulator (ingmar medical) connected to a pb ventilator (medtronic) set in psv, we simulated a massive number of different respiratory cycles. each simulated cycle represented a unique combination of compliance and resistance of the respiratory system, duration and intensity of the muscle pressure (pmus), positive end-expiratory pressure (peep) and pressure support levels. using least squares regression methods, the flow waveform was fitted according to the equation of motion of the respiratory system to determine the compliance and resistance of the respiratory system, and the pmus. the hypothesis used (alone or in combination) to constrain the system were: linearity of pmus at the onset of the inspiratory effort, nullity of pmus at the end of insufflation, and nullity of pmus during expiration. thus, nine methods were built and tested. calculated and actual peak pmus values were compared using the bland-altman method. the nine methods of pmus assessment were evaluated using different simulated cycles. by limiting the analysis to selected cycles with a predefined applicability criterion (intrinsic peep less than cmh o), a limited inspiratory effort (peak pmus less than cmh o) and a high quality of fitting (r > . ), the method using the three hypothesis together to constrain the system was characterized by a bias of . cmh o and limits of agreement of - . and . cmh o. however, when widening the analysis to all the simulated conditions, no method allowed an accurate estimation of the peak pmus : the best one exhibited a bias of - . cmh o and limits of agreement of − . and . cmh o. conclusion: among the nine machine learning methods tested, some provided an accurate estimate of the respiratory effort in selected cycles but none allowed such accuracy across all simulated conditions. this incites to assess automated methods using a more complex physiological and physical model. compliance with ethics regulations: not applicable. rationale: there is a growing interest in esophageal pressure monitoring in mechanically ventilated patients. esophageal pressure can be measured with a specific nasogastric catheter equipped with esophageal balloon and connected to a pressure transducer. it is used as a surrogate for pleural pressure and may be considered as a corner stone in advanced care of ventilated patients to better assess lung and chest wall mechanics and easily detect patient-ventilator asynchronies. however, this promising technique is still seldom used in clinical practice. trained icu nurses may perform oesophageal pressure measurements which may help facilitate its implementation in the usual patient care. this study aimed at assessing whether a specific educational program to train nurses to perform esophageal pressure monitoring allowed reliable measurements. this was a prospective monocenter study performed in an academic icu. written informed consent was obtained from the nurses before inclusion in the study. the specific educational program consisted of a -min online theoretical course, a -h group theoretical teaching and a -min simulation training on a mannequin. then each participating nurse performed three esophageal pressure measurements (using nutrivent ® catheters and an icu monitor connected to arterial line pressure transducers system) on three different mechanically ventilated paralysed patients under supervision. a knowledge assessment was performed with a short written mcq test. the skill evaluation was by two trained experts. concretely the trained nurses performed an esophageal pressure measurement without assistance. their ability to control the esophageal balloon position by an occlusion test, to measure the inspiratory and expiratory airway and transpulmonary pressures and to calculate of respiratory system, lung and chest wall compliances was assessed at the bedside using a standardized evaluation form. we present here the preliminary results of the first nine included nurses. the written knowledge assessment was considered as rationale: several modalities of ventilatory support have been proposed to gradually withdraw patients from mechanical ventilation. we conducted this study to compare t-piece and pressure support ventilation (psv) ( cmh and peep ) in the process of weaning of mechanical ventilation in burns. patients and methods: it was a prospective randomized trial in burn icu in tunisia during months. mechanically ventilated patients who met standard weaning criteria were included [ ] . patients were randomized into two groups: group under t-piece and group under psv. duration of the test: - min. the tolerance of the vs test should be judged on clinical criteria. stopping the test if occurred: agitation, tachypnea > cycles/ min, tachycardia > / min, spo < %. successful withdrawal was defined as the ability to maintain spontaneous respiration for h after extubation. results: thirty patients were included, randomized into two groups. the mean age was ± years with a ratio sex of . the average tbsa was ± %. the cause of mechanical ventilation was essentially a face neck burned ( %). the following table shows the weaning outcome of both modalities. eighty percent of succeeded extubation for both groups (n = / ). the cause of failure of extubation was secretion retention and clutter in majority of cases followed by neurological and cardiac distress. the duration of mechanical ventilation does not influence the outcome of the weaning test (p < . ), with a mean of duration of ± days. conclusion: our study did not show any difference between the two weaning modalities in the matter of outcome of extubation. the choice of weaning test of mechanical ventilation is to be judged by the clinician according of the state of his patient. compliance with ethics regulations: not applicable. rationale: when expiratory tidal flow does not go up after increasing expiratory driving pressure expiratory flow limitation (efl) occurs. it is thought that efl heralds airway closure (ac). we investigated the role of chest wall elastance (ecw) in both efl and ac in acute respiratory distress syndrome (ards) patients. our hypothesis was that the lower the ecw to lung elastance (el) ratio the higher the likelihood of efl and ac. patients and methods: twenty-five moderate to severe ards patients were prospectively included in two centers. mechanical ventilation was delivered in volume-controlled mode with tidal volume ml/kg predicted body weight at positive end-expiratory pressure cmh o in semi-recumbent position. airway (paw) and esophageal (pes) pressures and flow were continuously recorded during min by a data logger (biopac ). then, end-expiratory and end-inspiratory occlusions were performed for s, then respiratory system was slowly inflated at constant flow. finally, patient was allowed to breathe out freely to atmosphere by using a three-way stop lock by-passing expiratory valve. ac and airway opening pressure (aop) were determined according to chen et al. ( ) . efl was assessed by the atmospheric method ( ) . dynamic elastance of chest wall (edyn,cw) and lung (edyn,l) were obtained from least square linear regression method over consecutive breaths. static elastance (est,cw and est,l) were determined by classic formulas and also by taking into account aop (est,cw_aop and est,l_aop, respectively). the performance of ecw/el ratio to predict efl and ac was assessed by the area under receiver operating characteristic (aucroc) curve. results: efl was observed in patients ( %) and ac in ( %). median aop was . cmh o ( % ci . - . ) . aucrocs for ecw/el ratios to detect efl and ac are shown in table . edyn,cw/edyn,l ratio was better to detect efl than est,cw/est,l ratio with edyn,cw/edyn,l ≤ . % sensitivity and % specificity. correction for aop made the performance of est,cw/est,l ratio as good as that of the edyn ratio. ac was poorly predicted by edyn and est ratios but its prediction greatly improved with aop correction. however, with the est,cw/ est,l_aop the critical ratio was . (sensitivity %, specificity %) and . (sensitivity and specificity %) for predicting efl and ac, respectively. conclusion: efl and ac are frequent in ards at peep cmh o. edyn,cw/edyn,l ratio lower than best predicted efl occurrence. once ac is taken into account est,cw/est,l ratio greater than accurately predicts ac. efl and ac are two distinct phenomena. compliance with ethics regulations: yes. rationale: anesthesia outside the operatingroom (aoor) in a pediatric environment was giving increasingly increasing indications and a lot of progress because of its interest in carrying out diagnostic and/or therapeutic explorations: % of the acts of anesthesia are performed outside the operating room. the objective of our study is: to clarify the importance and the frequency of the practice of the ahbo, to define its particularities, as well as an evaluation of the ratio: benefit/risk in order to reduce the morbidity and mortality. patients and methods: we report in this study the experience of the service of the resuscitation mother-child on the gestures of aoor. this is a prospective observational study, spread over a period of months: from / / to / / , dealing with acts performed for endoscopic digestive and bronchial procedures, cures in dermatology and radiotherapy, and medical imaging (ct and mri). results: of the procedures performed: were performed for ct, for mri, for arteriography and for endoscopic digestive procedures, for bronchoscopies, for radiotherapy treatments, for laser treatments in dermatology. anesthesia techniques use intravenous induction in % of cases using: hypnotics (propofol, midazolam, ketamine), morphine (remifentanyl, fentanyl), inhalation induction in % of cases (sevoflurane, halothane) and curare for cases of bronchoscopy (rocuronium). this anesthesia was marked by the occurrence of accidents in order of frequency: cardiac in % of cases (tachycardia, hypotension and rhythm disorders), and then respiratory in % of cases. the most serious accidents were admitted in reality and are represented by cases, of which required an intubation (bronchoscopy), a case of cardiorespiratory arrest recovered, cases of severe hypoxia associated with bradycardia and which required the ventilation with the mask (radiotherapy), and cases of bronchospasm requiring the deepening of the anesthesia (absence of tci). a good knowledge of the patient and the intervention, and difficulties specific to each specialty is necessary, as well as a preanesthetic consultation. the aoor must obey the same safety rules as in the operating theater and that in terms of: equipment, monitoring (integrate the capnograph to respiratory monitoring whenever deep sedation and when the continuous control of vas is difficult), anesthetic technique (tcbi) and post-procedure wakefulness management that must meet the same requirements as the sspi, especially for prolonged sedation. compliance with ethics regulations: yes. umbilical vein catheterization through wharton's jelly: a possibility for a fast and safe way to deliver treatments in the delivery room? suzanne borrhomée hôpital rené dubos, france correspondence: suzanne borrhomée (suzanne.borrhomee@gmail. com) ann. intensive care , (suppl ):p- rationale: a fast and safe venous access can be a critical issue in the delivery room during neonatal cardiopulmonary resuscitation, or before endotracheal intubation. here, we describe a new method to inject drugs using the umbilical vein, directly punctured through wharton's jelly. this method was performed in newborns between november and may . umbilical vein was identified and punctured easily and a reflux was obtained in all patients. the first step was antisepsis, and then the umbilical vein was punctured. the puncture was made approximately to cm above the navel. after checking for blood reflux, the nurse injected the treatment. the cannula was left in the vein during the injection and removed as soon as the intervention was over (intubation was performed, or the heart rate had increased). results: here, we report ten cases of emergency injection in the delivery room using this method: -four cases of cardiopulmonary resuscitation using this method to deliver epinephrine. cardiac massage was performed on all patients.-six cases of intubations in the delivery room using this method to administer the premedication. in all patients, the umbilical vein was identified easily. the equipment was the one usually used for venous injection in our unit and was manipulated and handled with ease. venous access was obtained in a matter of seconds, and blood reflux was observed in all patients. the treatments were efficient in all but two patients, which was imputable to the method in one patient. discussion: although this method has been known in our nicu for several years, there has been no publication regarding this method in neonates. inserting an umbilical vein catheter in the delivery room has been validated for resuscitation but this technique is lengthy and requires some sterility conditions that makes it even longer, and thus non-fitting for an emergency tracheal intubation. our method is fast and can be performed easily with no specific training. the whole manipulation procedure, from the beginning of the puncture to the end of the flush-out takes to s. we only identified few specific risks related to this method, mostly infectious, and the risk of drug diffusion. we describe a new route for administration of drugs in the delivery room that was successfully used in nine neonates. umbilical vein needle catheterization is not only safe and efficient, but is also fast and easy to perform without any special training. compliance with ethics regulations: yes. rationale: liver transplantation (lt) is the only option for children with end stage liver disease. recent advances in surgical procedure and immunosuppression have permitted a better patient and long term graft survival. however, acute cellular rejection remains a frequent complication occuring in to % of the cases according to different studies. it is more likely to occur during the first weeks post lt. many predictive factors of acute rejection have been described in litterature and results differ from one study to another. pediatric studies regarding this topic are few. the aim of this work is to study acute cellular rejection prevalence in the days following lt and to determine predictive factors. rationale: sedation practices for pediatric magnetic resonance imaging (mri) are highly heterogenous. the main challenge is to keep children immobile while being alone in a traumatizing environment for a long time. clinicians have to ensure hemodynamic and respiratory stability in this isolated environment while minimizing sedation neurologic adverse effects. in this series, we report the potential usefulness, feasibility, efficacy and safety of dexmedetomidine sedation for pediatric mri. patients and methods: a single center retrospective review of six children sedated with dexmedetomidine for mri in an emergency context. all children were hospitalized in the pediatric intensive care unit of a university hospital at the time of mri. results: data on six patients aged months to years is reported. five patients received dexmedetomidine by intravenous route (bolus of - µg/kg over min, followed by a continuous infusion of µg/ kg/h). one child received dexmedetomidine by intranasal route ( µg/ kg with atomization device). one child experienced bradycardia that did not require any intervention. very few movements were recorded during the mris for which images were rated as good quality. conclusion: dexmedetomidine seems a promisingly useful sedation agent for pediatric mri, thanks to its efficient sedative properties and good tolerability without respiratory compromise. compliance with ethics regulations: yes. rationale: computational models, or virtual patients, could be used to teach cardiorespiratory physiology and ventilation, determine optimal ventilation management as well as forecast the effect of various ventilatory support strategies. currently, there is no virtual patient specifically designed for modelling children cardiorespiratory system. thus, our research team has developed a cardiorespiratory simulator for children called "simulresp©". according to summers et al., the quality of a physiologic model is evaluated by three specific criteria: qualitatively, which relates to the model's ability to provide directionally appropriate predictions; quantitatively in steady states and in dynamics, which is the ability of the model to provide accurate predictions in steady state situations as well as dynamic transitions. the purpose of this study was to evaluate the quality ofsimulresp© according to these criteria. this study consisted in a prospective evaluation of the simulresp©'s predictions with simulated healthy subjects. the tests were performed with patients from to years old ( , , , , , years), with different characteristics; gender (m, f) and weight ( th, th and th percentile). blood gas values (ph, pco , po and spo ) were simulated for several virtual healthy patients with different characteristics. this study was conducted for both spontaneously breathing and mechanically ventilated patients. simulresp©'s quality and reliability were evaluated in terms of accuracy, robustness, repeatability and reproducibility. results: simulresp©'s validation procedures are ongoing. we intend simulresp© to be accurate when simulating healthy spontaneously breathing patients. but we hypothezised that simulresp© would not be able to simulate accurate blood gas values of mechanically ventilated patients conclusion: simulresp© is a promising computational model that will serve to perform calibration and validation procedures of clinical decision support systems and help clinican to determine optimal respiratory support strategies at bedside. further calibration procedures are yet required. compliance with ethics regulations: yes. the isthmic surgical tracheostomy, which was performed in the operating room by otolaryngologist under general anesthesia. the cutaneous incision was transversal in all cases.the choice of the cannula was adapted to the age, and the decanulation was carried out according to the evolution of the underlying disease. complications associated with tracheotomy are diverse, and common complications are such as careassociated pneumonia ( . %), tracheostomy tube obstruction ( . %), accidental decannulation ( . %), pneumothorax ( . %) and cases of tracheal stenosis ( . %). the mortality rate amounted to . %, where in most cases was due to the poor prognosis of the underlying diseases. the main factors of evolution are the patient's previous condition, cranial trauma, guillain-barré syndrome, tracheostomy time, prolonged tracheal intubation and the presence of complications. conclusion: regardless of the indication, the tracheotomy is an act of survival whose usefulness and effectiveness are certain. rationale: aspiration pneumonia (ap) is a frequently suspected complication of drug overdose requiring mechanical ventilation (mv) and admission to intensive care unit (icu). in the absence of reliable biomarkers for distinguishing between aspiration pneumonia and aspiration pneumonitis, antibiotic therapy is frequently prescribed. latest studies suggest that a care protocol could better select patients requiring antibiotic therapy. the objective was to determine the impact of a care protocol on the antibiotic prescription among patient admitted to icu for toxic coma with mv. we conducted a prospective observational cohort study in four icu. we included all patients admitted for toxic coma with mv. in the university-affiliated icu, a care protocol was applied. in the three others icu, physicians declared that they did not follow formalized conduct within the service and did as usual. results: we included patients in care protocol group and in control group. the mean saps ii was . (± . ) with a mean glasgow coma scale score at . (± . ) before intubation. within the total population, patients ( %) had a pulmonary bacteriologic sample (pbs), mostly because purulent tracheobronchial aspirate and new infiltrates on the chest x-ray (respectively . % and . % of the population with a bacteriological sample). among the patients with a bacteriological sample, ( %) were culture positive. the incidence of probabilistic antibiotherapy did not differ between the care protocol group (n = ) and the control group (n = ) . there was no difference for the incidence of pbs ( in each group). the others secondary outcomes did not differ either (table ) . conclusion: our study does not show that a care protocol allows a reduction of antibiotic prescription among patient admitted to icu for toxic coma with mv. our incidence of antibiotic prescription is lower than the previous studies. the absence of difference can be explain by two reasons: some of the physicians of the control group had been trained in the university-affiliated icu in the last years and may follow a management approach similar to that of the control group; despite our precautions, the existence of the study could have modify the practices in the control group. compliance with ethics regulations: yes. rationale: pancreatic surgery is associated with high morbidity, mostly due to infectious complications, so that many centers introduce post-operative antibiotics for all patients. such systematic prescriptions are not consensual and often rely on local practices. the aims of the study were to describe the occurrence of surgical site infection (ssi) and the antibiotic (atb) prescription after pancreatic surgery, and to determine the risk factors of post-operative surgical site infection, in order to better define the clinical indications for the prescription of antibiotics after major pancreatic surgery. patients and methods: all patients undergoing a scheduled major pancreatic surgery from january to november were included in the study. patients were classified in four groups according to the occurrence of a surgical site infection and to the post-operative antibiotic prescription as follows (ssi+/atb+; ssi-/atb+; ssi+/atb-, ssi-/ atb-). in addition, risk factors (fever and pre-operative biliary prosthesis) associated with the occurrence of a surgical site infection and with the antibiotic prescription, were analyzed using a logistic regression model. results: data from patients ( pancreaticoduodenectomies and splenopancreatectomies) were analyzed and classified as presented in the table. thirty patients ( . %) experienced a surgical site infection and ( . %) received post-operative antibiotics. we did not find any difference on post-operative antibiotic prescriptions ( . % versus . %, p = . ) between patients who developed a surgical site infection and those who did not. amongst the patients who were not prescribed antibiotics post-operatively, ( . %) did not develop a surgical site infection while ( . %) did. in-icu mortality did not differ between infected and non-infected patients ( versus %, p = . ). post-operative fever was different between ssi+ and ssi-( . versus . %, p < . ), while the prevalence of pre-operative biliary prosthesis was similar ( . versus . %, p = . ). amongst patients who did not develop a surgical site infection, antibiotic prescription was not associated with fever (p = ), but associated with a higher prevalence of preoperative biliary prosthesis ( . versus . %, p = . ). conclusion: non-systematic antibiotic prescription after major pancreatic surgery allowed to appropriately spare antibiotics in ( %) patients at the cost of under prescription in ( . %) patients. these results suggest that systematic post-operative antibiotic prescription could be excessive. fever appears to be a relevant clinical sign for individual-based prescription, whereas the presence of a biliary prosthesis does not. compliance with ethics regulations: yes. ( , ) . however, there is little evidence to support those recommendations ( ) . we aimed to describe care paths of pm with sepsis in french hospitals and to assess outcomes depending on their hospital trajectory. we conducted a retrospective analysis of the french medico administrative (pmsi) database of consecutive patients with pm and sepsis admitted to french hospitals, between and . only the first hospital admission was considered. cases were identified using a combination of a diagnosis code for pm plus a diagnosis code for organ failure or a procedure code for organ support. hospital trajectories were determined from the first admission to death or discharge, taking into account all potential transfers. costs and endpoints were determined at the end of patients' trajectories. five groups of patients were defined, according to care pathways: direct icu admission ( sticu); secondary icu admission, after initial admission to another unit including wards (ward ndicu) rationale: new-onset atrial fibrillation (af) is a common complication in patients with sepsis and is associated with increased mortality and morbidity rates. this condition results from a complex chain of events in response to infection, involving immunologic, humoral and cellular process and sympathetic overactivity. landiolol, the new injectable beta-blocker, with high beta selectivity and minimal impact on arterial blood pressure, may have beneficial effects in such a context. in this study, we aimed to investigate whether landiolol decrease the newonset of atrial fibrillation in a mice model of endotoxin-induced sepsis. patients and methods: thirty c bl/ male mice were randomly allocated to the following groups: sham (administration of µl of isotonic saline intraperitoneally-ip), septic (administration of µl of isotonic saline with mg/kg of lipopolysaccharide-lps-of e. coli o :b ip) and septic + landiolol (administration of isotonic saline with mg/kg of lps and, two hours later mg/kg of landiolol ip). four hours later, an attempt of af occurrence was triggered by a transesophageal electric pacing at fixed rate (as previously reported) in all mice previously anesthetized by isoflurane %. ekg was continuously recorded. results: ten mice per group (mean weight: ± g) have been included and analyzed. among the sham group the mean heart rate was at bpm versus bpm in the septic group. among the septic + landiolol group the mean heart rate was at bpm (p < , ). after transesophageal stimulation, none mice in the sham group had af, seven mice ( %) in the septic group had an af, and mice ( %) in the septic + landiolol group had an af. landiolol decreased the incidence of new-onset, sepsis-induced atrial fibrillation in mice (p = . ). conclusion: landiolol seems to have a protective effect against sepsis-induced af in mice. however, the mechanisms, including sympathetic activation and inflammasome pathways, should be investigated before drawn definitive conclusion regarding to efficiency of landiolol to prevent new-onset af during sepsis. compliance with ethics regulations: yes. - mg/l at or h, proportion of patients with a vancomycin serum concentration < mg/l, previously associated with resistance emergence and assessment of mortality and test of cure. results: a serum vancomycin concentration between - mg/l was reported in out of included patients ( %). a serum vancomycin concentration < ml/l and > mg/l were reported in patients ( %) and patients ( %), respectively. vancomycin serum concentrations during follow-up are shown in fig. . in multivariate regression analysis, a longer time between admission and initiation of vancomycin was the only parameter associated with a serum vancomycin out of this target, while acute kidney injury (aki) was associated with a lower incidence of subtherapeutic concentration. acute kidney injury rate was significantly higher in patients with a serum vancomycin concentration > mg/l. discussion: an adequate therapeutic target of serum vancomycin concentration was reached in % patients with nearly % < mg/l, which was similar to previous studies. aki and rrt requirement were higher in patients with serum vancomycin concentration > mg/l, whereas it is hardly to know whether it is a cause or a consequence. conclusion: these findings highlight the importance of a larger loading dose, vancomycin monitoring and measured creatinin clearance to improve vancomycin dosing protocol. compliance with ethics regulations: yes. rationale: suicide is a global phenomenon and one of the leading causes of death in the world. tunisia ranks second in the suicide rate in the maghreb, with . cases of suicide per , inhabitants. the aim of this study was to reconstruct the state of suicidal subjects before the act in order to identify their psychiatric profile. patients and methods: a -year prospective observational singlecenter ( -bed intensive care unit) study including all patients hospitalized for suicide attempt (sa). psychiatric evaluation of patients and contact with their families were done before intensive care unit discharge. results: seventy-one patients were enrolled with female predominance (sex ratio . ). mean age was ± years. familial or personal history of mental illness were found in ( %) and cases ( %) respectively. personal mental disorders were depression ( %), bipolar disorder ( %), schizophrenia ( %) and border line personality disorder ( %). twenty-five per cent had prior sa. sixty-three per cent were single, % married and % divorced. the common methods of suicide included drug ( %), chloralose ( %) and pesticide ( %) poisoning. mean igs ii and apache ii scores were ± and ± respectively. on admission, % of all patients were in coma, % had shock and % developed aspiration pneumonia. mechanically ventilation was done in % of all cases with mean duration of days. the mean length of stay in intensive care unit was days. mortality rate was %. psychiatric evaluation and contact with families deduced that the main precipitating factors for suicide were traumatic events. in fact: relationship problems (familial, marital or breakups), school failure and mourning were found in %, % and % of all cases respectively. reactional sa accounted for %. rationale: poisoning is a worldwide problem, associated with high morbidity and moratlity. in tunisia, the rate of fatal poisoning has been increasing in the last years, with emergence of new toxic substances. regardless of the toxic, fatal poisining is considered as a non natural death, that requires medico-legal investigation, to assess whether it is suicidial, crimnal or accidental death. this study aimes to determine the epidemiological characteristics of the cases of fatal poisoning in south, to identify the toxics used in oder to deduce the preventive measures. patients and methods: we conducted a retrospective study of all cases of fatal poisoning recorded in the forensic department of habib bourguiba university hospital in sfax, tunisia, over a -years period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . results: during the study period, cases of fatal poisoning were autopsied. the number of victims recorded per year varied between and cases with an average of cases per year. the average age was years with extrems ranging from months to years. nearly half ( . %) were younger than years. a male predominance was noted with a sex-ratio of . . the majority of victims were single, loweducated and from rural origin. personal antecedent of psychiatric pathology was found in . % of cases. psychotic disorders (schizophrenia) and depression were the most common pathologies. in our study we noticed that death occured every weekday without significant difference between days. however, the frequency of fatal poisoning was slightly higher in cold seassons ( . %). in . % of cases, victims were found dead at home. accidental fatal poisoning was the most common ( %). no criminal cases have been observed. we noted a male predominance in accidental forms and a female predominance in suicidal forms. carbon monoxide poisoning was the most common ( cases) followed by the organophosphorus poisoning which was noted in cases. conclusion: decreasing the mortality rate from poison ingestion requires increasing public awareness about poisons and improving emergency service equipment and health personnel training. compliance with ethics regulations: yes. severe acute poisoning by organophosphate pesticides: report of cases at the oran hospital and university center mourad goulmane hospital and university center of oran, oran, algeria correspondence: mourad goulmane (goulmane.mourad@univ-oran . dz) ann.intensive care , (suppl ):p- rationale: organophosphate pesticides are synthetic organic pesticides widely used in agriculture mainly as an insecticide, nemacid or acaricide. these are the agricultural products, the most incriminated in poisoning in our context. the objective of this work was to determine the clinical, paraclinical, and progressive characteristics of this poisoning in a resuscitation environment. patients and methods: retrospective study of cases admitted to intensive care (january -december ). inclusion criteria were clinical, para-clinical, therapeutic and progressive. results: cases were identified: women and men, mean age = . ± years. the suicide attempt was the main reason for the intoxication ( cases). the glasgow coma score averaged ± . the central syndrome was present in % of our patients, followed by muscarinic syndrome % and nicotinic syndrome in % of cases. therapeutic management consisted of mechanical ventilation in % of cases, the use of vasoactive drugs in % of cases and the administration of antidotal treatment in % of cases. the overall mortality was . %. conclusion: organophosphate pesticides intoxication is a real health problem in algeria. it is a serious condition dominated by the respiratory and neurological distress that causes most deaths. it concerns in our context especially young women who ingest the product for the purpose of autolysis. the diagnosis is based on the clinical and dosage of cholinesterase activity in the plasma. treatment combines symptomatic measures that rely primarily on respiratory and neurological resuscitation to antidotal treatment. the clinical course in this type of intoxication is generally favorable under treatment with regression of signs in a few days. mortality is high in our context, so it should be considered a diagnostic and therapeutic emergency. the commercial availability of these products is worrisome, justifying the use of a broad prevention program to inform the public and authorities of the danger of organophosphate pesticides compliance with ethics regulations: not applicable. . the clinical examination revealed that five patients met the criteria for serious intoxication with the following signs: coma in four patients requiring the use of mechanical ventilation, seizures (n = ), rhabdomyolysis (n = ), shock (n = ), toxic takotsubo (n = ) and hepatocellular failure (n = ) leading to patient's death. the use of mechanical ventilation was necessary in patients. the analysis of the severity factors did not show a statistically significant association between severity, age (p = . ), sex (p = ) and chronic consumption of psychoactive substances (p = . ). on the other hand, we did not find a statistically significant association between serious intoxication, the number of tablets ingested (p = . ), the apacheii score (p = . ) and the average length of stay (p = . ). conclusion: ecstasy acute poisoning is becoming more common in our country and can potentially be very serious regardless of age, sex, medical history or number of tablets ingested. on the other hand, the concentration of nmda could be the only factor to be taken into consideration upon admission. compliance with ethics regulations: yes. quarter of early trauma-related mortality, in some series. early identification of poor outcome predictors could be valuable to guide the most appropriate care. we aim to determine factors associated to mortality in patients with severe non-penetrating chest trauma admitted to the icu. patients and methods: this is a prospective cohort study, including all patients with isolated severe blunt chest trauma (abbreviated injury scale ais > ) admitted to the intensive care unit of a university hospital, over a one-year period. the primary objective was to analyse risk factors associated to death and poor outcome using univariate and multivariate analysis. results: one hundred-thirty patients were admitted to the icu for blunt chest trauma among them were diagnosed with severe isolated chest trauma and were included. the mean age was at ± , mean iss at ± and mean tts at ± . twenty-eight ( %) patients were diagnosed with acute respiratory distress syndrome, ( %) with post-traumatic acute kidney injury and fourteen ( %) with post-traumatic pulmonary embolism. the mean length of icu stay (los) was at ± days and mean number of days on ventilator was at ± days. thirty-two ( %) patients underwent elective tracheostomy for prolonged intubation. thirty-seven patients ( %) developed infections, among them thirty ( %) were diagnosed with pulmonary infection and seven ( %) with non-thoracic infections. overall mortality had an incidence of . % ( patients rationale: early hyperglycaemia in traumatic brain injury (tbi) is a part of the stress response. it is an important indicator of severity and a reliable predictor of prognosis. we aimed to describe the epidemiological, clinical and paraclinical characteristics and to assess the prognostic impact of this hyperglycaemia on the tbi. we conducted a retrospective study in the intensive care unit (icu) of our hospital between and . were included all patients with tbi and blood glucose > mmol/l at the first h post-trauma. results: during the study period, patients were hospitalized in our icu with tbi. early hyperglycemia (> mmol / l) was found in patients ( . %). in univariate analysis, glycaemia > . mmol/l (= mg/dl) at admission was significantly associated with mortality (p = . ). we observed that glycaemia > . mmol/l at h , > . mmol/l at h , > . mmol/l at h and > . mmol/l at h was significantly associated with mortality (p = . ; p < . ; p = . and p = . , respectively). the risk factors significantly associated with mortality were age > years (p < . ), saps ii > (p < . ), initial shock (p < . ), glasgow coma scale (gcs) < / (p < . ), coma period > days (p = . ). the ct scan lesions statistically associated with mortality were: subdural hematoma (p < . ), cerebral oedema (p < . ), intra cerebral haemorrhage (p = . ), cortical contusion (p = . ), contusion of cerebral trunk (p = . ), contusion of the corpus callosum (p = . ), thalamus contusion (p = . ). in multivariate analysis, independent risk factors statistically associated with mortality were age > years old (or = . ic [ . - . ]; (p = . )), glycaemia > . mmol/l at admission (or = . ic [ . - . ]; (p = . )),gcs < / (or = . ic [ . - . ]; p < . ), intracerebral hematoma (or = . ic [ . - . ]; p = . ). we recommend a mandatory control of the blood glucose levels during a tbi with a target between . and . mmol/l in the acute phase. compliance with ethics regulations: not applicable. the fat embolism syndrome (fes) is a set of clinical, biological and radiological signs resulting in the obstruction of microcirculation by micro-droplets of insoluble fats.the clinical signs of the fes are not very specific, the diagnosis is difficult and the risk of misunderstanding this syndrome is very real.the fes appears after a trauma, often few days later. however, it sometimes occurs without previous trauma; and it is particularly difficult to recognize in these cases. the aim of this work is to define the epidemiological profile, the clinical and para-clinical features of this syndrome and its therapeutic management. rationale: sedative and analgesic treatment administered to critically ill patients with mechanical ventilation need to beregularly assessed to ovoid complications of oversedation mainly in elderly patients. our objective is to evaluate our sedation practice in the elderlyin our unit patients and methods: it was a prospective observational study, including elderly patients over years of age without acute brain injury requiring sedation more than h of hospitalization in the intensive care unit of our university hospital between april and december . thirty patients were included. the aged was . years, the sex ratio was . . respiratory distress was the most common reason for hospitalization %. the most accepted diagnoses were the decompensation of copd in % of cases and septic shock in % of cases. the saps ii averaged ± points, sofa averaged ± . points. renal failure was found in patients ( %), hepatic impairment was noted in patients ( %), hypoproteinemia was marked in patients ( %). midazolam was used in % of patients. it was in combination with fentanyl in % of cases and remifentanyl in % of cases. the median ramsay score . ± . on the first day of sedation and . ± . on the second day of sedation. the median rass scale was − . ± . on the first day of sedation and − . ± . on the second day of sedation. the median bps scale . ± . on the first day of sedation and . ± . on the second day of sedation. the mean wake up time was ± , days. neuromyopathy of resuscitation was suspected in seven patients ( %), withdrawal syndrome was observed in two patients ( %) and acute cognitive dysfunction in two patients ( %). the median duration of sedation was . days ± . days, the median duration of mechanical ventilation was . ± . days, the median length of stay was . ± . days. ventilator-associated pneumonia was diagnosis among % of patients. the mortality in intensive care was %. conclusion: sedation analgesia in the elderly person should be adapted according to age, ideal weight and renal and hepatic function by decreasing the initial doses. it should be evaluated by the recommended scores by setting a sedation objective according to the pathology. compliance with ethics regulations: not applicable. rationale: more than original articles are newly indexed in pub-med every day. journal club (jc) is one way to cope with this abyssal amount of medical information. we aimed at ( ) describing journals and articles analyzed during our jc sessions ( ), reporting the proportion of published articles being analyzed during jc sessions and ( ) assessing the clinical impact on our daily practices for each journal. patients and methods: a retrospective analysis of prospectively collected data over a -year period from to in a universityaffiliated icu. jc sessions were scheduled weekly and participants were free to choose and expose orally an article recently published in any medical journal (general, icu or non-icu specialized). clinical impact of a journal was retrospectively and independently assessed by two attending intensivists (dc, hm) and was defined by the ratio of articles considered as having a direct impact on our daily practices over the number of articles of the same journal read during the same period. results: from august to august , jc sessions were held and articles-mostly original (n = / ; %)-from journals were analyzed, accounting for . % of the articles ( . % of the original articles) referenced in pubmed during the same period. median number of articles exposed per session was [ ] [ ] [ ] [ ] . median number of doctors attending each session was [ ] [ ] [ ] (attendings: [ ] [ ] , fellows: [ ] [ ] , residents: [ ] [ ] ). general, icu and non-icu specialized journals accounted for %, % and % of the exposed articles, respectively. most of the reported articles dealt with intensive care (n = , %) especially infectious diseases (n = / ; %), hemodynamics (n = / ; %) or icu-organization (n = / ; %). compared to general and non-icu specialized journals, the proportion of read-over-published articles was higher for icu-specialized journals ( . % vs. . % vs. . %, respectively; p < . ). among original articles, only ( . %) [interventional (n = / ; %); observational (n = / ; %) studies] were considered as having a clinical impact on our daily practices. compared to icu and non-icu specialized journals, general journals had a higher clinical impact ( . % vs. . % vs. . %, respectively; p = . ). data regarding the most read general, icu and non-icu specialized journals are detailed in table . in a french university-affiliated icu with regular jc sessions, the proportion of read-over-published articles and the clinical impact of medical journals appear minor. in the ocean of medical literature, general medical journals appear more worth reading by intensivists than icu-specialized journals. compliance with ethics regulations: yes. rationale: the world's population is aging and the and over's age group is growing fast (+ . % per year). this aging population is impacting intensive care units with exponential rates of elderly patients ( . % in , % in ) , associated with significant mortality (from % to %). the evolution and the prognostic factors of these elderly patients in intensive care are therefore a public health issue for optimal management. patients and methods: we included all patients aged and over who were operated and admitted to surgical resuscitation in our center, with a duration of stay greater than h, from april to july . the data collected were: general characteristics of this population, mortality in intensive care, at day and at months and the prognostic factors guiding their evolution in intensive care and at months. results: of the patients included in our study, mortality was . % in intensive care, . % at day and . % at months. the prognostic factors in the intensive care unit were the average dose of noradrenaline at day (threshold at . mg/h), the sofa score at day (threshold at points) and the igs score (threshold at points). the prognostic factors at months were ventilatory autonomy on day (spontaneous ventilation, non-invasive ventilation, invasive ventilation), the reason for admission to intensive care (acute respiratory distress or septic shock) and the fragility score (clinical failure scale with a threshold at ). conclusion: the mortality of patients aged and over is influenced by prognostic factors easily obtained daily at patient's bed. these prognostic factors could be an aid for the resuscitation teams to evaluate the relevance of the care undertaken in elderly or even very elderly patients admitted in an acute situation. compliance with ethics regulations: not applicable. assessing patient safety culture perception in the intensive care unit in tunisia oussama jaoued, chaoueh sabrina, sik ali habiba, wael chemli, gharbi rim, fekih hassen mohamed, elatrous souheil hôpital taher sfar, mahdia, tunisia correspondence: oussama jaoued (oussamajaoued@gmail.com) ann. intensive care , (suppl ):p- rationale: in tunisia health care system, patient safety has become a priority of quality assessment. the aim of our study was to describe the safety culture perception of the intensive care unit staff. patients and methods: the safety attitude questionnaire (saq-icu) was distributed to all intensive care unit staff by email. the questionnaire explores safety culture domains: "team work", "safety climate", "job satisfaction", "stress recognition", "perception of the hospital and intensive care unit management" and "work condition". results: eighty participants responded to the questionnaire, % of them were women. participants were doctors in . %. the coordination between physicians and nurses was very good only in %. thirtynine participants thought that the workload was high and % like their work. medical errors are handled appropriately in % of cases and it was difficult to discuss errors in % of cases. the hospital is a good place to work in % of participants, % of participants were less effective at work when there were tired. the hospital did a good effort of training new personal in % of cases. the number of medical staff was lower than expected in % of cases. half of participants would feel safe being treated as patients in their respective units. all domains explored by saq-icu could be improved according to attendants. conclusion: safety culture perception among intensive care unit staff had several deficiencies, mainly the working conditions, the ignorance of medical error reporting procedures and the lack of communication. rationale: the simplified acute physiology score ii (saps ii) is an icu scoring system used to predict the mortality risk in patients presenting at the icu. however the majority of critically ill patients present initially at the ed and their transfer to the icu may be delayed for hours. therefore, the ability to accurately assess mortality risk at ed may have a great impact. the purpose of this study was to evaluate the performance of saps ii in predicting early and late mortality in ed patients. patients and methods: this prospective study was conducted at the ed during a -month period. data for adult ed patients were evaluated. saps ii score was used to predict early and late mortality rates at -h and -day respectively. discrimination was evaluated by calculating the area under the receiver operating characteristic curve (auroc). results: during the study period patients were enrolled. the mean age was ± years, % of the patients were men. the mean saps ii was . the early mortality rate was % and late mortality rate was %. saps ii was efficient in predicting early mortality, with an auroc of . ( % ci . - . ). however, it demonstrated no value in predicting late mortality with an auroc of . ( % ci . - . ) conclusion: in this study, saps ii score was accurate in predicting early mortality, however this tool appears less suitable for predicting late mortality. compliance with ethics regulations: yes. oussama jaoued, chaoueh sabrina, sik ali habiba, yosri ben ali, fekih hassen mohamed, elatrous souheil hôpital taher sfar, mahdia, tunisia correspondence: oussama jaoued (oussamajaoued@gmail.com) ann. intensive care , (suppl ):p- rationale: the aging of the population increased the number of hospitalizations in icu. the aim of our study was to determine the impact of hospitalization of patients over the age of on morbi-mortality and consumption of care (omega score). patients and methods: this is a retrospective study carried out in the icu in the hospital of taher sfar in mahdia over a period of years. all patients hospitalized in the icu were included in this study. two groups of patients were individualized: g : patients over years old, g : patients under years old. results: during the study period, patients ( < years old and ≥ years old) with a mean age ± years and with a mean sapsii ± were included. the common reason for hospitalization was acute respiratory failure in % of cases. comparing the two groups, the severity score sapsii was higher among patients older than years ( ± vs ± , p < . ). the use of mechanical ventilation was more common in the first group ( % vs. %, p < . ). the incidence of nosocomial infections was similar in both groups ( % in the group g and % in group g , p = . ) and the use of renal replacement therapy was also similar in tow groups ( % in the g group and % in the g group, p = . ). the duration of mechanical ventilation and length of stay were similar between the two groups. workload evaluated by the omega score was higher in the first group ( rationale: icu outcome depends on quality of pre-icu care. we aimed to assess the chain of care of deteriorating ward patients (dwp), through evaluation of preadmission severity and delays before admission, and association with outcome. patients and methods: retrospective observational study in a single center ( beds general hospital) for year-may th of to . all adult patients admitted in the icu from the wards were included, except for scheduled surgery, or unexpected event in the operative theater. preadmission severity was assessed through levels of national early warning score (news ): group with news inferior to , group with news between and , and group with news superior to . these scores were established from vital signs during the h before icu admission. patterns of patients, including sofa and saps , knaus index, charlson comorbidity score, cause of admission and technics used in the icu, length of stay in the icu and in the hospital, limitations of life-supporting care, and mortality at and days after icu stay. satistical analysis was performed through chi and fisher tests on qualitative parameters, and with kruskal-wallis, student and mann-whitney tests for quantitave data. results: sixty-eight patients were studied: in group , in group and in group . most patients (all except ) had not respiratory rate monitoring before icu admission. icu mortality was associated with rising preadmission severity (group : . %; group : . %; group : . %). base patterns (charlson comorbidity score, knaus index) did not differ between the groups, and . % of patients presented with sepsis. main causes of admission were respiratory ( . %), hemodynamic ( %) or neurologic ( . %) failures. all patients admitted after cardiac arrest resuscitation ( patients) belonged to group . acute severity scores (sofa and saps ) followed preadmission severity. limitation or withdrawing of life support in the icu was higher in group ( . %) than in groups ( %) and ( . %) . median delay between first news equal or superior to and icu admission was h, and h between news equal or superior to . diffrences in delays were not associated with outcome. discussion: our study outlines weaknesses in the chain of care of dwp. emphasis should be put on respiratory rate monitoring and better assessment of severity. rationale: access to critical care is controversial in older patients for reasons: lack of available icu-beds and speculation on induced costs. in contrast, admission of young patients aged or under is infrequently questioned even though they develop catastrophic multiple-organ failure requiring full care. in addition, emotive reaction triggered in staff by these patients often represents a heavy psychological burden when icu-stay is < h. information on the epidemiology, clinical information and induced costs regarding such patients is lacking. patients and methods: this study retrospectively assessed the records of patients aged or under, and admitted from january to august . cost-related expenses charged to care-payers were obtained from our medical information department. data (number, percentages or medians) were reported and discussed by comparison with those of nonagenarians during the same period. results: of , icu-admissions, were aged or under ( %), of whom ( . %) died within the icu, with ( %) dying within h of admission despite full intensive care. the latter represent our study population ( . % of the screened population). the median age was . years , male gender was prevalent ( %). half the patients (n = , %) were referred from the emergency department, ( . %) from hematology, from oncology ( . %), from medical intermediate care units ( . %), and one from digestive surgery ( . %). the first diagnosis at admission was septic shock (n = , . %), followed by post-anoxic encephalopathy (n = , . %), coma (n = , . %), acute respiratory failure (n = , . %) and cardiogenic shock (n = , . %). sapsii was . all patients were ventilated and infused norepinephrine. two patients underwent ecmo, and others mars. mean (± sem) retribution per stay was , ± €, and mean retribution per "day of stay" €. discussion: full care of these icu-patients, with early mortality has a financial impact similar to that of nonagenarians at , ± , €; the cost per "day of stay" is therefore on average % higher than that of nonagerians (mean length of stay: . days), and, in our experience, % higher than that of average patients. conclusion: icu-patients aged or under represent a small percentage of admissions and display half our overall mortality: one third of them die within h of admission with a not insignificant financial impact for cost-payers. septic shock is the first cause of referral, followed by unexpected cardiac arrest. compliance with ethics regulations: yes. rationale: severity scores in patients with sepsis are useful for triaging and predicting mortality. mortality in emergency department sepsis (meds) score is validated in patients with sepsis in the emergency department. curb- is validated in patients with communityacquired pneumonia but not in sepsis. curb- is a simple bedside tool that has many common elements with new sepsis identification score-q sofa. the study aimed to assess the accuracy of curb- score in predicting icu admittance and mortality compared to meds score. patients and methods: this prospective study was conducted at the ed during a -month period. we enrolled all adult patients with sepsis admitted to the ed. meds and the curb- scores were calculated at admission. patients were studied using curb- score and their icu admission and in-hospital mortality were ascertained. results: a total of patients were enrolled. the mean age was ± years. % of the patients were men. % of patients had a curb- score ≥ points with a mean meds score of %. among these patients, % were admitted to icu and % died. the curb- score,was efficient in predicting both icu admittance and in-hospital mortality with an auroc of . ( % ci . - . ) and . ( % ci . - . ), respectively. conclusion: a higher curb- score was correlated with higher rates of icu admittance and mortality in patients with sepsis due to any cause. compliance with ethics regulations: yes. abderrahim achouri, hadil mhadhbi, khedija zaouche, hamida maghraoui, radhia boubaker, kamel majed university hospital center rabta of tunis, tunis, tunisia correspondence: abderrahim achouri (achouryabderrahim@gmail. com) ann. intensive care , (suppl ):p- rationale: sepsis is a major cause of mortality. in other hand, preexistent chronic diseases seem to worsen outcomes among critically ill patients. the acknowledgement of this fact may motivate studies in this type of situations in order to improve survival in sepsis. on that purpose, our study tried to check the impact of chronic pre-existent illnesses on outcomes in this type of emergency patients. patients and methods: we have included patients in whom the sepsis- definition was met throughout emergency department admission cases for infection. in this study, considered outcomes were in-hospital mortality, shock occurence and the use of mechanical ventilation. results: we collected patients admitted to ed for sepsis. mean age was years ± with bornes of and . men were % of the patients. cormorbidities were: insulin dependent diabetes mellitus in . % of patients, non insulin dependent diabetes mellitus in . %, chronic obstructive lung disease in . %, chronic renal failure in . % with % in chronic replacement therapy from total patients, coronary artery disease in . %, with stent in . % and . % with aortic coronary graft from total patients, arterial hypertension in %, chronic heart failure in . %, atrial fibrillation in . %,. death occurs in . % of total patients, septic shock in % and the use of mechanical ventilation in . %. we did not find any association between comorbidity and the use of mechanical ventilation, but association with in-hospital mortality was found in pre-existent coronary artery disease (p = . ) and in patients with coronary artery stent (p = . ). odds ratio (or) was respectively . ( % ic = [ . - . ]) and . ( % ic = [ . - . ] ). we found significant association between chronic heart failure and shock (p = . ) with or = . ( % ic = [ . - . ] ). discussion: the small size of our sample may enlimit the contibution of other comorbidities on outcomes in sepsis such chronic renal failure, especially with renal replacement therapy and diabetes mellitus. whereas, we can conclude that cardiac diseases have the most important impact on outcomes in sepsis. outcomes in sepsis can be affected by comorbidities, especially cardiac diseases. therefore, that needs large studies to check it. compliance with ethics regulations: yes. micafungin population pk analysis in critically ill patients receiving continuous veno-venous hemofiltration or continuous veno-venous hemodiafiltration nicolas garbez , litaty mbatchi , steven c. wallis , laurent muller , jeffrey lipman , jason a. roberts , jean-yves lefrant , claire roger chu nîmes, nîmes, france; university of queensland, brisbane, australia correspondence: nicolas garbez (nicolas.garbez@umontpellier.fr) ann. intensive care , (suppl ):p- rationale: to compare the population pharmacokinetics (pk) of micafungin in critically ill patients receiving continuous veno-venous hemofiltration (cvvh, ml/kg/h) to those receiving equidoses of hemodiafiltration (cvvhdf, ml/kg/h + ml/kg/h). critically ill patients in septic shock undergoing continuous renal replacement therapy (crrt) and receiving mg micafungin once daily were eligible for inclusion. total micafungin plasma concentrations were analyzed using pmetrics ® . probability of target attainment (pta) was calculated from monte carlo simulations using -hour area under curve/minimum inhibitory concentration (auc - /mic) cut-offs (c. parapsilosis), (all candida species) and (c. non parapsilosis). daily dosing regimens of , and mg were simulated for the first days of treatment. results: eight patients were included in the study. micafungin concentrations were best described by a two-compartmental pk model. no covariate, including crrt modality (cvvh and cvvhdf), was retained in the final model, confirmed by internal validation. the mean parameter estimates (standarddeviation) were . ( . ) l/h for clearance, . ( . ) l for the volume of the central compartment, . ( . ) /h and . ( . ) /h for rate constants. the standard mg daily dosing was unable to reach % of pta for all candida species except c. albicans on the second day of therapy (fig. ) . conclusion: there was no difference in micafungin pk between equidoses of cvvh and cvvhdf. a dose escalation to mg is suggested to achieve the pk/pd target of candida species with mics exceeding . mg/l in this population. these "off-label" dosing regimens should be further investigated in clinical trials knowing the favourable toxicity profile and the post-antifungal effect of micafungin in order to ensure efficacy and to prevent the emergence of resistance due to an inadequate initial antifungal dosing regimen. compliance with ethics regulations: yes. rationale: sepsis is an important cause of morbidity and mortality in hospitalized patients. recognizing and responding to patients who experience clinical deterioration remains challenging in daily practice. our purpose was to assess the ability of the quick sequential organ failure assessment (qsofa) score to identify, among patients reviewed by an intensivist, those at risk of adverse outcomes. patients and methods: retrospective cohort of patients with suspected infection reviewed by an intensivist in a university-affiliated hospital between january and june . outcomes of interest were hospital mortality and a combined criterion of hospital mortality or icu stay of days or more. results: during the study period, patients were reviewed by an intensivist, of whom ( . %) had suspected infection according to the sepsis- criteria. at the time of review, ( . %) patients with suspected infection were qsofa positive (≥ ) and ( . %) were qsofa negative ( - ). following the review, ( . %) patients were admitted to the icu, among whom ( . %) had a prolonged stay (≥ days). in-hospital mortality was . %, and . % of the patients met the combined criterion of in-hospital mortality or prolonged icu stay. qsofa positive patients required more frequently mechanical ventilation ( . % vs. . %, p = . ) and vasopressor support ( . % vs. . %, p < . ) than qsofa negative patients. moreover, qsofa positive patients had higher hospital mortality than qsofa negative patients ( . % vs. . %, p = . ). for the prediction of in-hospital mortality, a positive qsofa had a predictive positive value (ppv) of %, and a negative predictive value (npv) of %. for the prediction of in-hospital mortality or prolonged icu stay, a positive qsofa had a ppv of % and a npv of %. conclusion: hospitalized patients with suspected infection for whom a review by an intensivist was requested, are at high risk of hospital mortality. although the accuracy of qsofa for identifying patients at risk of adverse outcomes is limited, its integration in a multimodal risk assessment approach may help distinguish the subset of patients who will benefit from an escalation of care. compliance with ethicsregulations: yes. rationale: according to the sepsis- consensus, sepsis is identified as an increase of at least points in the sepsis-related organ failure assessment (sofa) score in patients who presented infection. the quick sofa or qsofa is considered as a predictive tool of sepsis and mortality when it is equal to points or more. systemic inflammatory response syndrome (sirs) criteria are of limited utility because of their low sensitivity. hyperlactatemia, as known is a determinant of tissue hypoperfusion. our objective was to evaluate the prognostic value of sofa > , sirs > , qsofa > and lactate level > mmol/l in infected patients. nine-month prospective cohort study. patients aged years or older who had a proven or suspected infection were included. sofa score, sris criteria, sofa q and lactate levels were determined within the first h of infection. the primary endpoint was hospital mortality at days. the predictive power of the studied parameters was determined using using the area under the receiver operating characteristic curve (auroc). results: a cohort of cases was studied with mean age at . years. bacterial pneumonia was the most common infection site ( %). in the first h of onset of infection the medians [iqr - ] of the sofa, sris, and sofa scores and lactate levels were respectively [ ] [ ] [ ] [ ] [ ] [ ] [ ] , [ ] [ ] , [ - ] and . [ . - . ] . the progression to severe septic status was observed in patients ( %) and norepinephrine was introduced in cases. median length of stay was days [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and mortality was %. overall, the accuracy in predicting mortality of the studied parameters was poor. an increase of sofa score by at least points had greater accuracy with auroc = . [ . - . ], sensitivity = % and specificity = %. conclusion: in infected patients, the sofa score had greater prognostic accuracy than the sirs criteria, the qsofa score or the lactate level. these results suggest that sirs, qsofa, and high lactate level may be useful in screening for sepsis, but this utility is limited in predicting mortality. compliance with ethics regulations: yes. rationale: quick sequential organ failure assessement (qsofa) has been validated for patients with presumed sepsis and others in general emergency department (ed) population. however, it has not been validated in specific subgroups of patients with a high mortality. the aim of this study is to evaluate the ability of qscore to predict prognosis in patients with decompensated liver cirrhosis. patients and methods: this is a retrospective study, conducted over a period of years from january to december . consecutive patients with decompensated cirrhosis, admitted in our department are included. data of all patients were collected and the qsofa score was calculated at admission. the main study endpoints were length of stay, complications and in-hospital mortality. results: a total of patients diagnosed with decompensated cirrhosis were enrolled. mean of age was years ( - ). sex ratio was . . hcv ( %) was the main etiology of cirrhosis. the reasons of hospitalization were: oedema with ascitic syndrome in % of cases, digestive haemorrhage ( % of cases), fevers ( % of cases), and hepatic encephalopathy was present in % of cases. the mean duration of stay was days ± . in-hospital mortality rate was % and mean score qsofa was . .the qsofa score was significantly correlated with length of stay (p = . ) and complications(p = . ) but not with in-hospital mortality (p = . ). conclusion: the qsofa score was not useful for predicting in hospital mortality in patients with decompensated liver cirrhosis but it was significantly correlated to the length of stay and complications. compliance with ethics regulations: yes. angioedema associated with thrombolysis for ischemic stroke: analysis of a case-control study clara vigneron , aldéric lécluse , thomas ronzière , sonia alamowitch , olivier fain , nicolas javaud médecine interne, centre de référence associé sur les angioedèmes à kinines (créak), hôpital saint-antoine, aphp, paris, france; neurologie, chu angers, angers, france; neurologie, chu pontchaillou, rennes, france; neurologie, hôpital saint-antoine, aphp, paris, france; urgences, centre de référence associé sur les angioedèmes à kinines (créak), hôpital louis mourier, aphp, colombes, france correspondence: clara vigneron (claravigneron@hotmail.fr) ann. intensive care , (suppl ):p- rationale: bradykinin-mediated angioedema is a complication associated with thrombolysis for acute ischemic stroke. risk factors are unknow and management is discussed. the aim of this study was to clarify risk factors associated with bradykinin-mediated angioedema after thrombolysis for acute ischemic stroke. patients and methods: in a case-control study conducted at a french reference center for bradykinin angioedema, patients with thrombolysis for acute ischemic stroke and a diagnosis of bradykinin-mediated angioedema, were compared to controls treated with thrombolysis treatment without angioedema. two matched control subjects were analyzed for each case. results: thrombolysis-related angioedema were matched to control subjects. the sites of attacks following thrombolysis for ischemic stroke mainly included tongue ( / , %) and lips ( / , %). the upper airways were involved in ( %) cases. three patients required mechanical ventilation. patients with bradykinin-mediated angioedema were more frequently women ( ( %) vs. ( %); p = . ), had higher frequency of prior ischemic stroke ( ( %) vs ( %); p = . ), hypertension ( ( %) vs. ( %); p = . ), were more frequently treated with angiotensinconverting enzyme inhibitor ( ( %) vs. ( %); p < . ) and were more frequently hospitalized in intensive care unit ( ( %) vs. ( %); p = . ). in multivariate analysis, factors associated with thrombolysisrelated angioedema were female sex (odds ratio [or], . ; % confident interval [ci], . - . ; p = . ) and treatment with angiotensin-converting enzyme inhibitors ([or], . ; % [ci], . - . ; p < . ). discussion: because of theretrospective case-control design and the lack of the total number of thrombolysis for ischemic stroke, the incidence of this complication could not be evaluated in our study. previous studies reported an incidence of . to . % of angioedema in patients treated with a thrombolytic therapy for acute ischemic stroke. our case-control study permits for the first time to analyse more cases to evaluate associated risk factors of this rare complication. conclusion: this case-control study points out angiotensin-converting enzyme inhibitors and female sex as risk factors of bradykininangioedema associated with thrombolysis for ischemic stroke. compliance with ethics regulations: yes. rationale: patients with inflammatory bowel disease (ibd), frequently treated by immunosuppressive drugs, are more susceptible to be admitted to the intensive care unit (icu). however, outcome and predictive factors of mortality are little known. therefore, we aimed to assess the outcome and prognostic factors for critically ill ibd patients. patients and methods: we retrospectively studied data of consecutive ibd (i.e. crohn's disease and ulcerative colitis) patients admitted in icus between and . in-icu and one-year mortalities were estimated and predictive factors of in-icu mortality were identified by univariate and multivariate analysis. results: seventy-six patients (male: %, median age: . [ . - . ] years, charlson index: [ . - . ]) entered the study. ibd type was largely represented by crohn's disease ( . %) and its localization was mostly extensive: l ( . % of crohn's disease) or e ( % of ulcerative colitis) according to the montreal classification. twenty-seven patients ( . %) were treated with corticosteroids and ( %) with immunosuppressive therapy (azathioprine: . % and anti-tnfα: %). reasons for admission were shock/sepsis ( . %) and acute respiratory failure ( . %). icu diagnoses were infection ( %), ibd flare-up ( . %) or both ( . %), and pulmonary embolism ( . %). at admission, sofa score was [ . - . ] and . fifty-three patients ( . %) required mechanical ventilation, ( . %) vasoactive drugs, and ( . %) renal replacement therapy. twenty-three patients underwent emergency surgery ( . %) and six urgent endoscopic treatment ( . %). in-icu and one-year mortality rate were . % and . %, respectively. prognostic factors of in-icu mortality were sofa score (hr . , % ci [ . - . ], p < . ) and azathioprine treatment before icu admission (hr . , % ci [ . - . ], p < . ) (fig. ) . previous immunosuppressive treatment with anti-tnf did not alter the prognosis and even the type of ibd. conclusion: our study showed that more than % of ibd critically ill patients were discharged alive from the icu and a majority of them survived after one-year ( . %). we also found that sofa score and previous azathioprine immunosuppressive treatment worsened icu outcome. higher severity of the acute event affected short-term prognosis and should be taken into account for best icu triage and management. intensivists should pay particular attention to patients treated by azathioprine. compliance with ethics regulations: yes. fig. outcome of ibd patients admitted to the icu according to precious treatment with azathioprine status all aps patients with any new thrombotic manifestation(s) admitted to icus. results: one hundred and thirty-four patients (male/female ratio: . ; mean age at admission: . ± . years), who experienced caps episodes, required icu admission. the numbers of definite, probable or no-caps episodes (fig. ) , respectively, were: ( . %), ( . %) and ( . %). no histopathological proof of microvascular thrombosis was the most frequent reason for not being classified as definite caps. overall, / ( . %) episodes were fatal, with comparable rates for definite/probable caps and no caps ( % vs. . % respectively, p = . ). the kaplan-meier curve of estimated probability of survival showed no between-group survival difference (log-rank test p = . ). discussion: our results suggest that the caps criteria do not sufficiently encompass all the parameters responsible for thrombotic aps patients' disease severity in the icu. the absence of items referring to organ dysfunction/failure in the caps criteria probably limited their ability to predict mortality. albeit useful for the retrospective classification and comparison of patients, the caps criteria may be too stringent and not yet ready-to-use for the management of icu patients. for physicians outside expert aps centres, the absence of caps criteria could be misleading and lead to rejection of the diagnosis for near-caps patients, thereby preventing them from receiving the appropriate aggressive treatment they indeed require. we think that, when confronted with a critically-ill thrombotic aps patient, caps criteria should be interpreted with caution and should not be the only elements taken into account to decide the intensity of the therapeutic management. rationale: % of resuscitation patients develop anemia during their stay, it can worsen the prognosis, prolong the length of stay and lead to transfusions that can be the cause of complications. the objective of our work is to specify the incidence of anemia in our unit, its etiologies and its therapeutic management. patients and methods: we conducted a descriptive and analytical retrospective study within the surgical emergency resuscitation department of ibn rochd university hospital of casablanca, over a period of years from to . we included all anemic patients. statistical analysis was performed with spss statistics . p < . was considered significant. results: we included patients with an estimated incidence of %, the average age was years, the sex ratio h / f was . . % of admissions were for traumatic pathology and % postoperative digestive surgery. % had hypotension at admission and the mean temperature was . % .the onset of anemia and its depth were related to length of stay with . % of patients who were anemic beyond the th day of hospitalization with a hemoglobin level that became < . g / dl beyond the th day. % of the patients had a normochromic normocytic anemia becoming microcytic with the lengthening of the duration of stay. ferritinemia dosed in % of patients and was normal. % of our patients had exclusive parenteral nutrition while % had an enteral / parenteral combination. % were transfused in red blood cells (rbc) and % of patients were transfused more than once. % received between and rbc units. in patients who received transfusion episodes costing euros, the transfusion was inappropriate. the total cost of the transfusion was estimated at around , euros. % were supplemented with oral iron with an increase in hemoglobin in % of them. % of the patients came out of the intensive care unit with a hemoglobin level < g/dl/l. the mortality rate of our patients was % with as predictive factors in multivariate analysis, hyperthermia, coagulopathy, the transfusion appears as a factor of good prognosis. the prevention of blood spoliation and the fight against inflammation and nosocomial infection remain the pillars of the management of anemia in intensive care but in view of our results and the protective role of transfusion it would be interesting to see again the transfusion thresholds in our context. compliance with ethics regulations: yes. (fig. ). discussion: we described a series of patients with severe acute viral myopericarditises associated with anti-rnapol autoantibodies, an association that has never been reported previously. the fortuitous association of these autoantibodies with acute myopericarditis is highly unlikely. acute myocarditis is a very rare disease with a reported incidence of / , inhabitants. anti-rnapol -antibody detection is also very rare: . % positive tests (including the patients in this series) out of samples during a -year period in our immunology laboratory. this % proportion of patients with proven influenza-virus infections suggest that such severe infections could trigger anti-rnapol autoantibody production. however, influenza is a common disease and anti-rnapol autoantibodies are very rare. furthermore, no anti-rnapol autoantibodies were detected in the patients with severe influenza-related ards. last, anti-rnapol autoantibodies remained detectable several months after the viral infection had been cured. conclusion: this previously unknown association between severe acute viral myopericarditis and anti-rnapol autoantibodies is probably not fortuitous. anti-rnapol antibody detection in acute myopericarditis patients could imply individual susceptibility to severe viral infection. further studies are needed to investigate the pathophysiological mechanisms involved in this entity and potential specific therapeutic strategies. fig. relative frequencies of digestive manifestations in critically ill tma patients rationale: arrhythmia-induced cardiomyopathy has been recognized for several decades, but most severe forms, i.e. cardiogenic shock and refractory cardiogenic shock requiring mechanical circulatory support, were rarely described in adults. in this retrospective study, we described patients admitted in our tertiary care center for non-ischemic acute cardiac dysfunction (or worsening of previously known cardiac dysfunction) and recent onset supraventricular arrhythmia who developed cardiogenic shock requiring veno-arterial ecmo (va-ecmo). results: in a years period, patients had va-ecmo for acute non ischemic cardiac dysfunction and recent onset supraventricular arrhythmia (table ). fourteen ( %) patients had known nonischemic cardiomyopathy and ( %) known paroxystic atrial fibrillation. cardiogenic shock was the first manifestation of the disease in patients. atrial fibrillation was the main cause of arrythmia ( % of cases). at ecmo implantation, sofa score was [ - ], inotropic score , lvef % [ - ] and lactate level was [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] mmol/l. twelve patients had sustained successful reduction after amiodarone and/or electric shock, all were weaned from ecmo and survived without transplantation nor long term assist device. among the patients with failure of reduction, underwent an atrio-ventricular ablation while on ecmo and had atrial tachycardia ablation; all were weaned from ecmo and survived. among the remaining patients without reduction and without ablation procedure, only the patients who were bridged to heart transplantation or left ventricular assist device survived. in univariate analysis, factors associated with unfavorable outcome were previously known heart disease, heart rate, renal replacement therapy, nt-probnp level, failure of rhythm reduction after amiodarone load and/or electric shock. among the patients who recovered and survived ( with successful reduction and with successful ablation), lvef increased from [ - ]% before ecmo implantation to [ - ]% at long term follow-up. discussion: this is the largest cohort of arrhythmia induced cardiomyopathies on va-ecmo and the first description of atrio-ventricular node ablation with favorable outcome in this setting. conclusion: arrhythmia induced cardiomyopathy is probably underrecognized and should be considered in any patient with nonischemic acute cardiac dysfunction and recent onset supraventricular arrhythmia. recovery is possible in the most severely ill patients on va-ecmo, even with severe left ventricular dilation. aggressive rate control by av-node ablation may be warranted in case of failure of reduction, and may allow recovery and favorable outcome. compliance with ethics regulations: yes. rationale: diagnosis of sepsis is a major challenge in intensive care units and is associated with a high morbidity and mortality. sepsis identification is even more difficult in patients with extracorporeal membrane oxygenation (ecmo) because of many confounding factors. the primary objective was to study the ability of c-reactive protein (crp) and procalcitonin (pct) values measured at ecmo support initiation (day ) to predict the occurrence of early sepsis in patients undergoing venoarterial ecmo (va-ecmo) or venovenous ecmo (vv-ecmo). the secondary objectives were to study the association between these biomarkers and mortality rate during ecmo support and in-hospital mortality rate. furthermore, we investigated the relationship between early sepsis and mortality. patients and methods: we performed a retrospective, monocentric study in the cardiovascular intensive care unit of the university hospitals of lille, france. between november , and december , , we included patients over years old, who underwent an ecmo support for a medical or surgical indication, and for whom biomarkers (crp and pct) levels were available for at least the first days of admission. biomarkers and blood cultures were daily assessed for the first ecmo support days. early sepsis was defined by sepsis diagnosis in the first days after circulatory assistance initiation. in-hospital mortality rate was censored at days. after univariate analysis, a cox multivariate regression model was used to assess if the association between biomarkers levels and early sepsis or mortality rate was independent. a kaplan-meier survival plot was used to describe the association between early sepsis and mortality. results: among patients included, underwent va-ecmo and underwent vv-ecmo. an early sepsis diagnosis was made in . % of va-ecmo patients and in % of vv-ecmo patients. pct and crp levels on day were significantly associated with early sepsis diagnosis (fig. rationale: fluids are one of the most prescribed drug in intensive care, particularly among patient with circulatory failure. yet, very little is known about their pharmacodynamic properties and this topic has been left largely unexplored. several factors may impact the haemodynamic efficacy of fluids among which the infusion rate. the aim of this study was to investigate the influence of the rate of fluid administration on the fluid pharmacodynamics, in particular by studying mean systemic pressure (pms). we conducted a prospective observational study in patients with septic shock to compare two volume expansion strategies. a fluid bolus, ml of normal saline were administered and several haemodynamic variables were recorded continuously: cardiac output (co), arterial pressure (ap), mean systemic pressure (pms, estimated from ci, pvc and map). infusion rate was left at the discretion of the attending physician. a "slow" and a "fast" groups were determined based on the median of the infusion duration. fluids effect was measured by the area under the curve (auc), maximal effect (emax) and time to maximal effect (tmax) for each haemodynamic variable. the effects of fluid on psm disappeared in one hour on average. compared to patients of the "slow" group, those of the "fast" group had a shorter tmax and a higher emax for pms (p = . and . respectively). the auc for pms was identical between group, while in case of similar effect of infusion rates, it should be larger in the "slow" group. regarding co, tmax was also shorter in the "fast" than in the "slow" group (p = . ). the decreasing slope from maximal effect was comparable between groups, for pms as for co. the effect of a ml fluid bolus with normal saline in septic shock patients vanished within one hour. a faster infusion rate increased the maximal and total effect of the fluid bolus and shortened the delay to reach the maximal effect. rationale: significant hypotension following spinal anesthesia is a common issue in everyday clinical practice. toavoid this potentially harming situation, an empirical fluid administration is usually performed before the procedure. inferior vena cava (ivc) ultrasound has been demonstrated effective in guiding fluid therapy in critical care patients. the purpose of this study was to evaluate the ivc ultrasound guided volemic status optimization in order to decrease post-spinal hypotension rate. patients and methods: in this prospective, controlled, randomised study, consecutive patients were recruited and patients were randomly assigned to a control group, consisting of pre-anesthesia empirical fluid administration (itt), an ivc ultrasound group in which fluid management was based on an ivc ultrasound evaluation, and a passive leg raising test (plrt) group in which volume optimization was performed following the above mentioned test. primary outcome was the hypotension rate reduction after spinal anaesthesia following fluid optimization therapy between the groups. secondary outcomes were the total fluid amount administered, the total vasoactive drug amount used and the time needed to realize the whole anaesthetic procedure in all three groups. results: % reduction in hypotension rate ( % ci - %, p = . ) was observed between the echocardiography group and the control group, and there was a reduction of hypotension rate by % (ci % - %, p = . ) between the echocardiography group and the plrt group. the total fluid amount administered was significantly greater in the ultrasound group than in the control group ( ml; sd ml, versus ml; sd ml, p = . ). the total amine consumption was % in control group, % in ivc group and % in plrt group. an increased of total study time was observed for the echocardiography group min (sd min) in comparison with the control group min (sd min) and ptlr group min (sd min), (p < . ). the study showed a faint but positive trend toward the use of ivc-ultrasound to identify patients in spontaneous breathing needing fluid optimization before spinal anesthesia compliance with ethics regulations: yes. rationale: we performed a systematic review and a meta-analysis of studies investigating the ability of the end-expiratory occlusion (eexpo) test to predict preload responsiveness, through the changes in cardiac output (co) or its surrogates, in adult patients. this meta-analysis was prospectively registered on prospero (crd- ). we screened pubmed, embase and cochrane database to identify all original articles published between and evaluating the ability of the eexpo test to predict a significant increase in co or surrogate, compared to the one induced by a subsequent volume expansion or by passive leg raising (plr). the meta-analysis determined the pooled area under the receiver operating characteristics curve (auroc) of eexpo testinduced changes in co to detect preload responsiveness, as well as pooled sensitivity and specificity and the best diagnostic threshold. subgroup analysis and sensitivity analysis were planned to investigate potential sources of heterogeneity. results: thirteen studies ( patients) were identified and included in the analysis. nine studies were performed in the intensive care unit and four in the operating room. preload responsiveness was defined according to co changes induced by fluid administration in studies (fluid-induced increase in co ≥ % or ≥ %) and according to co changes induced by plr in one study. the duration of the respiratory hold ranged between and s. for the eexpo test-induced changes in co, the pooled sensitivity and specificity were [ - ]% and [ - ]%, respectively, while the pooled auroc curve was . ± . (fig. ) . the corresponding best diagnostic threshold was . ± . %. when changes in co were monitored through pulse contour analysis compared to other methods the accuracy of the test was significantly higher ( ( ). continuing (decrease to % of peak level) or modification (decrease < %) of antibiotic therapy was guided by a serum pct assay from the third day of treatmentand every h until antibiotic was stopped. this last was stopped when pct levels had decreased of % from the initial value. results: a total of patients had been diagnosed as sepsis (n = , %) and septic shoc (n = , %). mean age was years ± . an average ubs and absi score of % and . the average length of stay in icu was days. patients were assigned into two groups: group a (favorable evolution, n = ); group b (unfavorable evolution, n = ). the therapeutic attitude according to the kinetics of the pct are presented in the table . we found a significant difference between patients with unfavorable evolution compared to those with a favorable evolution (in whom we stopped antibiotics) (p < . ), in terms of hemodynamic state, pct concentration and renal clearance. pctguided antibiotic treatment has been proven to significantly reduce length of antibiotic therapy in our patients. the average duration of antibiotic was . ± days. conclusion: pct measurement may help with the decision to initiate antibiotic therapy in low risk acuity of infection and allows more judicious antibiotic use by reducing antibiotic exposure. compliance with ethics regulations: not applicable. rationale: reducing the risk of severe hypoxemia during endotracheal-intubation (eti) is a major concern in intensive care unit but little attention was paid to co variations during this period. we conducted a prospective observational study to describe transcutaneous co (ptcco ) throughout intubation in patients who received preoxygenation with standardoxygen therapy (sot), non-invasive ventilation (niv), or high flow nasal cannula oxygen therapy (hfncot). patients and methods: patients over years undergoing eti in icu were continuously monitored for ptcco during intubation and the following h under mechanical ventilation (mv). haemodynamics and respiratory parameters were also recorded as well as arterial partial pressure of co (paco ) to evaluate reliability of the transcutaneous measure. results: two hundred and two patients were included in the study. we found a strong correlation between ptcco recorded at preoxygenation and the last paco available before intubation (r = . , p < . ). in % of patients ptcco values recorded at initiation of mv were out of - mmhg ranges. ptcco recorded at eti, at initiation of mv, min and h of mv were significantly higher than ptcco during preoxygenation (p < . by anova). variations of ptcco were significantly different according to the preoxygenation method (p < . for interaction in anova). lastly, a decrease in ptcco higher than mmhg within half an hour after the beginning of mv was independently associated with postintubation hypotension (pih) (odds ratio = . , % confident interval . - . , p = . ). conclusion: ptcco is a valuable tool to record paco variation in patients requiring invasive mechanical ventilation and could be useful to prevent pih. compliance with ethics regulations: yes. rationale: intubation in intensive care unit (icu) is a critical procedure which leads to serious adverse event in to % of cases. several recent trials were conducted to help physicians to choose medications, devices and modality of intubation. especially, videolaryngoscope (vl) led to several publications in the last few years, with increasing tools marketed and spread use (difficult airway management, routineintubation). we designed an online survey to take a picture of intubation process and devices availability in france. toolbox. it was positioned as a first line laryngoscope for every intubation in critically ill patients to reinforce the vl skill training. present study was performed using prospectively collected data from a continuous quality improvement database about airway management in a -beds french teaching hospital medical icu. all consecutive intubation procedure performed with vl from september to june were included. "first attempt success" group and "first attempt failure" group were compared by univariate and multivariate analysis in order to analyze the first attempt intubation success rate according to the level of operators' expertise, identify factors associated with first pass intubation failure and describe the intubation related complications. results: we enrolled consecutive endotracheal intubations. overall first attempt success rate was ( %). comorbidities, junior operator, the presence of cardiac arrest and coma were associated with a lower first attempt success rate. the first attempt success rate was less than % in novice operators ( - previous experiences with vl, independently of airway expertise with direct laryngoscopies) and % in expert operators (greater than previous experiences with vl) (fig. rationale: tracheostomy in intensive care unit (icu) has many advantages. but only patient comfort and shorter icu and hospital stay were demonstrated. the timing of this procedure is still debated. the aim of this study was to determine the impact of early tracheostomy on prognosis. we performed a retrospective study in a medical icu ( beds unit) from january to november . the technique of tracheostomy was exclusively surgical in the operating room made by the surgeon. the primary endpoint was mortality in icu. the secondary outcomes were post-tracheostomy incidence of ventilator acquired pneumonia, duration of mechanical ventilation and length of stay in icu. these criteria were assessed in relation to timing of the tracheostomy defined as early when performed before day of mechanical ventilation. results: forty-two patients were enrolled during the study period. mean age of patients was ± years. median length of stay in icu was of days. mortality rate was of %. comparing the two groups, early vs late tracheostomy, no difference was found with respect to mortality ( % vs. %, p = . ), vap occurrence ( % vs. %, p = . ), post-tracheostomy duration of mechanical ventilation ( ± d vs. ± d, p = . ), or length of stay in icu ( ± d vs. ± d, p = . ). in multivariate analysis, the only factor independently related to mortality was the sofa score patient on tracheostomy day with p = . and or = . (ci % [ . - . ] ). conclusion: tracheostomy in the intensive care unit remains a justified alternative despite the discordant data in the literature. in our study, the delay of the procedure didn't interfere with the evolution. however, the patient severity as attested by sofa score at the day of tracheostomy, was the only independent prognostic factor. those results should be confirmed by other large prospective studies. compliance with ethics regulations: not applicable. sabah benhamza, mohamed lazraq, youssef miloudi, abdelhak bensaid, najib el harrar réanimation de l'hôpital du août, casablanca, morocco correspondence: sabah benhamza (benhamzasabah @gmail.com) ann. intensive care , (suppl ):p- rationale: many unknowns remain as to the place of tracheostomy in intensive care. reluctance to perform a tracheotomy is numerous, especially when pre-exists chronic respiratory failure, but some data suggest benefits. we report in this work our experience in tracheotomy in the intensive care unit of the august hospital, casablanca. patients and methods: this is a retrospective descreptive study over years (january to january ) including all patients that have been tracheostomized in the intensive care unit of the august hospital . results: during the study period, patients were tracheostomized with a prevalence of . % in years, the predominance was male (sex ratio . ). the average age was ± years old. the indication for tracheostomy was prolonged ventilation in % of cases, extubation failure in % of cases, and intubation failure in % of cases. tracheostomy was performed on average on the th day of intubation. all patients were tracheostomized in the operating room by ent surgeons. the main complications attributable to tracheotomy were hemorrhage of the tracheostomy orifice in patients ( %) immediately resumed, cases of subcutaneous emphysema ( %), case of pneumothorax ( %), cases of orifice infection ( %). no patient died of a tracheostomy related cause. the tracheotomy in intensive care is still a subject of debate especially concerning the time of its realization. however it seems to reduce the duration of mechanical ventilation, facilitates the care and also the ventilatory weaning. compliance with ethics regulations: yes. rationale: hfnco is a frequently used device providing heated and humidified high flow oxygen with several advantages: decreased work of breathing, decreased dead space, increased end expiratory lung volume (eelv), more stable fio . the increase in eelv is relying of the positive expiratory effect generated by the device. the level of generated pep seems however to largely depend on whether the mouth is open or not. this study was aimed to assess the impact of mouth opening on eelv increase induced by hfnco using electric impedance tomography. patients and methods: the following hfnco trial was proposed to healthy subjects who used hfnco on a regular basis for patients care. oxygen flow was set successively during min periods at , and l/min (optiflowtm; fisher & paykel healthcare, auckland, nz). these three conditions were tested in semi recumbent and supine position chosen at random. measurement started in supine position with no flow (baseline) and each period was separated from the following by a wash out period on min during which the subject could breath normally with no supplemental oxygen. electric impedance tomography (pulmovista ® , dräger medical gmbh, lündbeck, germany) was performed applying a electrodes belt placed between the th and th intercostal space, including a reference electrode located on the abdomen. as no spirometer was used, the data of eelv computed on the eit device were expressed as percentage of variation of the value measured in supine or semi recumbent position with no flow. demographic data were expressed as median and extreme values. comparisons were performed using u mann whitney test. [ . - . ] accepted to participate to the study. when subjects received hfnco with open mouth (whatever position) no modification of eelv was observed (table ) . conversely, a significant increase in eelv was noted with closed mouth, whatever position. in the semi recumbent position the increase in eelv was even more important with l/min. conclusion: electrical impedance tomography illustrates the impact of mouth closure on eelv increase among healthy subjects receiving hfnco. compliance with ethics regulations: yes. rationale: in stable copd patients, nasal high flow oxygen (nhf) use can be associated with reduction in respiratory rate (rr) and minute ventilation (mv). in thesepatients, paco remains stable or decreases under nhf. this suggests a possible dead space reduction related to a washout effect of nhf. the aim of this study was to assess the physiological effects of nhf in hypercapnic patients with acute copd exacerbation. patients and methods: crossover study in hypercapnic patients suffering from acute copd exacerbation and treated with intermittent non-invasive ventilation (niv). nhf l/min or standard oxygenotherapy (stand o ) were randomly administered during h between niv treatments. rr, tidal volumes (vt), mv and corrected mv (cormv = mv x paco / ) variations were recorded during the last min of each study period using a respiratory inductive plethysmography vest. blood gas analysis was performed at the end of each oxygen administration period. visual analogic dyspnea score (vas) quoted from to was assessed by the patient after and min. results given as median [iqr] . wilcoxon tests were used to compare data between stand o and nhf. results: twelve patients were included and data could be recorded in ( (fig. ). dyspnea scores were not different between the modalities. conclusion: in case of acute copd exacerbation, using nhf between niv treatments was associated with paco and rr decrease. mv concomitantly decreased suggesting a deadspace volume reduction related to a washout effect of nhf. corrected mv decreased in all the patients except one. these results suggest that nhf could be used to deliver oxygen between niv treatments to copd patients suffering from acute exacerbation and could contribute reducing paco . compliance with ethics regulations: yes. rationale: the role of atypical micro-organisms in acute exacerbation of chronic obstructive pulmonary disease (copd) that require mechanical ventilation is poorly none. the aim of this study was to determine the role of atypical pathogens in severe acute exacerbation of copd. patients and methods: in this prospective study we included all patients admitted for acute exacerbation of copd requiring mechanical ventilation. atypical pathogens (chlamydophila pneumoniae and mycoplasma pneumoniae) were searched by serological diagnosis and by culture of sputum samples. in this study we included patients aged ± years. sixty-eight percent of sputum culture were considered significant. six cultures were positive with different microorganisms. neither chlamydophila pneumoniae nor mycoplasma pneumoniae were found. the prevalence of chlamydophila pneumoniae was . % (positive igg serum). the demographic characteristics was similar between patients with and without positive culture. the rate of noninvasive ventilation (niv) failure was % in positive serology group versus % in negative serology group (p = . ). the mortality was similar in both groups. in multivariate logistic regression analysis only positive serology (or = . ; % ic [ . - . ], p = . ) was an independent factor of niv failure. conclusion: a positive serology of chlamydophila pneumoniae was a predictive factor of niv failure without an impact on the morbidity and mortality of copd patient treated with mechanical ventilation. compliance with ethics regulations: yes. rationale: emergency departments (ed) receive a growing up number of patients with acute exacerbation of chronic obstructive pulmonary disease (copd) .non-invasive ventilation (niv) could be a good alternative to achieve a respiratory support, avoiding as much as possible the complications of invasive ventilation. the study aimed to assess the clinical outcomes of using niv in acute exacerbation of copd at ed and to identify whether clinical variables present at admission are predictive of niv failure. we conducted a prospective study conducted at the ed over a period of one year. data of all patients admitted for acute exacerbation of copd for all causes and requiring non-invasive ventilation were collected. niv failure was defined as need for endotracheal intubation or death. results: during the study period, a total of patients with a mean age of years (± ) were included. acute exacerbation of copd was due to bronchitis in %, to pneumonia in % of cases. % of patients had no apparent etiology of acute exacerbation of copd. bilevel positive airway pressure was performed on all patients, during a mean period of h (± ). clinical niv success was observed in patients ( %). the predictors of niv failure were advanced age, tachycardia, and hypercapnia. conclusion: the efficiency of niv in the management of acute exacerbations of copd at ed is well documented. this is further supported by our study which showed a clinical success in % of patients with acute exacerbation of copd. compliance with ethics regulations: yes. rationale: non invasive ventilation (niv) is often performed in elderly patients with acute respiratory failure (arf) at emergency department (ed). this technique may be subject to many difficulties, due to the presence of frequent co-morbidities. the aim of this study was to identify the predictive factors of niv failure in elderly patients with arf at ed. patients and methods: this was a retrospective study conducted at ed on year and months including patients aged more than years and who required the use of niv for an arf. all data were collected and analyzed using the spss software. patients were divided into two groups: niv failure and niv success. niv failure was defined by inhospital mortality, requirement of intubation or hospitalization at intensive care unit. results: during the study period, a total of elderly patients that required niv for arf were included. median age was years (min = , max = ) and sex ratio was . . the median charlson index was (min = , max = ). the etiological diagnoses of arf were acute decompensation of chronic obstructive pulmonary disease ( %), acute heart failure ( %), pneumonia ( %) and pulmonary embolism ( %). the arf was hypercapnic in % of cases and nonhypercapnic in %. niv failure concerned %. predictive factors of niv failure were clinical signs of right heart dysfunction (p < . ), c reactive protein (p = . ), initial ph (p = . ) and kidney dysfunction (p < . ). conclusion: in our study, niv failure in elderly patients with arf at ed was influenced by clinical signs of right heart dysfunction, c reactive protein, initial ph and kidney dysfunction. these clinical and biological factors could be useful to identify the most critical elderly patients and to better guide therapeutic decisions. compliance with ethics regulations: yes. rationale: the interest of ecco r in the management of very severe acute asthma exacerbations is still unclear. since it could help to control respiratory acidosis and /or to limit dynamic hyperinflation, its clinical benefits are uncertain, even in mechanically ventilated patients. the rexecor observatory is a prospective ecco r cohort in the great paris area. tencases of severe asthma treated by ecco r were retrospectively reviewed. mainly, arterial blood gases (abg), duration of ecco r and imv were collected and in-icu mortality were assessed. data are reported as median (iqr). results: ten patients ( men, age: (ic: - ) years, bmi: . (ic: . - . ) kg/m , fev- : . (ic: . - . ) l, ( (ic: - ) %), saps : . (ic: . - . ) points) were included. one patient suffered from cardiac arrest before admission and one had pneumothorax at icu admission. nine patients were under imv (started on the day of admission for ). before ecco r, patients received systemic corticosteroids, paralyzing agents, epinephrine and salbutamol. two patients suffered from pneumonia. ecco r was started (ic: - . ) days after intubation. venous vascular access was achieved via the right internal jugular route in patients and via the femoral route in . the hemolung device was used in patients, the ila activve in and the prismalung in . abg before and after day of ecco r are reported in table . duration of ecco r was (ic: . - ) days and patients were weaned from imv under ecco r. for the remaining patients, duration of imv after ecco r was (ic: - . ) days. icu stay was . (ic: - . ) days. the only one niv patient was not intubated. ecco r as stopped in patients because of complications (one hemolysis, one internal bleeding and one membrane clotting). one patient died in icu after limitation of life-sustaining therapy decision. we report a preferential use of ecco r in imv patients, contrasting with a marginal use in only one niv patient to prevent intubation. the mortality rate was low, in line with previous case series of severe acute asthma with ecmo or ecco r support. more studies are needed ( ) to better delineate the pathophysiological benefits of ecco r in asthma patients and ( ) to confirm strong clinical benefits. compliance with ethics regulations: not applicable. rationale: acute exacerbations of chronic obstructive pulmonary disease (aecopd) are the most important events characterizing respiratory illness progression. their management often needs noninvasive or invasive ventilation (iv). data of literature confirm that the mortality of aecopd requiring iv is high but are discordant about prognostic factors. the aim of our study was to describe the epidemiologic and clinical features of patients admitted for aecopd requiring iv, the treatment and the evolution in intensive care unit in order to deduce the independent factors of mortality. patients and methods: a -year retrospective analytic observational single-center study including patients hospitalized for aecopd requiring iv. results: fifty-eight patients were enrolled. mean age was ± years with sex-ratio of . . eighty one percent were smokers and % were classified gold stage . history of intensive care hospitalization and prior iv were found in % and % of all cases respectively. mean apache ii score was ± . the predominant precipitating factor for aecopd was respiratory tract infection ( % of all cases). twenty two percent of all patients presented septic shock. iv was initiated on admission in % of all cases and after noninvasive ventilation failure in % of all cases. forty-eight per cent of all patients developed septic shock as evolutionary complication. mortality rate was %. in univariate analysis: male gender (p = . ), duration of respiratory disease progression (p = . ), annual exacerbations frequency (p < − ), gold stage (p = . ), prior iv (p < − ), duration of symptoms before hospitalization (p = . ), apache ii score (p = . ), ph (p = . ), shock on admission (p = . ) and septic shock as evolutionary complication (p = . ) were predictors of mortality in our study. besides; shock on admission (p = . ) and as evolutionary complication (p = . ) were the two independent prognostic factors in multivariate analysis. conclusion: vital and functional prognosis of aecopd requiring iv depends on the severity of the underlying respiratory illness, the severity of the exacerbation and the quality of an early management. this emphasizes the importance of controlling modifiable risk factors including smoking cessation, basic treatment improvement and early appropriate treatment of these exacerbations. compliance with ethics regulations: yes. medical background, biological parameters, death-rate and outcome of patients have been compared. results: in total, patients have been included in the "hlh" population. death-rate in intensive care unit was % in the "hlh" group compared to % in the "not hlh" group (p = . ). we used more extrarenal cleansing in the "hlh" group ( % vs. %, p < . ), the duration of assisted ventilation was longer ( . days vs. . days, p < . ), as well as the duration of extrarenal cleansing ( . days vs. . days, p < . ) and those of amines ( . days vs. . days, p = . ). the average time of hospitalization was significantly longer in the "hlh" group ( . days vs. . days, p < . ). the secondary hlh to sepsis in intensive care unit, not well known and understudied, seems to have a different profile and a more serious outcome but no change in death-ratehas been found considering the pairing with the sofa. further studies are needed to plan a better therapeutic strategy within this population. compliance with ethics regulations: not applicable. serum and peritoneal exudate concentrations after high doses of ß-lactams in critically ill patients with severe intra-abdominal infections: an observational prospective study lisa leon, philippe guerci, elise pape, nathalie thilly, amandine luc, adeline germain, anne-lise butin-druoton, marie-reine losser, julien birckener, julien scala bertola, emmanuel novy chru nancy, vandoeuvre les nancy, france correspondence: lisa leon (lisaleon @gmail.com) ann. intensive care , (suppl ):p- rationale: critically ill patients with severe intra-abdominal infections (iais) requiring urgent surgery may undergo several pharmacokinetic alterations that can lead to ß-lactam under dosage. the aim of this study is to measure serum and peritoneal exudate concentrations of ß-lactams after high doses and optimal administration schemes. patients and methods: this observational prospective study included critically ill patients with suspicion of iai who required surgery and a ß-lactam antibiotic as empirical therapy. serum and peritoneal exudate concentrations were measured during surgery and after a h steady-state period. the pharmacokinetic/pharmacodynamic (pk/ pd) target was to obtain ß-lactam concentrations of % ƒt> x mic (minimum inhibitory concentration) based on a worst-case scenario (highest ecoff value) before bacterial documentation (a priori) and redefined on the mic of the isolated bacteria (a posteriori). results: forty-eight patients were included with a median [iqr] age of [ - ] and a saps ii score of . septic shock occurred in % of cases. the main diagnosis was secondary nosocomial peritonitis. piperacillin/tazobactam was the most administered ß-lactam antibiotic ( %). prior to bacterial documentation, patients ( . %) achieved the a priori pk/pd target. iai was documented in patients ( %). enterobacteriaceae were the most isolated bacteria. based on the mic (n = ) of isolated bacteria, % of the patients achieved the pk/pd target ( % ƒt> xmic). in the fig. we presented serum ß-lactams pk/pd target attainment and observed total concentrations of piperacillin-tazobactam at each timepoint in serum and peritoneal exudate. in critically ill patients with severe iais, high doses of ß-lactams ensured % ƒt> xmic in % of critically ill patients with severe iais within the first h. a personalized ß-lactam therapeutic scheme with a pk/pd target based on local ecology should be warranted. compliance with ethics regulations: yes. rationale: intensive care unit acquired bloodstream infections (icu-bsi) are frequent, and associated with high morbidity and mortality rates. the objective of our study was to describe the epidemiology and the prognosis of icu-bsi in our icu (cayenne general hospital). secondary objectives were to search for factors associated to icu-bsi caused by esbl-pe, and those associated with mortality at days. patients and methods: we retrospectively studied icu-bsi in the medical-surgical intensive care unit of the cayenne general hospital, during months (january to june ). we assessed survival at days from the diagnosis of icu-bsi. results: icu-bsi was diagnosed in . % of admissions giving a density incidence of . icu-bsi/ days. the median delay to the first rationale: necrotizing soft tissue infections (nsti) are a heterogenous group of severe infections. among them, group a streptococcal (gas) infection represent a subgroup that could benefit from specific therapies targeting the toxinic pathway, such as intravenous immunoglobulins or clindamycin. nevertheless, previous trials evaluating these treatments suffered from a low rate of gas infection among the study population. early identification of patients at high risk of gas infection would allow for assessing targeted treatment strategies. patients and methods: we conducted a secondary analysis of a previously published cohort of patients admitted to our tertiary center for surgically proven nsti between and . admission characteristics and microbiological documentation based on surgical samples, blood cultures or subcutaneous puncture were recorded. we compared patients with a documented gas infection to all other patients regarding admission characteristics. a generalized linear regression model was used to identify admission characteristics associated with a subsequent documentation of gas infection. results: among patients, ( %) had a gas infection, which was monomicrobial in ( %) cases. admission characteristics associated with gas infections by univariate analysis were nsaid treatment before admission ( ( . %) for gas infections vs ( . %) for others, p = . ) and leukocytosis as a continuous variable ( , /mm [ , - , ] vs. , [ - , ], p = . ). those inversely correlated with gas infections were immunodeficiency ( ( %) vs. ( . %), p = . ), and an abdominoperineal topography ( ( . %) vs. ( . %), p > . ). after multivariate analysis only immunodeficiency (or = . [ . - . ], p = . ) and an abdominoperineal infection (or = . [ . - . ], p = . ) remained associated with the absence of gas infection. using these criteria allowed for identifying subgroups of patients with increased likelihood of gas infections: from % overall (n = ) to % for non-abdominoperineal infections (n = ), % for patients without immunodeficiency (n = ) and % for both non abdominoperineal infections in patients without immunodeficiency (n = ). a sensitivity analysis for monomicrobial gas infections yielded similar results with the addition of younger age and non-nosocomial infections as predictors. conclusion: upon admission, the absence of immunodeficiency and of an abdominoperineal infection in nsti patients were covariables associated with gas infection. compliance with ethics regulations: yes. rationale: sickle-cell disease is the most common genetic disorder in the world. a complication of this disease is the acute chest syndrome (acs) which is associated with a high risk of death. respiratory tract infections are often mixed up and the introduction of betalactam antibiotics is recommended. glomerular hyperfiltration is common and responsible of a high risk of underdosing. this study compares cefotaxim continuous infusion to intermittent bolus in adult patients with acs. patients and methods: this observational retrospective monocentric study included acs admitted in intensive care unit and treated by cefotaxim with at least one plasmatic dosing between may and august . results: thirty patients received bolus administration while the others received continuous infusion. we observed patients ( %) and patients ( %) with a cefotaxim trough level ≥ mg/l in the bolus and continuous group, respectively (p < . ). the median residual concentration was mg/l [ - ] and . mg/l [ . - . ] in the bolus and continuous group, respectively (p < . ). there was no toxic effect induced by overdosing of cefotaxim. conclusion: compared to intermittent bolus infusion, continuous cefotaxim administration maximizes the pharmacokinetics parameters by obtaining a plasmatic concentration times above the minimal inhibitory concentration of usual germs associated with acs. continuous infusion of time-dependant antibiotics seems to decrease the risk of underdosing in patients with sickle cell disease. compliance with ethics regulations: not applicable. (n = , %), followed by esophageal varices rupture (n = , %), ulcer bleeding (n = , %) and diverticular hemorrhage (n = , %). infectious diseases were diagnosed in three patients ( %), including one clostridium colitis, one erosive gastritis with helicobacter pylori and one esophageal candidiasis. conclusion: gib is associated with a high mortality rate in immunocompromised patients, especially in patients with hematological malignancies. specific malignant lesions were the main etiology and may be difficult to treat. comparison with critically ill non-immunocompromised patients with gib will help physicians to provide specific therapeutic strategies in this population. compliance with ethics regulations: yes. risk factors for delayed defecation and impact on outcome in critically ill patients: a multicenter prospective non-interventional study benoît painvin ,* , arnaud gacouin , antoine roquilly , claire dahyot-fizelier , sigsimond lasocki , chloe rousseau , denis frasca , philippe seguin anesthésie-réanimation/chu rennes, rennes, france; réanimation médicale/chu rennes, rennes, france; réanimation chirurgicale/ chu nantes, nantes, france; réanimation chirurgicale/chu poitiers, poitiers, france; anesthésie-réanimation/chu angers, angers, france; centre investigation clinique/chu rennes, rennes, france; anesthésie-réanimation/chu poitiers, poitiers, france; réanimation chirurgicale/chu rennes, rennes, france correspondence: benoît painvin (painvinbe@gmail.com) ann. intensive care , (suppl ):p- rationale: delayed defecation is very common in intensive care units (icu) and it increases length of mechanical ventilation (mv), icu length of stay (los) and possibly mortality. the objective of this prospective multicenter study was to determine risks factors for constipation in icu and to evaluate their impact on mortality. patients and methods: it was a prospective multicenter non-interventional trial performed in university icus in france from january to october . all patients ≥ years old who had an expected los of days and mechanically ventilated for at least days were eligible. defecation was defined as the time of the first stool passage. results: patients were included in the analysis. a stool passage was observed in % of the patients during their icu stay with a mean delay of ± days. in multivariate analysis, risk factors for delayed passage of stool were non-invasive ventilation use and time spent under invasive ventilation whereas alcoholism, laxative treatment (before and after icu admission) and nutrition ≤ h favoured passage of stool (table ) . no relations between constipation and mortality were found. conclusion: we highlighted new and important independent factors for constipation in critically ill patients leading to a better prevention of this phenomenon.. compliance with ethics regulations: yes. rationale: community peritonitis is a frequent medical-surgical emergency of the adult, acquired by the patient in a non-hospital setting. careful multidisciplinary care is essential, involving surgeons, anesthetists, microbiologists and radiologists. the objective of our study is to determine the bacteriological aspects of intra-abdominal sepsis, to describe their sensitivity profiles and to propose treatment regimens for the management of community peritonitis. we conducted a descriptive retrospective study spanning a period of two years from january to january involving cases of community abdominal sepsis operated in the operating room of surgical emergencies of our hospital. we included in our study adult patients admitted for suspected or confirmed abdominal sepsis who had undergone bacteriological examinations on the abdominal collections. samples taken are sent directly to the bacteriology laboratory for bacteriological analysis of the results. the studies showed the mean age is . years old, with a sex ratio of . . we found positive results mainly of peritoneal origin with a percentage of . % peritonitis, dominate by intestinal peritonitis . % followed by the appendicular origin . % then peritonitis by perforation of ulcer. the most incriminated organism in intraabdominal sepsis is e. coli with a percentage of . % of the total germs found, followed by streptococcus spp . %, enterococci . %, non-fermenting bgn composed mainly of pseudomonas aeruginosa . %, staphylococci . % and acinetobacter baumanii . %. note also the presence of bacteroides fragilis is %. e. coli had a very low sensitivity profile for amoxicillin/clavulanic acid ( . %), unlike ceftriaxone, gentamicin, amikacin and ertapenem, which had a sensitivity of . %, respectively. . %, %, . %. conclusion: knowledge of the bacterial ecology of intraabdominal sepsis is important in the choice of probabilistic antibiotherapy, pending bacteriological findings. no data are yet available about nutritional management and risk of malnutrition in tunisian medical intensive care units (icu). the purpose of this study was to describe nutritional management in medical intensive care patients and to evaluate the risk of malnutrition. patients and methods: we conducted a prospective observational cross-sectional study in medical icus all around the tunisian country on the th september . all participant units received a questionary form about routine nutritional management and data of all patients hospitalized in icu on the study day. collected data were: demographic characteristics, reason for admission, severity scores and subjective evaluation of nutritional status on admission, type and volume of nutritional support on the study day and the day before, nutritional status, nutric score and biological data on the study day, reasons for nutritional interruption and other supports prescribed. results: thirteen icu all around tunisia participated to the study. no icu had a nutrition team and only one had a written nutrition protocol. four icus evaluated systematically the nutritional status on admission. all icus were aware and practiced early enteral nutrition in patients unable to maintain oral intake with a systematic supplementation of oligoelements and minerals. neither target energy nor protein intake were calculated. on the study day, patients were hospitalized with an occupation rate of %. mean age was ± years. mean body mass index was ± and % of patients were judged well nourished. enteral nutrition support was prescribed on admission in % of cases with a mean caloric intake of ± kcal/day. the mean caloric target on the study day was ± kcal/day with a mean caloric intake of ± kcal/day and a mean caloric gap of ± kcal/day. the mean nutric score and body mass index on the study day were ± and ± respectively. twenty patients were judged malnourished by the nutric score and twenty two by clinical evaluation. a good correlation was found between nutric score and clinical evaluation of nutritional status (k = . ). conclusion: tunisian icus don't have nutrition team or nutritional written protocol. early enteral feeding and supplementation is common. a good correlation exists between nutric score and clinical nutrition status evaluation. compliance with ethics regulations: yes. rationale: whether more intensive glycemic control (gc) is beneficial or harmful forcritically ill patient has been debated over the last decades. gc has been shown hard to achieve safely and effectively in intensive care. the associated increased hypoglycemia and glycemic variability is associated with worsened outcomes. however, modelbased risk-based dosing approach have recently shown potential benefits, improving significantly gc safety and performances. the stochastic targeted (star) gc framework is a model-based controller using a unique risk-based dosing approach. star identifies modelbased patient-specific insulin sensitivity and assesses its potential variability over the next hours. these predictions are used to assess hypoglycemic risks associated with a specific insulin and/or nutrition intervention to reach a specific target band. this study analyzes preliminary clinical trial results of star in a belgian icu compared to the local standard protocol (sp). the mean age in our series was . years with a male predominance (sex ratio = . ). the main revealing symptoms were epigastralgia, weight loss and vomiting. subtotal gastrectomy was performed in . % of cases and total gastrectomy in . % of cases. curative resection could only be performed in . % of cases. operative mortality was . % and morbidity was . %. the main factor influencing operative mortality was age greater than years. in univariate analysis the main prognostic factors; tumor size, degree of parietal invasion, presence of ganglionic invasion, presence of more than ganglia invaded, presence of metastases, locally advanced tumor, tumor stage and curative nature of resection. patient-related factors such as age associated blemishes and biological factors have a significant influence on the patient's prognosis. the prognosis of gastrectomies, although it has improved overall, remains mediocre. the only way to improve the prognosis remains the early diagnosis with an effective surgical management and the introduction of an adapted resuscitation. compliance with ethics regulations: yes. efficacy of multiple second line agents in refractory status epilepticus in a pediatric intensive care unit lea savary, claire le reun chu tours, tours, france correspondence: lea savary (lea.savary@hotmail.com) ann. intensive care , (suppl ):p- rationale: convulsive status epilepticus (cse) is the most common neurological emergency in children. refractory status epilepticus (rse) occurs whenseizures are not controlled with first-and secondline agents. in adults, rse requires pharmacological induced coma. in pediatric patients, association of second line treatment is often used to avoid general anesthesia although there is currently no data on the efficacy of this association. we performed a monocentric retrospective study to assess the efficacy of multiple second line agents in pediatric rse. all children admitted to clocheville hospital (tours) between january and december with a diagnosis of rse were included. our population was divided into two groups: need of general anesthesia (midazolam+) or not (midazolam-). results: children were included ( in group midazolam+, in group midazolam−) during the study period. among the patients with multiple second line agents, % did not need general anesthesia (n = ). in group midazolam+, cse was % longer in patients treated with multiple second line agents ( rationale: drowning is an acute respiratory failure resulting from immersion or submersion in a liquid. patients and methods: we report cases of drowning collated in the pediatric reanimation department during a period from to . the aim of our retrospective study was to analyze and compare the different epidemiological, clinical, parcalinical, therapeutic and evolutionary of drowning in our study. results: our study contains boys and girls, with a sex ratio (m/f) of , in an age between months and years. for cases studied, no one was classified stage i, . % classified stage ii, % stage iii, and . % stage iv. all cases collected by ou service were victim of accidental drowning, . % were secondary to the lack of parental supervision. among cases, had respiratory complications, cases of hydroelectrolytic disorders, case with infectious complications, cases of neurological and cases of cardiac or hypothermic complication. in our study, cases recovered well and cases died. the survival of the drowned person depends on the speed and efficiency of the intervention, which in thefirst place is prehospital, thus ensuring the first actions at the scene of the accident, which will have repercussions on the hospital care. this has an equal share in the improvement of the victim's prognosis. compliance with ethics regulations: not applicable. epidemiology of severe pediatric trauma following winter sport accidents in the northern french alps emilien maisonneuve , nadia roumeliotis , pierre bouzat , guillaume mortamet chu grenoble, grenoble, france; chu sainte-justine, montréal, canada correspondence: emilien maisonneuve (emilienmaisonneuve@orange. fr) ann. intensive care , (suppl ):p- rationale: this study describes the epidemiology of severe injuries related to winter sports (skiing, snowboarding and sledding) in children, and assesses potential preventive actions. we did a single-center retrospective study in our pediatric intensive care unit in the french alps. we include all patients less than years old, admitted to the intensive care unit following a skiing, snowboarding or sledding accident from to . results: we included patients (mean age . years and % were male); of which ( %), ( %) and ( %) had skiing, snowboarding and sledding accidents, respectively. the average iss (injury severity score) was . the major lesions were head (n = patients, %) and intra-abdominal (n = patients, %) injuries. compared to skiing and snowboarding, sledding accidents affected younger children ( vs. years, p < . ); most of whom did not wear a helmet ( % vs. %, p < . ). severity scores were similar amongst winter sports (iss = for skiing, for snowboarding and for sledding accident, p = . ). rationale: best strategies for the management of severe pediatric traumatic brain injury (tbi) are still not clearly established and wide variations among professional practices have been reported in the literature. unfortunately, these variations in practice have an impact on the patient's outcome. the objectives of this work were to assess the adequacy of professional practices to the guidelines for the management of severe head injury and to assess the level of agreement of respondents in the absence of guideline. patients and methods: a practice survey was conducted in frenchspeaking hospitals in canada, belgium, switzerland and france from april st to june th, . the survey was conducted as a progressive clinical case with questions based on guidelines and the literature from to . the questions related to the assessment and management of tbi during the acute and intensive care phase. results: seventy-eight questionnaires were included. the adherence to guidelines was good, with items out of obtaining an adherence rate of more than % regardless of the annual number of tbi managed by the centre. there was strong agreement among clinicians on the intracranial pressure (pic) (> %) and cerebral perfusion pressure (> %) thresholds used according to age. guidelines for indication of pic monitoring were almost perfectly followed in the case of glasgow score < and abnormal brain ct scan (n = , %). on the other hand, the natremia and glycemia thresholds and the role of transcranial doppler were not consistent. strong adherence to recent recommendations was achieved: seizure prophylaxis with levitracetam (n = / , %) and capnia threshold (n = , %). assessment of o pressure in brain tissue (n = , %) and autoregulation (n = ; %) was not a common practice. conclusion: overall, practices for the management of tbi appear to be standardised. variations persist in areas where there is a lack of literature and guidelines in paediatrics, so clinicians seem to refer to adult guidelines. compliance with ethics regulations: yes. choubeila guetteche chu constantine, constantine, algeria correspondence: choubeila guetteche (cguetteche@gmail.com) ann. intensive care , (suppl ):p- rationale: ingesting a coin cell is a common household accident in children, which can have serious consequences. the goal is to determine prognostic factors to improve management and reduce complications. patients and methods: we conducted a retrospective study including children under admitted in pediatric intensive care between january and may for ingestion of button cells, with epidemiological, clinical and paraclinical data collection. results: twenty-six children boys ( %), and girls ( %) were included, with an average age of months ( - ), increased incidence in recent years. clinical signs indicative were dysphasia with hyper-sialorrhea in cases, cervical pain in one case, respiratory distress in one case, the cell was located in the upper third of the esophagus in cases, third average in cases, third inferior in cases, the mean time before extraction was h. complications: cases of mediastinitis, cases of oesotracheal fistula, a case of perforation. conclusion: the young age of the child, the diameter of the battery, and especially the time of care are risk factors for the occurrence of complications, the prevention passes through the education of the general public and creation of channel of taking into account fast charge. compliance with ethics regulations: not applicable. yacine benhocine university hospital center nedir mohamed, tizi-ouzou, algeria correspondence: yacine benhocine (yacine @yahoo.fr) ann. intensive care , (suppl ):p- rationale: inhalation of foreign bodies is a common and serious accident in children, especially between and years old. at this age, children use their mouth to explore their environment. asphyxia is the immediate risk and respiratory sequelae may appear secondarily. the severity of this incident has been considerably reduced due to the progress of the instrumentation and anesthesia which condition the smooth running of the therapeutic act. aim: to evaluate the anesthetic modalities of the extraction of the foreign bodies of the airways in children, in order to optimize our care with a maximum of security. a prospective, mono-centric, descriptive study from january to november of patients treated for inhalation of foreign bodies in the airways. study population wasdefined by: age, sex, hospitalization context, physical and radiological examination data, anestheticmanagement. results: the average age of the patients was . months, the male predominated ( %), and the hospitalization context was polymorphic. general anesthesia was necessary in all cases, sevoflurane mainly for narcosis; the combination of an opioid in . % of cases and a curare in . %. spontaneous ventilation is desirable, but % was manually broken down intermittently between extraction attempts. cases of desaturation, bronchospasm, bradycardia, and pneumothorax have been reported. . % had a good evolution. discussion: the results of the epidemiological data are consistent with those of the literature. the penetration syndrome is very revealing. the chest x-ray is the key examination, the diagnosis is often based on indirect signs. in case of asphyxia by foreign body enclosed above or between the vocal cords, laryngoscopy and oxygenation is the first step to perform. in other cases, a rigid bronchoscopy is performed under general anesthesia; inhalation induction with sevoflurane is the technique of choice for many experienced authors. controlled ventilation is used in the majority of cases because spontaneous ventilation is not often not possible. the heterogeneity of anesthetic practices accounts for the multiplicity of clinical situations. conclusion: the inhalation of a foreign body is a diagnostic and therapeutic emergency. extraction of the foreign body takes place under general anesthesia, which is difficult and at risk. compliance with ethics regulations: yes. non-invasive neurally adjusted ventilatory assist (nava) in infants with bronchiolitis: a retrospective cohort study alex lepage-farrell, sally al omar, atsushi kawaguchi, sandrine essouri, philippe jouvet, guillaume emeriaud chu sainte justine, université de montréal, montréal, canada correspondence: alex lepage-farrell (alex.lepage-farrell@umontreal.ca) ann. intensive care , (suppl ):p- rationale: bronchiolitis is one main reason for admission to pediatric intensive care unit. most infants are successfully managed with nasal cpap or high-flow nasal cannula, but about a third of these patients are not sufficiently supported and require an alternative support. non-invasive neurally adjusted ventilatory assist (niv-nava) improves patient-ventilator interactions and could therefore improve the effectiveness of non-invasive support. our hypothesis is that niv-nava is feasible in infants with bronchiolitis and that it reduces the respiratory effort. patients and methods: we retrospectively studied all patients under years of age with a clinical diagnosis of bronchiolitis ventilated with niv-nava in our pediatric intensive care unit, between october and june . patients characteristics, respiratory and physiologic parameters, including diaphragmatic electrical activity (edi) were extracted from an electronic medical database (data collected every s). respiratory effort was estimated using the modified wood clinical score for asthma (mwcas) and the inspiratory peak edi, and -h periods before and after niv-nava initiation were compared (wilcoxon rank test). the study was approved by the local research ethics committee. results: during the study period, patients were admitted with bronchiolitis; infants ( boys) with a median ( th- th percentile) age of ( - ) days were treated with niv-nava after a failure of other non-invasive support methods, and all were included. twentyfive subjects ( %) had at least one comorbidity. the interfaces used were predominantly face masks ( %). the maximum ventilatory settings were nava level of . ( . - . ), peep of ( - ) cmh o, fio of % ( - ) and maximal pressure of ( - ) cmh o. total duration of non-invasive ventilation was ( - ) hours, including ( - ) hours in niv-nava. as detailed in the table , mwcas significantly decreased after niv-nava initiation, from . ( . - . ) to . ( . - . ), p < . . a decrease in inspiratory peak edi was also observed, which was particularly clinically relevant in infants with high baseline edi (> mcv). capillary blood ph and pco also significantly improved after niv-nava introduction. six patients ( %) needed escalation to endotracheal intubation. conclusion: this study confirms the feasibility of niv-nava in infants with bronchiolitis after failure of first line non-invasive support, with a low failure rate. niv-nava initiation was followed by a decrease in respiratory effort and an improvement in blood gases. this observational study supports the needs for prospective interventional trial. compliance with ethics regulations: yes. rationale: the use of blood transfusion is frequent in pediatric intensive care units and has increased significantly since . considered as therapeutic, it requires an assessment of the benefit / risk balance before making the transfusion decision. the aim of our study is to describe the transfusion practices in the pediatric resuscitation department of the ehs canastel, algeria. patients and methods: a retrospective observational study over a -month period from january of any blood transfusion performed in hospitalized patients, in the pediatric intensive care unit. we studied : the age, the sex, the history of blood transfusion, the indication of transfusion, the haemodynamic and respiratory parameters, the transfusional accidents, the length of stay in intensive care, the evolution after a blood transfusion. results: these included transfusion patients out of hospitalizations during the -month period, mean age was months.all patients had no transfusion history, % of patients had their anemia admission and % developed it during their stay. the reason for hospitalization was respiratory distress in %, convulsive condition in %, polytrauma in %, and head trauma in %. the indication of the transfusion was placed on a hb inferior or equal to g / dl in % of cases, in % on an hb superior to g / dl in addition to the clinical criteria of intolerance to anemia; in % of the cases no clinical or biological criteria found, the nature of the blood products was of the red cell in % of the cases and of the plasma concentrate in / of the cases and pfc in %. % received a+, % of a-, % of b+, % of o+ and % of o-. % of the patients had a transfusion-like reaction at min after the start of the transfusion; % of the patients were under artificial ventilation and % were under hemodynamic support, % under diuretic.the average length of stay was days; the favorable outcome was % of the patients after the transfusion with an increase in the hb level beginning, % of the patients had complications of their pathology and the death in % of the cases. conclusion: current transfusion practices in children often do not reflect the implementation of our current knowledge of the need for transfusion. hence the need to review the protocols and practice other transfusion alternatives to avoid complications and improve the quality of care. compliance with ethics regulations: not applicable. rationale: bacterial multi drug resistance is medical actuality nowadays, because of its morbidity and mortality especially in intensive care, it constitutes a real problem in our hospitals. we conducted a retrospective descriptive study, to identify bacterial drug resistance profile of patients with cross infections in the department of intensive care in august hospital. this study included patients hospitalized between st january and st december . the data was collected from medical records of this unit as from the register of the bacteriology service of ibn rochd university hospital. results: patients were hospitalized in the resuscitation service, of which had nosocomial infection, an incidence of . %. the mean age of the patients was years with male predominance (sex ratio . ), the average stay in intensive care was days. the site of infection was pulmonary in % of cases, blood in % of cases, urinary in % of cases, central catheter in %, neuro-meningeal in . % of cases. the germs isolated were: acinetobacter baumanii in . % of cases, pseudomonas aeroginosa in . % of cases, klebsiella pneumonia in . % of cases, enterococcus feacalis in . % of cases, e.coli in . % of cases and staphylococcus aureus in % of cases. acinteobacter baumanii showed resistance rates of up to % for the impenem and % for amikacin. regarding pseudomonas, it was resistant to impenem in % of cases and in % of cases to amikacin. compared to klebsiella, resistance to imipenem was % and % for amikacin. the mortality rate of infected patients was % conclusion: in the light of this work, we found that important emergence of multidrug resistance bacteria in intensive care unit is related to not only the immunocompomised state of patients but also to daily bad practices of health professionals such as the misuse of antibiotics. compliance with ethics regulations: yes. overnight culture of escherichia coli, klebsiella pneumoniae, staphylococcus aureus and pseudomonas aeruginosa, was also sequenced. results: twenty-four samples and the pc were analyzed. amplicon sequence analyses found similar results with the two primer pairs in % of cases. cultured pathogen was found in % ( / ) for human primer pair and in % ( / ) for earth primer pair. for each eta, ngs revealed bacteria unknown as pathogen globally identified as oropharyngeal flora in conventional microbiology (table ) . alpha diversity decreased for all vap patients overtime, average shannon . ( ; . ) versus ( . ; . ), and was higher in upper respiratory tract (os) versus lower respiratory tract (eta): average shannon . ( . ; . ) vs. . ( . ; . ) (ns). conclusion: this pilot study highlights the impact of s rdna amplification procedures (especially oligonucleotide sequences) used on the results in microbiome research. concordance between ngs and bacterial culture, as well as similar evolution of the alpha diversity than previously described ( ), enables us to validate our methodology using the "gut primers" pair f- r. these findings allow furthers major studies on the pulmonary microbiome of icu ventilated patients including comparison according to the occurrence of a vap or not. compliance with ethics regulations: yes. rationale: in the field of intensive care only few studies have explored bacterial microbiota whereas virome remained hardly considered. it appears essential to describe both evolution in mechanically-ventilated patients to improve the pathophysiological understanding of ventilator-associated pneumonia (vap) development. to date no study had been simultaneously conducted on lower respiratory tract with a single nucleic acid extraction before metagenomics analysis of bacterial microbiota and virome. we conducted a preliminary study to validate our methodology based on a common automated extraction of nucleic acids. patients and methods: twelve mechanically ventilated patients were selected: five who developped (vap) and seven controls (c) who did not. endotracheal aspirate (eta) were collected between intubation and day (or dvap for vap patients). conventional bacterial microbiology and multiplex respiratory viruses pcr were also performed. total nucleic acids were extracted using nuclisens easymag extractor. for the bacterial microbiota, region v of the s rrna genes was amplified. for the virome, the nextera dna xt kit (illumina) and rna seq trio kit (nugen) protocols were used to prepare viral dna and rna libraries. libraries underwent paired-end sequencing on the illumina miseq (bacteria) or nextseq- (virus) platform. after bioinformatics analysis we compared the performance of metagenomics analysis with conventional bacterial culture and other common viral detection methods. results: for culturable bacteria, concordance between conventional microbiology and sequencing was found in % ( / table . our preliminary results confirm the feasability of exploring both bacterial microbiota and virome on the same sample using a common extraction method. data from metagenomics were highly concordant with conventionnal detection methods for known pathogenic viruses and bacteria in lower tract respiratory sample and enables identification of other microorganisms. this is the first step for a large cohort study that aims to compare evolution of global lung microbiome in patients at risk of vap and assess how bacteria and virus interplay. compliance with ethics regulations: yes. references . clancy department of medical and toxicological critical care, lariboisière hospital one microorganism was isolated in . % and two in . % of cases. the main isolated microorganism were enterobacteriaceae in . % of patients. they were esbl-producers in . % of cases. initial antibiotic therapy was appropriate in . % of cases. factors independently associated with esbl-pe as the causative microorganism of icu-bsi were esbl-pe carriage prior to icu-bsi the sensitivity of esbl-pe carriage to predict esbl-pe as the causative microorganism of icu-bsi was . %, and specificity was . %. mortality at days was . % in the general population in multivariable analysis, there was no parameter which was independently associated to mortality at day from the occurrence of icu-bsi. conclusion: icu-bsi complicates . % of admission to icu and was associated with % in-hospital mortality assessing and applying individualized treatment for group a streptococcal necrotizing soft-tissue infection is possible service de réanimation médicale intensive care decompressive craniectomy in traumatic brain injury: about cases karama bouchaala sex ratio of . . the mean (sd) length of stay in icu was . ± . days. the mean glasgow coma score (gcs) (sd) was . ± . and gcs ≤ in . %. sofa score > was found in patients ( . %) and sapsii score ≥ in patients ( . %). the cerebral ctscan at admission showed acute subdural hematoma (asdh) in ( . %), cerebral oedema ( . %) and cerebral contusions ( %) teaching: fresenius medical care; patent or product inventor: gml czech republic banydeen rishika: no conflict of interest baptiste amandine: no conflict of interest baptiste olivier: no conflict of interest barbar saber davide: no disclosure barbier françois: no disclosure barbierlouise: trainings, teaching: ethicon, astellas; invitation to national or international congresses: sandoz, astellas barnerias christine: no disclosure baron aurore: no disclosure baron elodie: no conflict of interest barr att -due andreas: no disclosure barrau stephanie: no disclosure barraud damien: no disclosure barraud helene: no disclosure barrois brigitte: no conflict of interest baruchel andré: no disclosure bastide marie anaïs: no conflict of interest baudel jean-luc: no conflict of interest baudin florent: invitation to national or international congresses: dr baudin has received speaking fees from maquet critical care (epnv teaching: drager; invitation to national or international congresses: msd; hill rom beganton frankie: no conflict of interest begot erwan: no disclosure beinse guillaume: research support/scientific studies: association pour la recherche contre le cancer ion and fresenius kabi bensaid abdelhak: no disclosure bensardi fatimazahra: no disclosure benyamina mourad: no disclosure benzerara laurent: patent or product inventor: aphp benzerdjeb nazim: research support/scientific studies: amarape, icap; consultancy, expert: alphasights, msd; trainings, teaching: msd beqiri erta: no disclosure bÉranger agathe: no conflict of interest berard emilie: no conflict of interest berdai adnane: no disclosure berger patrick: no disclosure bernal william: no disclosure bernardin gilles: no disclosure berrada lina: no conflict of interest berthaud romain: no conflict of interest berthet guillaume: no conflict of interest berti enora: no conflict of interest bertoli sarah: no disclosure bertrand pierre-marie no conflict of interest besbes lamia: no disclosure besbes mohamed: no conflict of interest besch camille: invitation to national or international congresses: abbvie no conflict of interest boisseau chloé: no disclosure boissel nicolas: no disclosure boissier florence: no conflict of interest boivin alexandra: no conflict of interest bonacorsi stéphane: no conflict of interest bongiovanni filippo: no conflict of interest bonnardel eline: no conflict of interest bonnefoy-cudraz eric: no disclosure bonnet sixtine: no conflict of interest bonnevie tristan: research support/scientific studies invitation to national or international congresses: fresenius kabi and fresenius medi-calcare bucur petru: no disclosure buetti niccolo: research support/scientific studies: swiss national science foundation research grant and bangerter rhyner foundation supporting my postdoc bui hoang-nam: no disclosure burelli gabrielle: no conflict of interest burgel pierre-régis: no disclosure burghi g: no conflict of interest bustarret olivier: no conflict of interest butin-druoton anne-lise: invitation to national or international congresses expert: astra-zeneca; invitation to national or international congresses expert: hamilton medical; invitation to national or international congresses: hamilton medical chemli wael: no conflict of interest chenouard alexis: no conflict of interest cherkab rachid: no conflict of interest chevret sylvie: no disclosure chhun stephanie: no conflict of interest chiche jean-daniel: no disclosure chicoisneau maxence: no conflict of interest chlilek abdelaziz: no disclosure chocron richard: consultancy, expert: aspen chommeloux juliette: no conflict of interest chomton maryline: no conflict of interest chosidow olivier: no disclosure chouchana laurent expert: biotest; invitation to national or international congresses: sanofi research support/scientific studies: fresenius medical care; consultancy, expert: fresenius medical care; invitation to national or international congresses: xenios novalung, heilbronn, germany dachraoui fahmi: no disclosure dahoumane redouane: no conflict of interest dahyot-fizelier claire: no disclosure daix thomas: no conflict of interest daly foued: no conflict of interest damonti lauro: no conflict of interest dantan etienne: no conflict of interest darmon michaël: research support/scientific studies: msd no disclosure das vincent: no disclosure daubin cedric: no conflict of interest daubin delphine: no conflict of interest daudon michel: no disclosure daufresne pierre: no conflict of interest dauger stéphane: no conflict of interest daviet florence: invitation to national or international congresses: sandosz de courson hugues: no conflict of interest de jong audrey: trainings, teaching: baxter, medtronic; invitation to national or international congresses teaching: cardiosleep delhaes laurence: no disclosure delignette marie-charlotte: no conflict of interest dellamonica jean: trainings, teaching: medtronic; invitation to national or international congresses: msd, general electrics delpierre clément: no conflict of interest delville marianne: no conflict of interest demailly zoé: research support/scientific studies: srlf demarest elsa: no disclosure demaret pierre: no conflict of interest demiselle julien: no conflict of interest demondion pierre: no conflict of interest demoule alexandre: research support/scientific studies: drager, philips; consultancy, expert: baxter, respinor, lungpacer; trainings, teaching: fisher & paykel, hamilton, baxter; invitation to national or international congresses: fisher & paykel denis manon: no conflict ofinterest depeyre fanny: invitation to national or international congresses: pfizer deplante yvon: no conflict of interest dequin pierre-françois: research support/scientific studies: medimmune combioxin ferring pharmaceuticals a/s asahi kasei pharma america corporation derauglaudre lucie: no conflict of interest derbel karim: no disclosure derkaoui ali: no disclosure dervin krystel: no conflict of interest desaive thomas: no conflict of interest desguerre isabelle: research support/scientific studies: ptc inc, avexis; consultancy, expert: avexis, ptc inc, biogene; trainings, teaching: roche, ptc inc, avexis; invitation to national or international congresses: sarepta, biogen, avexis, biomarin desnos cyrielle: no conflict of interest desroys du roure françois: no conflict of interest detollenaere charles: no conflict of interest devaquet jérôme: invitation to national or international congresses expert: lungpacer; invitation to national or international congresses: lungpacer dreyfuss didier: research support/scientific studies: grant from french ministry of health drouot xavier: no disclosure du cheyron damien: no conflict of interest dubÉ bruno-pierre: consultancy, expert: novartis, gsk dubert marie: no conflict of interest dubost baptiste: no conflict of interest dubost jean-louis: no conflict of interest duburcq thibault: no conflict of interest duchemann boris: consultancy, expert: bms, msd, roche; invitation to national or international congresses no conflict of interest frÉrou aurélien: no conflict of interest fritz caroline: no disclosure fromentin mélanie: research support/scientific studies: msd; invitation to national or international congresses: msd frouin antoine: no conflict of interest frugier alexandre: no disclosure gaboriau louise: no conflict of interest gaci rostane: invitation to national or international congresses: bard gacouin arnaud: no disclosure gaddas mehdi: no conflict of interest gaillard arnaud: trainings, teaching: zoll medical gaimard sophie: no conflict of interest gainnier marc: no conflict of interest galbois arnaud: no conflict of interest galerneau louis-marie: invitation to national or international congresses: agir À domicile galicier lionel: consultancy, expert: novartis, eusapharma; trainings, teaching: baxalta, pfizer; invitation to national or international congresses no conflict of interest ichaÏ philippe: no conflict of interest imen sioud: no conflict of interest ioos vincent: no disclosure iserin franck: no disclosure issa nahema: no conflict of interest jaber samir: consultancy, expert: drager, fisher-paykel; medtronic; baxter xenios fresenius; invitation to national or international congresses: drager no conflict of interest jacq gwenaëlle: no conflict of interest jacquet emmanuelle: research support/scientific studies: unicancer (esme and storm studies invitation to national or international congresses: pfizer université laval-qc-ca labbe vincent: no disclosure labro laura: no disclosure lacaille florence: no conflict of interest lacampagne alain: no disclosure lacan claire: no conflict of interest lacherade jean-claude: no conflict of interest ladjemi maha-zohra: no conflict of interest lafon charles: no conflict of interest lafon marie-edith: no disclosure lafon thomas: no conflict of interest lagache laurie: invitation to national or international congresses advertising documents: philips; trainings, teaching: novartis, gsk, astra zeneca, boeringher; invitation to national or international congresses: chiesi, astra zeneca, sos oxygene, novartis, boeringher lamoth frédéric: consultancy, expert: gilead, msd, basilea; invitation to national or international congresses: msd expert: norgine; trainings, teaching: fujifilm, boston scientific lebreton guillaume: no disclosure lebrun-vignes benedicte: research support/ scientific studies: novartis; consultancy, expert: ansm lebuffe gilles: no disclosure leclerc maxime: no conflictof interest lÉcluse aldéric: research support/scientific studies: pgrx avc study; consultancy, expert: bms-pfizer, boerhinger ingelheim, bayer; invitation to national or international congresses: bms-pfizer, boerhinger ingelheim ledoux didier: no disclosure lefebvre francois: no conflict of interest macloughlin ronan: research support/scientific studies: aerogen ltd no conflict of interest mari arnaud: no conflict of interest marie damien: no conflict of interest marijon eloi: no disclosure mariotte eric: consultancy, expert: sanofi-aventis marjanovic nicolas: no disclosure marjanovic zora: no disclosure maroni arielle: no conflict of interest marot benoit: no conflict of interest marque sophie: no conflict of interest marti teaching: zambon, chiesi; invitation to national or international congresses no conflict of interest matusik elodie: no conflict of interest mauchien benedicte: no conflict of interest maury eric: research support/scientific studies: doran international, drager; trainings, teaching: vygon maxime virginie: no conflict of interest mayaux julien: invitation to national or international congresses stock shareholder: tanderev; patent or product inventor: tanderev mercat alain: research support/scientific studies: fisher-paykel, general electric; consultancy, expert: faron pharmaceuticals no disclosure merhabene takoua: no conflict of interest merle jean-claude: no disclosure mesotten dieter: no conflict of interest messaadi amenallah: no conflict of interest messika jonathan: invitation to national or international congresses: cslbehring; fisher&paykel metaxa victoria: no disclosure metogo mbengono junette arlette: no conflict of interest meunier anne: no conflict of interest meurice jean-claude: no disclosure meybeck agnes: consultancy, expert: janssen, gilead; invitation to national or international congresses teaching: msd no conflict of interest morimont philippe: no conflict of interest moro-sibilot denis: no disclosure mortamet guillaume: no conflict of interest mosbah nabil: no conflict of interest moschietto sebastien: no conflict of interest moucadel virginie: research support/scientific studies: biomérieux moulaire rigollet valérie: no disclosure mouliade charlotte: no conflict of interest moulin florence: no disclosure mounir yousfi: no conflict of interest mourabit karima: no disclosure mourvillier bruno: trainings, teaching: msd research support/scientific studies: aerogen; advertising documents: aerogen; patent or product inventor: aerogen musiari michele: no conflict of interest n'guyen quang-thang: no conflict of interest n'guyen tran: no disclosure nabil mosbah: no disclosure naccache lionel: no disclosure naimi skander: no conflict of interest nakaa sabrine: no disclosure nallet-amate megan: no conflict of interest natalis eloïse: no disclosure naudin jérôme: invitation to national or international congresses: novartis nay mai-anh: no conflict of interest nemlaghi safaa: no conflict of interest neofytos dionysios: research support/scientific studies: msd; consultancy, expert: msd, gilead, pfizer; invitation to national or international congresses: gilead, pfizer nesseler nicolas: no conflict of interest neviere remi: no disclosure nguyen alexandre: no disclosure nguyen khoa thao: no conflict of interest nicolau-travers marie-laure: no disclosure niÉrat marie cécile: no conflict of interest nieszkowska ania: no disclosure nigeon olivier: no conflict of interest nitel gautier: no conflict of interest nodea elena madalina: no conflict of interest noel marine: no conflict of interest nogier marie-béatrice: no disclosure noorah zaid: no disclosure nouira wiem: no conflict of interest noumeir rita: stock shareholder: softmedical noury norbert: no conflict of interest novy emmanuel: research support/scientific studies: msd; invitation to national or international congresses: pfizer expert: air liquide medical system ollivier veronique: no conflict of interest onimus thierry: no conflict of interest oppenheimer anne: invitation to national or international congresses: gedeon richter orkisz maciej: no conflict of interest orliaguet gilles: research support/scientific studies research support/scientific studies: oxynov; patent or product inventor: oxynov patrier juliette: no conflict of interest paugam catherine: no disclosure paul marine: no conflict of interest paul-bellon rachel: no disclosure paulo nicolas: no conflict of interest pavot arthur: invitation to national or international congresses: fresenius medical care france pehlivan jonathan: no conflict of interest peigne vincent: invitation to national or international congresses: air liquide pÉju edwige: no conflict of interest pene frédéric: consultancy, expert: alexion pÉpin-lehalleur adrien: invitation to national or international congresses: chiesi pere morgane: no conflict of interest pereira bruno: no disclosure perez didier: no disclosure perez pierre: no disclosure perez yonatan: no conflict of interest perier françois: no disclosure perin nicolas: no conflict of interest biomerieux robin emmanuel: no conflict of interest robin nicolas: no disclosure robineau olivier: no disclosure roch antoine: no disclosure roche anne: no conflict of interest roger claire: consultancy, expert: pfizer, fre-senius medical care; invitation to national or international congresses: msd,pfizer rolle amélie: no conflict of interest rondeau eric: no disclosure ronziÈre thomas: no disclosure roquilly antoine: no disclosure rosselli sylvène: no disclosure rouby jean-jacques: no disclosure rouis sana: no conflict of interest rouleau stéphane: no conflict of interest roulet sylvie: no disclosure roulland charlotte: no disclosure roumeliotis nadia: no conflict of interest rousse natacha: no disclosure rousseau anne-françoise: invitation to national or international congresses no disclosure sagnier anne: no disclosure saillard colombe: trainings,teaching: amgen, novartis; invitation to national or international congresses no conflict of interest schmidt aline: no disclosure schmidt matthieu: consultancy no disclosure schultz marcus: no conflict of interest schwebel carole: invitation to national or international congresses: pfizer scicluna brendon: no disclosure sculier jean-paul: no conflict of interest see perrine: no conflict of interest seghboyan jean-marie: no disclosure seguin amelie: no conflict of interest seguin philippe: consultancy, expert: lfb; invitation to national or international congresses: astellas sejourne caroline: no conflict of interest sellami walid: no conflict of interest sendid boualem: research support/scientific studies: allfun project, fp european commission; invitation to national or international congresses: pfizer senhadji lahcen: no conflict of interest serbouti rita: research support/scientific studies: fresenius medical care; consultancy, expert: fresenius medical care; trainings, teaching: fresenius medical care; invitation to national or international congresses: fresenius medical care serfaty lawrence: no disclosure sÉrie mathieu: no conflict of interest shaw geoffrey m.: no conflict of interest shi rui: no conflict of interest shimi abdelkrim: no disclosure shojaei maryam: no disclosure si-tahar mustapha: consultancy, expert: cynbiose respiratory; stock shareholder: cynbiose respiratory siami shidasp: no conflict of interest silva daniel: research support/scientific studies: fresenius medical care france; consultancy, expert: fresenius medical care france; invitation to national or international congresses: xenios novalung, heilbronn no conflict of interest sirault bruno: no disclosure sirodot michel: no disclosure slama michel: no disclosure slim amine: no disclosure smielewski peter: no disclosure soares marcio: stock shareholder: epimed solutions teaching: gilead; invitation to national or international congresses: pfizer spagnoletti marco: no conflict of interest steckelmacher claire: no disclosure stockx luc: research support/scientific studies: phenox, medtronic; consultancy no conflict of interest voiriot guillaume: research support/scientific studies: biomérieux, sos oxygène, janssen; consultancy, expert: biomérieux; invitation to national or international congresses: biomérieux von kietzell matthias: invitation to national or international congresses expert: aguettant; invitation to national or international congresses: vifor yacoubi wejden: no conflict of interest yager hélène: no conflict of interest yahya yosra: no conflict of interest yakini khalid: no disclosure yakouben karima: no disclosure yonis hodane: invitation to national or international congresses: lvl medical et pfizer younan romy: no conflict of interest youssoufa atika: no disclosure zacharia mahi: no disclosure zafrani lara: research support/scientific studies: jazz pharmaceuticals zambon olivier: no disclosure zaouak nadia: no conflict of interest zaouche khedija: no conflict of interest zarrougui wafa: no conflict of interest ze minkande jacqueline: no disclosure zeghdoud dalila: no disclosure zerbib yoann: no conflict of interest zerhouni amel: no conflict of interest zerhouni amine: no conflict of interest zerimech farid: no conflict of interest zerouali khalid: no disclosure zheng yi: no conflict of interest zimmerli stefan: research support/scientific studies: msd, pfizer, gilead; consultancy, expert: msd, pfizer; trainings, teaching: gilead; invitation to national or international congresses springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations farhat hached hospital, sousse, tunisia; yassminet regional hospital, ben arous, tunisia; habib bougatfa regional hospital, bizerte, tunisia; larabta hospital, tunis, tunisia; carthagene private hospital, tunis, tunisia; regional hospital of zaghouan, zaghouan, tunisia; regional hospital of tozeur, tozeur, tunisia; habib thameur hospital, tunis, tunisia correspondence: samia ayed (samia.ayed@yahoo.fr) ann. intensive care , (suppl ):p- geoffroy hariri, kyann hodjat-panah, laurene blum, jean-rémi lavillegrand, idriss razach, naike bige, jean-luc baudel, bertrand guidet, eric maury, hafid ait-oufella médecine intensive-réanimation, hôpital saint-antoine, paris, france correspondence: geoffroy hariri (geoffroyhariri@hotmail.com) ann. intensive care , (suppl ):p- rationale: hemolytic anemia (ha) is a common condition in intensive care unit but its diagnosis remains challenging. free hemoglobin (and heme) degradation leads to co release that can bind to hemoglobin to form carboxyhemoglobin (hbco). we hypothesized that hbco concentration could be used as a reliable diagnosis tool for ha. patients and methods: we performed a monocentric retrospective study in a -bed intensive care unit at st antoine hospital, paris, between and . all patients hospitalized for ha with arterial hbco dosage at admission were included. arterial hbco was measured in routine in our department with an il system ph/ blood gas analyzer. demographic and biological data were collected. a group control of patients with non-hemolytic anemia (hb < g/ dl) (nha) was also included. finally, we analyzed patients outcome according to hbco changes during icu stay. results: between and , patients with ha were included. nha patients were included in the control group. patients with ha were younger than patients with nha ( [ ; ] vs. [ ; ] years old, p = . ) but admission sofa was not different between groups ( [ ; ] , vs. [ ; ] , p = ns). among patients with ha, % had thrombotic microangiopathy, % had autoimmune hemolytic anemia and % had sickle cell disease. at icu admission, ha patients had higher hbco level than patients with nha ( . [ . ; . ] vs. . [ . ; . ] %; p < . ). hbco was a reliable biomarker of hemolysis (auc . ( . ; . ) p < . ). an hbco level threshold at . % identify hemolysis with a sensitivity ( - ) % and a specificity ( - ) %. in ha group, hbco was negatively correlated to hb level (r = . ; p < . ). in ha patients, changes of hbco level during icu management were associated with outcome, decreasing in survivors ( . [ ; . ] vs. . [ . ; . ] ; p = . ) but not in non-survivors ( . [ . ; . ] vs. . [ . ; . ] %; p = . ). conclusion: carboxyhemoglobin is a reliable diagnosis and prognosis biomarker for hemolytic anemia in icu compliance with ethics regulations: yes. rationale: thrombocytopenia is the most commonly hemostatic disorder encountered in intensive care, present in to % of patients. the mortality associated with this thrombocytopenia, the numerous pathological contexts associated with resuscitation and the lack of a recommended management strategy led to the establishment of these guidelines. the aim of our study was to determine the incidence, causes and risk factors associated with the occurrence of thrombocytopenia, as well as the impact of thrombocytopenia on the mortality and length of stay in the icu ibn medical resuscitation unit. rochd de casablanca, over a period of months. patients and methods: this was a prospective study, carried out in the medical resuscitation department of ibn rochd university hospital in casablanca over a period of months. there were two groups: ''sick'' group with thrombocytopenia with a platelets count < , / mm , and a ''control'' group without thrombocytopenia. patients with previous platelet disorders, hematologic malignancies, and patients undergoing chemotherapy were excluded. of the patients included, episodes of thrombocytopenia were identified, anoverall incidence of . %. sepsis was incriminated times ( . %), followed by ards in patients ( . %), massive filling in patients ( . %), disseminated intravascular coagulation in patients ( . %), and massive transfusion in patients ( . %). the drug origin was incriminated in patients ( . %). it was due to quinolones and imipenem. the mortality rate was deaths ( . %) which was inversely proportional to the lowest platelet count in the thrombocytopenia group, compared to deaths ( %) in the control group. the mean duration of stay in the thrombocytopenia group was ± days with extremes ranging from to days. conclusion: thrombocytopenia was a common abnormality in the intensive care system, it occured in many pathological situations and was a factor of morbidity and excess mortality. the most common etiology in this study was sepsis. the diagnostic and therapeutic approach depended on the particular clinical context in which thrombocytopenia occurs. its onset may constitute a hematological emergency, particularly when there is a major mucocutaneous and / or visceral hemorrhagic syndrome, which necessitates a rapid etiological diagnosis, and the establishment of an effective treatment, both symptomatic and specific. compliance with ethics regulations: not applicable. marc pineton de chambrun , romaric larcher , frédéric pene , laurent argaud , alexandre demoule , rémi coudroy , elie azoulay , yacine tandjaoui-lambiotte , stanislas faguer , alain combes , charles-edouard luyt , zahir amoura sorbonne université, aphp, hôpital la pitié-salpêtrière, institut de cardiométabolisme et nutrition (ican), service de médecine intensive-réanimation, paris, paris, france; rationale: catastrophic antiphospholipid syndrome (caps), the most severe manifestation of antiphospholipid syndrome (aps), is characterised by simultaneous thromboses in multiple organs. diagnosing caps can be challenging but its early recognition and management is crucial for a favourable outcome. this study was undertaken to evaluate the frequencies, distributions and ability to predict mortality of "definite/probable" or "no-caps" categories of thrombotic aps patients requiring admission to the intensive care unit (icu rationale: septic acute kidney injury (s-aki) is a frequent complication in critically ill patients and is associated with high morbidity and mortality. it is well known that chronic kidney disease increases the risk of pulmonary embolism (pe), but few studies have investigated the relationship between acute kidney injury (aki) and pe occurrence in septic patients. the aim of this study is to determine whether patients with aki are at increased risk of developing pe. patients and methods: were included, in a prospective study conducted over months (january -june , ) in a medical surgical intensive care unit, all the patients older than years with septic shock at admission or during hospitalization. two groups were compared: patients with kidney injury (aki+ group) and patients without kidney injury (aki− group). we studied the occurrence of pe in these two groups. results: we included patients. the mean (sd) age was . ( ± ) years. sex ratio was . . thirty one ( . %) patients developed pe. the occurrence of pe was significantly higher in (aki + group) [ patients ( %) vs. patients ( %); p = . ]. the incidence of pe according to kidney injury severity was patients ( %) kdigo i, patients ( %) kdigo ii, patients ( %) kdigo iii. in the aki+ group, pe was significantly associated with increased sofa score at admission ( points vs. points; p = . ), lower platelets count ( , vs. , ; p = . ), higher lacatatemia at septic shock day [ . vs. . mmol/l; p = . ] and higher c reactive protein level [ mg/l vs. mg/l; p = . ]. in a multivariate analysis the pe risk factors in (aki+ group) were thrombopenia (odds ratio = . ; ci [ . - . ], p = . ) and c-reactive protein value (odds ratio = . ; ci[ . - . ], p = . ). discussion: the increased risk for pe with aki may be due to endothelial involvement, vascular injury and the related changes found in procoagulant proteins (increased levels of fibrinogen, factor vii, factor viii, von willebrand factor, and plasminogen activator inhibitor- ). in our study, lower platelet and higher c reactive protein level were found in patients with pe, suggesting the participation of disseminated intravascular coagulation. these factors may contribute to increase pe risk. conclusion: the risk of pe is higher in septic patients with aki than in those with normal kidney function. therefore, because of paucity of evidence, larger studies are needed to understand pe pathway in septic aki and to establish efficient prophylaxis protocols. compliance with ethics regulations: yes. and of these patients ( . %) required intensive care. the lasted were males ( %) and a majority ( %) were younger than years of age. in intensive care patients, only ( . %) had nosocomial infection, majority were community acquired infections ( . %) with ( %) pneumoniae, ( . %) profound abscess, pyelonephritis ( . %), ( %) meningitidis. patients( %) required mechanical ventilation for days ( % ci - ), length of stay in icu was days ( % ci - ) and mortality rate was %. conclusion: hmkp infections lead young patients in intensive care unit in one third of case with a majority of pneumoniae requiring mechanical ventilation and with a high rate of mortality. furthers studies are needed to investigate the role of this particular strain in severity. compliance with ethics regulations: yes. rationale: infections secondary to snakebite occur in a number of patients, and are potentially life-threatening. bothrops lanceolatus bites in martinique average thirty cases per year and may result in severe thrombotic and infectious complications. we aimed to investigate the infectious complications related to bothrops lanceolatus bite. patients and methods: a retrospective single-center observational study over seven years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) was carried out, including all patients admitted to the hospital due to bothrops lanceolatus bite. clinical and biological data were reported using the dx care, x-plore et cyberlab softwares of the emergency medicine and analyzed. one hundred and seventy snake-bitten patients ( males and females) were included. thirty-nine patients ( %) presented grade or envenoming. twenty patients ( %) developed wound infections. the isolated bacteria were aeromonas hydrophila ( cases), morganella morganii ( cases), group a streptococuss, and group b streptococcus (one case each). patients were treated empirically with third-generation cephalosporin (or amoxicillin/clavulanate), aminoglycoside and metronidazole combinations. outcome was favorable. the main factor significantly associated with the occurrence of infection following snakebite was the severity of envenoming (p < . ). our findings clearly point towards the frequent onset of infectious complications in b. lanceolatusbitten patients presenting with grade and envenoming. conclusion: infectious bite-related complications of bothrops lanceolatus account for approximately % of the cases, with a strong predominance for grade iii and iv. thus, based on the bacteria identified in the wounds; we suggest that empiric antibiotic therapy including third-generation cephalosporin should be administered to those patients on hospital admission. compliance with ethics regulations: yes. rationale: endocrine abnormalities have been reported with varying frequencies, following traumatic brain injury (tbi). few studies have examined the clinical features and outcomes of isolated acute thyrotropic hormone deficiencies after tbi. theaim of the study was to evaluate the early changes in thyrotropic hormone levels after traumatic brain injury (tbi) and to evaluate whether hormone changes are related to outcome patients and methods: we conducted a months long prospective cohort, including all patients admitted to a university hospital icu with moderate to severe traumatic brain injury (tbi), defined as a glasgow coma scale below twelve (gcs < ). blood samples for basal hormone values of thyroid-stimulating hormone (tsh) and free thyroxine (ft ) were obtained on days , , and . tsh serum concentrations were considered normal at > . mu/l; ft at > pmol/l. a thyrotropic insufficiency was defined as low ft and low tsh plasma levels. all patients were screened with a brain mri. patients were also monitored for neurological deterioration, including cognitive decline, convulsive seizures, increase in cerebral edema and brain herniation that were simultaneous to the diagnosis. results: during our study period's, trauma patients were admitted to our icu and met the inclusion criteria. on admission, our patients had a mean age at . ± , a mean injury severity score (iss) at ± , a mean abbreviated injury severity (ais) of the head at . ± . and a mean gcs at ± . of the patients a thyrotropic insufficiency was diagnosed in patients ( %) during the first days. the median delay to thyrotropic insufficiency diagnosis was days. in three of ( %), the thyrotropic insufficiency was nonrecovering during the patient's icu stay and was transient for the rest. none of the patients with acute thyrotropic insufficiency had direct hypothalamic or pituitary lesions on the brain mri. factors associated to the occurrence of acute thyrotropic insufficiency were: the ais of the head ( . ± . vs. ± . , p = . ), cerebral contusions ( % vs. %, p = . ), subarachnoid haemorrhage ( % vs. %, p = . ) and subdural haematoma ( % vs. %, p = . ). thyrotropic insufficiency was associated to neurological deterioration (p = . ) on the day of diagnosis but did not affect icu mortality ( % vs. %, p = . ). in this study, low pituitary-thyrotropic axis hormone levels were found in the acute phase of tbi and were associated to neurological deterioration but with no perceived effect on icu mortality. compliance with ethics regulations: yes. rationale: acute diabetes insipidus following head injury and its effect on patients outcome have not been sufficiently evaluated in large prospective studies. the aim of this study was to determine the incidence of acute cdi, delay of onset predictive factors and its impact on tbi patients. we conducted a prospective cohort, including all patients admitted to icu with moderate to severe tbi, defined as a glasgow coma scale (gcs) below twelve. for each tbi patient plasma sodium was measured daily, and if abnormally high, urine specific gravity and osmolality were measured. cdi was diagnosed using the seckl and dunger criteria. acute cdi was defined as cdi diagnosed in the first week following injury. all patients were screened with a brain mri. results: during our study's period, trauma patients were admitted to our icu, presented with moderate to severe tbi and were included. on admission, our patients had a mean age at . ± , a mean injury severity score (iss) at ± , a mean abbreviated injury severity (ais) of the head at . ± . and a mean gcs at ± . twenty-three percents ( patients) of the patients developed hypernatremia and % ( patients) were diagnosed with acute cdi. in of ( %), the cdi was nonrecovering. the median delay to develop transient cdi was h and for non-recoviring cdi was h (p = . ). none of the patients with acute cdi had direct hypothalamic or pituitary lesions. factors associated to the occurrence of acute cdi were: younger age ( ± vs ± , p = . ), neuro-surgery ( % vs. %, p < . ), hemorrhagic shock ( % vs. %), p < . ), cerebral edema ( % vs. %), p < . ), and fractures to the base of the skull ( % vs. %, p = . ). patients who developed cdi had a significantly higher mortality than those who did not ( of ( %) vs. of ( %), p < . ). there were no difference in terms of mortality between non-recovering and transient cdi ( % vs. %, p = . ), similarly the onset of cdi did not affect mortality ( h vs. h, p = . ). patients with acute cdi had poor glasgow outcome scale ( ± . vs. . ± . , p < . ) and longer icu los ( ± vs. ± , p = . ). conclusion: acute cdi is associated with higher mortality and poor outcome. therefore it is essential to diagnose and treat it promptly and correctly. compliance with ethics regulations: yes. acute glucocorticoid deficiency following traumatic brain injury mariem dlela, rania ammar zayani, abir bouattour, najeh baccouche, mounir bouaziz habib bourguiba hospital, sfax, tunisia correspondence: mariem dlela (mariem @gmail.com) ann. intensive care , (suppl ):p- rationale: published data demonstrates that long-term hypopituitarism could be common after traumatic brain injury (tbi).however, few studies focused on radiological, clinical, and repetitive endocrine assessment in the acute phase. the aim of the study was to evaluate the early changes in the adrenal axis following (tbi) and to evaluate whether hormone changes affect patient's outcome. we conducted a prospective study, including all patients admitted to a university hospital icu with moderate to severe traumatic brain injury (tbi), defined as a glasgow coma scale below twelve (gcs < ). each patient underwent sequential measurement of plasma cortisol (pc) on days , , and after tbi. we defined adrenal insufficiency as pc less than ng/ml. patients who received glucocorticosteroid therapy were excluded. outcome was measured by incidence of death, and glasgow outcome scale (gos) on day thirty. souhila sadat, dalila zeghdoud, dalila bougdal, kamel guenane ehs salim zemirli, alger, algeria correspondence: souhila sadat (sadatsouhila@hotmail.fr) ann. intensive care , (suppl ):p- rationale: the renewed interest in the pathophysiology of severe traumatic brain injury (tcg), allowed the understanding of the pathophysiological mechanisms leading to neuronal death.the non-invasive, easy, patient-based technical dtc allows evaluation of cerebral blood flow. purpose of the study: to determine the contribution of transcranial doppler (dtp) in the prevention of post-traumatic ischemia. patients and methods: a monocentric, observational, prospective study over a period of years, including tcg in the monitoring of cerebral blood flow (dsc) was provided by the dtc. we collected the following data: age, gender, lesion mechanism, lesion association, glasgow score at admission, time to perform the initial scan, time to perform the initial doppler, various abnormalities found at the initial dtp, the analysis of the level of map according to each situation of cerebral blood flow, the proposed therapies, the time to obtain a correct dtc. ( %), the statistical analysis showed no difference between the delay in setting up a hypohemia and the presence of a correct cerebral blood flow (p = . ), the statistical analysis of the map in the dtc group hypohemia compared to the correct dtc group objectified the absence significant difference between the two groups. the realization of dtp allowed therapeutic prioritization, the introduction of norepinephrine was in % of cases, osmotherapy in % of cases, optimization of sedation in . % of cases, the introduction of penthotal in . % of cases and the completion of decompressive in . % of cases. statistical analysis of mortality showed a significant difference in mortality (p = . ) in the hypohemic dtc group compared with the correct doppler . conclusion: ttc is an essential monitoring tool of cerebral hemodynamics, which may in prove the neurologic outiome of tcg. compliance with ethics regulations: yes. rationale: hyponatremia is a frequent electrolyte disturbance in hospitalized patients. it is particularly common in brain-injured patients with significantly elevated morbidity and mortality. the aim was to study the prevalence of hyponatremia in the acute phase of post-traumatic cerebral aggression, its degree of severity, its predictive factors as well as its prognostic impact in the population of post-traumatic brain injury. patients and methods: this is a retrospective study, carried out over a period of years about all traumatized head patients who developed hyponatremia during the first h of their stay. the descriptive part treated all patients who developed hyponatremia by detailing its different stages of severity.the analytical part treated the patients who developed a hypo-osmolar hyponatremia with a threshold of mmol/l retained to define the severity. during the study period, the incidence of hyponatremia in head trauma patients was . %. the occurrence of hyponatremia was associated only with the occurrence of early seizures (p = . ).severe hyponatraemia was associated with paroxysmal occurrence (p = . ), mass effect (p = . ), and hemostasis disorders. the multivariate study revealed that severe hyponatremia was associated with the glasgow score (p < . ) and pupillary changes (p = . ). on the other hand, it is the initial variation in serum sodium that was associated with both the severity of the initial neurological examination; glasgow (p < . ), saps (p = . ), pts (p = . ) and prism scores (p = . ), haemodynamic instability (p = . ) and neurovegetative disorders (p = . ). lesional features have also been found.regarding the prognosis, the occurrence of initial hyponatremia had a protective effect: a more favorable gos score p = . and a lower mortality (p = . ). a poor neurologic prognosis as well as a high mortality were associated with the most severe hyponatraemia and particularly with the initial variation of the sodium level (p = . ;). the mortality was . %. it was also particularly related to the initial change in sodium levels (p < . , . ). we concluded that there is no association between post traumatic early hyponatremia and the severity of the initial clinical presentation. however, the depth of hyponatremia and especially the initial change in sodium levels have been associated with more severe clinical pictures and a more limited prognosis. compliance with ethics regulations: yes. rationale: post-traumatic epilepsy (pte) is one of the complications described in the aftermath of headtrauma. its incidence is variable in the literature because of its clinical polymorphism. objectives of the study was to analyze the epidemiological profile (clinico-biological, radiological, therapeutic and evolutionary) of the patients having presented pte and to determine the risk factors for this pathology by comparing them with the rest of the traumatized brain patients. patients and methods: our study was retrospective. it was conducted in the intensive care unit (icu) of our university hospital between and . were included in our study all patients admitted to the service with brain injury and a glycaemia above mmol/l during the first h post-trauma. results: the incidence of pte was . %. ( among ) the average age was . ± . years. the sex ratio was . . the average of gcs was . ± . . three ( . %) patients had initial motor impairment. seizures were observed in ( . %) patients during the first h of hospitalization. the mean delay of occurrence of pte was ± . months. pte was diagnosed before the end of the first post-traumatic year in patients ( % of cases). the most commonly observed brain lesions were cortical brain contusions ( rationale: electrolytic disorders are common in neuro-resuscitation, especially dysnatremias and dyskalemias. hyponatremias are the most frequent, including the main etiologies: the syndrome of inappropriate secretion of antidiuretic hormone (siadh) and the "cerebral salt wasting" syndrome (csw). diabetes insipude of central origin secondary to a lack of dha secretion is the second most common disorder. patients and methods: it is a prospective study, analysing all the brains injured admitted to the a intensive care unit of chu hassan in fez, morocco. study spread over a -month period from / / to / / . the objective of the study is to detect the most frequent hydro-electrolytic disorders and to evaluate the therapeutic effectiveness of the service protocols. results: all these brains injured have caused he disorders over a period of time varying between d and d : * cases of hyponatremia ( %)/ cases of hypernatremia ( %), * cases of hypokaliemia ( %)/ cases of hyperkaliemia ( %), * cases of hyperchloremia, or %/ cases of hypochloremia ( %). * cases of diabetes insipidus, or . %. * cases without he disorder ( . %). the treatment for these disorders was: *for hypona; it reached mmol/l, initially corrected by a -hour water restriction, followed by an increase in the basic ration and furosemide boluses according to the ecv, even sodium loads for a single case of salt loss syndrome, while the main etiology remains the siadh. *for hyperna, it has reached mmol/l, evaluated by the extracellular volume, corrected by enteral tap water after calculation of the hydric deficit. if hperna is associated with polyuria greater than cc/kg/h; we speak of: *insipude diabetes, with polyuria up to cc/kg/h, compensated with potassium-containing solutions and blood ionogram monitored every h. desmopressin was used in titration, by bolus of . µg, with a diuresis objective between and . ml/kg/h. *for hypokalemia, up to . g/dl, observed mainly in the acute phase of brain aggression, corrected by increase in br for a k between . and g/l, and by potassium loads if k below . g/l. the evolution: deaths or . % ( cases of uncorrected diabetes insipidus), the restriction of disorders were corrected. conclusion: a knowledge of the hydroelectrolytic disorders encountered in this context is essential, as well as the implementation of a diagnostic and therapeutic protocol, which will reduce the time required to correct these disorders. compliance with ethics regulations: yes. . ] u/h). however, workload was increased under star ( vs. measurements per day), as expected from measurement interval difference between star ( -hourly) and the sp ( -hourly). conclusion: this unique patient-specific risk-based dosing approach gc framework was successful in controlling all patients safely and effectively. these preliminary results are encouraging and show gc can be achieved safely and effectively at lower target bands. in turns, these improved gc outcomes could improve patient outcomes. compliance with ethics regulations: yes. rationale: although its incidence has declined in recent years, gastric cancer remains common worldwide and is the leading cause of gastrectomy. his treatment is mainly surgical, but his prognosis remains poor. many studies on survival and prognostic factors have been carried out in foreign series. patients and methods: this is a retrospective study covering a period of three years from january to december interesting patients who had a gastrectomy and hospitalized in emergency resuscitation department surgical uhc ibnou rochd from casablanca. the statistical analysis of the different clinical, paraclinical and therapeutic data was carried out thanks to an exploitation sheet. rationale: gram-negative bloodstream infections (gnbsi) require timely appropriate antimicrobial therapy in intensive care units (icu) patients. conventional techniques usually take - h for antimicrobial susceptibility testing (ast). innovative approaches (accelerate pheno ™ system) provide pathogen identification in ~ h and ast including minimal inhibitory concentrations (mics) in ~ h. we report, in icu patients with gnbsi, results of implementation of the accelerate pheno ™ in our laboratory. we prospectively screened all gnbsi episodes reported in adult icu patients between september and september . to allow integration into the laboratory workflow, the accelerate pheno ™ was run on blood bottles positive before am (day ), in parallel with routine procedures: maldi-tof identification after short incubation on solid media (day ), β lacta (bio-rad ® ) test (day ) and disk diffusion method for ast (day+ ). for each episode, antimicrobial regimen was reassessed by a multidisciplinary team of bacteriologists, infectious diseases and icu physicians by the end of day . we measured: (i) concordance of accelerate pheno ™ results with conventional techniques, (ii) number of antibiotic adaptations on day and (iii) number of patients within the therapeutic range (free fraction over x mic and below concentration at risk of adverse events), based on real-time measurement of beta-lactams concentrations. results: of patients reported with gnbsi over the study period, were included. mean age was of ± . years, / were males. main sources of gnbsi were pulmonary (n = ) and digestive (n = ). bacterial identification of the accelerate pheno ™ was concordant with standard techniques in ( %): enterobacteriacae (n = ), pseudomonas aeruginosa (n = ). overall categorical agreement for ast was of % ( errors including very major errors). by the end of day , the antibiotic regimen was de-escalated in ( %) patients, which was appropriate in ( %). in cases, de-escalation was possible, but not fulfilled by icu physicians. twenty patients had beta-lactams concentrations measurements: were in the therapeutic range, below and over. conclusion: accelerate pheno ™ provided rapid and accurate results for most microorganisms isolated in blood cultures of icu patients with gnbsi. however, in a laboratory with routine maldi-tof early identification and β lacta test performed on day , the impact on early adaptation of the antibiotic regimen was evident in around patient over . compliance with ethics regulations: not applicable. jean-luc baudel , jacques tankovic , redouane dahoumane , jean-remy lavillegrand , razach abdallah , geoffroy hariri , naike bige , hafid ait-oufella , nicolas veziris , eric maury , bertrand guidet service bactériologie, hôpital saint-antoine, paris, france; service réanimation médicale, hôpital saint-antoine, paris, france correspondence: jean-luc baudel (jean-luc.baudel@aphp.fr) ann. intensive care , (suppl ):p- rationale: evaluation of the accurateness of the accelerate phenotest bc kit for rapid analysis ( . h for microorganism identification and additional hours for antibiotic susceptibility testing) of positive blood cultures from icu and hematology patients. patients and methods: from february to august , we included patients from the icu and hematology units with positive blood cultures. the following informations were collected : gender, age, duration of prior antibiotherapy, source of the infection, results obtained by conventional microbiological methods and by phenotest (data obtained and time to obtention of results). informed consent was obtained from all patients. results: blood cultures were analyzed in patients (m/f ratio . , age . ±, from the icu and from hematology). % of the patients were receiving antibiotics at the time of blood culture collection (mean duration : . days). the source of infection was unknown in % of cases, urinary in %, catheter-related in %, ascites in %, pneumonia in %. in cases ( %), there was a perfect match between phenotest and conventional results (identification and antibiotic susceptibility testing). in cases ( %), the bacterium responsible was not present in the phenotest panel. in cases ( %), phenotest identification was correct, but some discrepancies were observed regarding antibiogram. in cases ( %) phenotest identification was again correct but no antibiogram was available. in cases ( %), where two bacteria were present, phenotest could not identify one of them. in cases, phenotest did not provide bacterial identification because too few bacteria were present in the blood culture bottle. conclusion: the phenotest panel covered % of the bacteria implicated in this study. when the bacterium responsible was present in the panel, the results given by the phenotest correlated in % of cases with those of conventional methods. some rare discrepancies were observed regarding antibiotic susceptibility testing that have to be analyzed further. in the remaining % of cases, where too few bacteria or two different bacteria were present in the blood culture bottle, technical limitations did not permit to correctly identify microorganism(s) present or to obtain an antibiogram. compliance with ethics regulations: yes. mélanie fromentin, antoine bridier-nahmias, constance vuillard, jean-damien ricard, damien roux inserm umr iame infection antimicrobials modelling evolution, paris, france correspondence: mélanie fromentin (mel.fromentin@wanadoo.fr) ann. intensive care , (suppl ):p- rationale: studying human lower respiratory tract microbiota by using ngs (new generation sequencing) method is complex because of many unexpected biases due to dna extraction and amplification procedures. lung microbiota evolution under mechanical ventilation evolution may be highly informative to evaluate the actual risk of vap (ventilator-associated pneumonia) development. before starting a large study on the lung microbiome of ventilated icu patients, a methodological study was mandatory. patients and methods: five control and three vap patients were selected. endotrachealaspirate (eta) and oropharyngeal swab (os) were collected at icu admission for control patients and, days before and on the day of vap diagnosis for vap patients. after automated extraction of total dna, hypervariable region v of the s rdna genes was amplified with two different pairs of primers f- r: oligonucleotides from the earth microbiome project (earth primer pair) and from the gut microbiome project (gut primer pair), followed by sequencing on illumina miseq plateform. after bioinformatics analysis with mothur ® software, we compared the performance of ngs alongsideconventional bacterial culture. differences in alpha diversity (microbial diversity in a sample), expressed as the shannon index, across respiratory tract site (upper or lower) and across time (before and at vap time) has been investigated. a positive control (pc), rationale: colistin is used as a last-line treatment to combat multidrug-resistant (mdr) gram-negative bacilli (gnb). worryingly, colistin resistance in klebsiella pneumoniae, pseudomonas aeruginosa and acinetobacter baumannii is increasingly reported worldwide. we hereby report the prevalence of colistin resistance among gnb isolated from burn patients in tunisia. the study was carried out on strains of gnb isolated from microbiological samples of burn patients hospitalized in the intensive care unit between october and december . identification was performed by conventional methods. antimicrobial susceptibility was tested by disk diffusion method and the results were interpreted according to ca-sfm guidelines. minimum inhibitory concentration (mic) of colistin was determined using the eucast broth micro-dilution method (umic, biocentric ® ) results: pseudomonas aeruginosa was the most frequently isolated bacteria ( strains), followed by acinetobacter baumannii ( strains) and klebsiella pneumoniae ( strains). the most common sites of isolation were blood cultures ( %), catheters ( %) and skin samples ( %). most of p. aeruginosa isolates were multidrug-resistant with high levels of resistance to imipenem ( . %), ceftazidime ( %) and ciprofloxacin ( . %). however, all of them were susceptible to colistin. in fact, mics of colistin against all p.aeruginosa isolates were less than or equal to . mg/l. a. baumannii strains had high resistance rates to beta-lactams : % to ceftazidime and % to imipenem. only one strain was resistant to colistin with a mic equal to mg/l. all k. pneumoniae isolates were resistant to extended-spectrum cephalosporins. one third of these strains were resistant to imipenem and more than half ( . %) were resistant to amikacin. two strains were resistant to colistin with high mics (> mg/l). both were carbapenemase-producers, carrying oxa- and ndm carbapenemase encoding genes. conclusion: these data suggest that colistin-resistant or pan-drug resistant gnb clinical isolates are still relatively rare. however, they have important global public health implications because of the therapeutic problems they present, especially for vulnerable populations such as severely burned patients. hence the need to test colistin regularly in the laboratory and to set up a monitoring program for mdr pathogens. compliance with ethics regulations: yes. rationale: descending necrotizing mediastinitis (dnm) are medicosurgical emergencies whose forecast is closely related to the precocity of the therapeutic assumption. the purpose of our work is to profile these patients as well as the therapeutic and evolutionary aspects. patients and methods: retrospective study over years in the intensive care unit of the hospital august. all patients with dnm on cervicofacial cellulitis were included. results: cases were collected, % of cellulitis, incidence of . patients / year. average age , sex ratio of . . smoking, chronic alcoholism and diabetes are the most common antecedents. the favoring factors were: (poor dental conditions: % of cases, non steroidien anti-inflammatory drugs: %, diabetes: %). in % of cases the front door was dental. average time taken to take care of days. c-reactive protein and procalcitonin were positive in all patients. in % the chest x-ray was normal. all patients received tri-antibiotic therapy. intubation were difficult in all patients, we used nasofibroscope in % of cases and a rescue tracheotomy in one patient. only one patient had a cervico-thoracic surgical approach; for all the others she was cervical alone. streptococcus was the most isolated germ. the complications were (septic shock: %, ards: %). the average hospital stay was days with a mortality rate of %. conclusion: dnms are poorly prognostic. the best treatment remains prevention by better management of dental abscesses and tonsillar phlegmons. rationale: the initial, empirical antibiotic therapy of ventilator-associated pneumonia (vap) is often based on timing of its occurrence in relation to the onset of mechanical ventilation. this is due to reported differences between causal pathogens associated with early-onset (e-vap < - days of mechanical ventilation) compared to late-onset vap (l-vap ≥ - days of mv). e-vap is most often reported to be due to antibiotic-sensitive pathogens while l-vap is frequently attributed to antibiotic-resistant pathogens. however, there is emerging evidence that the isolated microorganisms may be similar regardless of onset time. the aim of our study was to compare the clinical outcomes of critically ill patients developing e-vap and l-vap and to compare the causative pathogens of the two groups. patients and methods: all the patients with the diagnosis of vap admitted between january and december were retrospectively included. vap was suspected on the basis of clinical and chest x-ray findings. the identification of the causative organisms was performed with endotracheal aspirate (eta) cultures. results: ninety patients developed vap. e-vap was observed in patients ( , %), whereas patients ( , %) developed l-vap. among patients with early-onset vap, % received antibiotics prior to the development of pneumonia, compared to % with late-onset vap (p = . ). otherwise, no differences (sociodemographic factors, antecedents, severity score, length of stay, length of mv) between the two groups were observed. the most common pathogens associated with e-vap were enterobacter species ( . %), pseudomonas aeruginosa ( . %) and oxacillin-resistant staphylococcus aureus (orsa , %). enterobacter species ( . %), acinetobacter baumannii ( . %) and pseudomonas aeruginosa ( %) were the most common pathogens associated with l-vap. no difference was noted in the contribution of multidrug resistant bacteria mdr ( % vs. %). hospital mortality was significantly greater for patients with l-vap caused by mdr ( %) compared to patients with e-vap ( %) (p = . ). conclusion: this classification is no longer helpful for empirical antibiotic therapy, since both early-onset and late-onset vap were caused by mdr bacteria. this justifies the need of intensive care unit-specific knowledge of causal agents associated with vap to reduce the rate of administration of inadequate antimicrobial therapy. compliance with ethicsregulations: yes. key: cord- - tapkjb authors: nan title: th escp-nsf international symposium on clinical pharmacy: clinical pharmacy tackling inequalities and access to health care. oslo, norway, – october date: - - journal: int j clin pharm doi: . /s - - - sha: doc_id: cord_uid: tapkjb nan pharmacy, sint maartenskliniek, ubbergen, pharmacy, radboud university medical centre, nijmegen, clinical pharmacy and toxicology, maastricht university medical centre, maastricht, netherlands please specify your abstract type: research abstract background and objective: according to literature adherence to statins ranges from to %. medication adherence is affected by both practical barriers and patient's beliefs about medication. however, physicians also have their beliefs about medication. several studies have shown that these beliefs also impact the decision of patients to agree with a particular treatment or not. as current published interventions on medication adherence (which focus predominantly on patients) are not or just partly effective, physicians' beliefs might be a promising target for interventions to improve adherence. however, there is currently no information available on physician's beliefs about statins and whether these beliefs affect patient's beliefs and adherence. therefore, the objective of this study is to examine whether physicians' beliefs about statins influence the beliefs and adherence of patients using a statin. setting and method: this cross-sectional study was conducted in gp practices and community pharmacies, between september , and march , . physicians' and patients' beliefs about statins were assessed with the beliefs about medicine questionnaire (bmq) specific. patients' adherence on statins was assessed with both the mars- and the morisky- questionnaires. please specify your abstract type: research abstract background and objective: nhs highland and nhs western isles are the most remote and rural health boards in the united kingdom, with high numbers of dispensing medical practices. a pilot is underway in dispensing practices with clinical pharmacists undertaking targeted medication reviews. a previous quantitative service evaluation demonstrated its value, with pharmaceutical care issues identified in almost all patients, the vast majority of which ( . %) were managed by the pharmacist without any need for general practitioner (gp) referral. the objective was to undertake a qualitative exploration of the service. setting and method: all patients and staff involved in the service were invited to participate. a semi-structured interview schedule was developed and piloted. telephone interviews were conducted with all consenting staff and a purposive sample of consenting patients recruited to the point of data saturation. interviews were audiorecorded, transcribed verbatim and analysed thematically. nhs ethics and research and development approvals were obtained. were the most confident with doacs (range from . to . %) please specify your abstract type: research abstract background and objective: patients are at risk of drug-related problems (drps) at transition points during hospitalization. the community pharmacist (cp) is often the first healthcare professional patients visit after discharge. cps lack sufficient information about the patient and so they may be unable to identify problems in medications, which may lead to dispensing the wrong drugs or dosage, and/or giving wrong information. we aim to assess the impact of a complex intervention comprising of medication reconciliation performed at discharge by a hospital pharmacist (hp) with communication between the hp and cp on drps during the days following discharge. setting and method: cluster randomized crossover trial involving medical and surgery care units (each unit corresponding to a cluster) in french hospitals during two consecutive -day periods, randomly assigned as 'experimental'(e) or 'control' c (usual care) periods. during the experimental period, the hp performed a medication reconciliation that was communicated to the patient's cp. main outcome measures: the primary outcome was a composite outcome of any kind of drp (prescription/dispensation, gap or patient) during the days following discharge assessed at day seven post-discharge by phone from patient and cp. the secondary outcomes were /unplanned hospitalizations assessed by phone contact at day after discharge and /the iatrogenic potential exposure scale from to for each patient established by a clinical team. analysis was conducted in intention to treat. results: hospitals corresponding to clusters enrolled patients ( e group v/s c group). no difference was observed on age, sex, autonomy, and number of drugs in home medication at admission and discharge. at day ; ( . %) patients in e group had at least one drp v/s ( . %) in c group (or . ; ic % [ . ; . ] p = . ). intervention was especially efficient for patient discharged from surgery unit (or . ic % [ . ; . ]) and aged less than years (or . ic % [ . ; . ] . although intervention decreased patient exposure to drp with high iatrogenic potential (from . to . % p \ . ), un-planned hospitalizations at day weren't different between groups ( . vs. . % p = . ). conclusion: medication reconciliation associated to communication between hospital and community pharmacists is efficient to decrease patient exposure to drp but not sufficient to decrease un-planed hospitalization. hp-pc : clinical pharmacists bridging health care levels by medication reviews in primary care katherine wendelbo *, , kristine lundereng namsos hospital pharmacy, central norway hospital pharmacy trust, namsos, levanger hospital pharmacy, central norway hospital pharmacy trust, levanger, norway please specify your abstract type: descriptive abstract (for projects) background and objective: nord-trøndelag county is sparsely populated and many inhabitants live far from the hospital. additionally, only half ( of ) of the municipalities have a local pharmacy. traditionally, namsos and levanger hospital pharmacies have performed quality audits of the implementation of drug administration procedures in primary care units. since , a service where clinical pharmacists participate in multidisciplinary medication reviews in municipalities throughout the county has been established. the objective of this poster is to describe the practical approach and design of the service. design: a descriptive report of an implemented clinical pharmacy service in primary care where clinical pharmacists, as part of multidisciplinary teams, perform medication reviews. results: medication reviews are performed on patients admitted to nursing homes and patients in home care, receiving help with handling of their drugs. primary care nurses prioritise patients (by selecting frail elderly with multiple co-morbidities and polypharmacy), usually five patients in each meeting. prior to the review, nurses collect medical information using a checklist including; diagnosis, drug-related symptoms, standard laboratory tests and an updated medication list. the clinical pharmacist receives de-identified medical information by postal mail or e-mail before the meeting. based on this information the pharmacist identifies possible drugrelated problems (drps) and provides recommendations on how to solve them. this is performed in a structured approach according to the integrated medicine management (imm) model. subsequently, the pharmacist visits the municipality and discusses the medication reviews in a multidisciplinary team meeting with nurses and physicians. in addition, the pharmacist gives lectures in a medication related topic (e.g. treatment of insomnia and anxiety, oral anticoagulants and cognitive side effects). following the meeting, the pharmacist reports the drps and suggested interventions to the multidisciplinary team, for further follow-up. during , totally medication reviews were performed in municipalities. in the same period, lectures were given by the clinical pharmacists. conclusion: this clinical pharmacy service enables multidisciplinary medication reviews even in municipalities with limited health professionals and resources. as a part of multidisciplinary teams, the clinical pharmacists contribute with medical competence. camille castel , arnaud de la blanchardière , vincent cattoir , guillaume saint-lorant *, pharmacy, infectious and tropical diseases, microbiology, chu caen, caen, france please specify your abstract type: research abstract background and objective: antimicrobial stewardship have clearly demonstrated their efficiency towards a more adequate use of antibiotics. since , the use of daptomycin, a ''critical last resort antibiotic'' has intensified in our hospital, occasionally outside the scope of its approved indications. this situation has led to the implementation of an antimicrobial stewardship and the drafting of local guidelines. the aim of this study is to analyse the evolution and pertinence of daptomycin prescriptions, after distribution of these guidelines within our institution. setting and method: a monocentric prospective study was conducted between july and november in a -bed university hospital. each daptomycin prescription recorded by pharmacy department was analysed by an infectious diseases specialist in the presence of the prescriber and considering local guidelines and the patient's clinical conditions. main outcome measures: the indicators chosen to determine prescription pertinence were: treatment indication, prescribed dose and other antibiotics associated with the daptomycin prescription. results: daptomycin prescriptions were analysed. observed indications were: sepsis ( %), infective endocarditis ( %), bone and joint infections ( %) and vascular prosthetic infections ( %). identified pathogens were: mrsa ( %), methicillin-resistant coagulase-negative staphylococci ( %), methicillin-sensitive staphylococcus aureus ( %), enterococci ( %) and methicillinsensitive coagulase-negative staphylococci ( %). daptomycin was prescribed as first-line treatment in % of cases. the mean dose was mg/kg/day [ - mg/kg/day] for a mean duration of days [ ; days] . local guidelines were followed in % of cases. daptomycin use was relevant for % of prescriptions. the irrelevant prescriptions triggered the modification or stoppage of antibiotic therapy in % of cases, respectively, generating an % decrease in consumption and an economy of over € for our institution. conclusion: this study shows the efficiency of antimicrobial stewardship in adequately using antibiotics, limitating ecological impacts, improving patient care and decreasing healthcare costs. it also shows that guidelines alone are insufficient to ensure a proper use of antibiotics. without a close prescription follow-up, constant reminders and sustainable evaluations, guidelines only affect a few prescribers. within the context of an ''antimicrobial crisis'', further development of guidelines and antimicrobial stewardship is essential to fight increasing bacterial resistances and requires a close collaboration between all healthcare professionals including pharmacists. interviews were transcribed verbatim and data were analysed using systematic text condensation. results: three major themes were identified: benefits, unrealised potential and criteria and barriers for success. ( ) benefits described by physicians included increased patient safety, increased awareness on drugs, and an ease of workload. drug interaction management was emphasized as one of the clinical pharmacists' most important work tasks, as well as being a resource for collaborating healthcare professions and to the patient himself. ( ) the clinical pharmacists expressed that they had an unrealised potential and could contribute to a greater extent in the multidisciplinary team than they did already. they mentioned education towards physicians and nurses, contribution in treatment decision-making and patient counselling as examples for possible extended work tasks. ( ) as criteria to succeed as a clinical pharmacist, physicians highlighted the importance of oral communication and physical presence on the wards. as barriers for integration in the team, the clinical pharmacists identified the physicians' lack of knowledge about the clinical pharmacists' skills as well as unclear expectations regarding their responsibilities. conclusion: physicians agreed that the clinical pharmacist represent a valuable contribution to the multidisciplinary team, where patient safety and drug interaction management are highlighted as main benefits. clinical pharmacists should to a greater extent educate healthcare professions in drug related topics and provide patient counselling. continuous effort on making the clinical pharmacist a natural part of the multidisciplinary team is crucial for the development of clinical pharmacy. by gathering perceptions from the collaborating professions as well as educating them on what clinical pharmacists can provide, we can develop a multidisciplinary team that enhances patient safety. hp-pc : assessment of dual antiplatelet therapy following acute coronary syndrome using grace and crusade sadeer fhadil * , paul wright, sotiris antoniou please specify your abstract type: descriptive abstract (for projects) background and objective: mortality and morbidity benefits of dual antiplatelet therapy (dapt) following acute coronary syndrome (acs) have been unequivocally demonstrated in a large body of evidence. with the availability of more potent antiplatelet agents, balancing ischemic and bleeding risks to prevent adverse outcomes is an on-going challenge, in particular, recognising that patients with high bleeding risk were excluded from clinical trials. grace and crusade scores stratify risk of mortality and in-hospital major bleeding post acs respectively. these tools should be used to support antiplatelet choice in light of newer more potent agents that equally pose a greater risk of bleeding. design: grace and crusade scores were calculated for patients presenting with acs. clopidogrel was recommended for patients with a high or very high crusade score (greater bleeding risk). ticagrelor was recommended for patients presenting with st-elevation myocardial infarction (stemi) or those with nsteacs with a grace score of intermediate or above (greater ischemic risk) and a crusade score of moderate or less (low bleeding risk). in either case, treatment was at the discretion of the clinician and patients received concomitant aspirin. a registry was collated of risk scores, diagnosis and choice of antiplatelet therapy. results: patients were included in the registry, of which ( %) presented with stemi and ( %) presented with nsteacs. of ( %) patients with a greater ischemic risk received ticagrelor as part of their dapt regime. advanced age, concomitant anticoagulation and those awaiting surgery were the most common reasons for patients with a greater ischemic risk to receive clopidogrel. ( %) had a high or very high crusade score. of these, ( %) received clopidogrel as part of their dapt regime. conclusion: risk stratification was streamlined using the data collection tool and useful to support choice of dapt. european society of cardiology (esc) guidance recommends use of established risk scores for prognosis and bleeding; however evidence to correlate to choice of dapt is lacking. outcome data is currently being reviewed and will provide further evidence to correlate choice of dapt to grace and crusade scores. please specify your abstract type: descriptive abstract (for projects) background and objective: in europe, approximately % of the patients with the human immunodeficiency virus (hiv) infection are co-infected with the hepatitis c virus (hcv). treatment recommendations in hiv/hcv co-infected patients are identical to those in patients with hcv mono-infection. however, potential drug-drug interactions (ddis) between antiretroviral agents and new direct-antiviral agents (daas) imply the need of a careful selection of the hcv treatment regimen. the aim of the present study was to evaluate the need of a change in the antiretroviral therapy (art) due to potential ddis in patients with hiv/hcv co-infection who started treatment for hcv with new daas. we also assessed the effectiveness of hcv treatment weeks after hcv treatment completion. design: we retrospectively registered clinical data about hcv and hiv management: hcv genotype, fibrosis metavir score, initial hcv viral load, hcv treatment and previous art regimen. we recorded the changes in art prior to starting hcv treatment and the reason of this switch (ddi, simplification or duplication of the therapy). results: between february and january , hiv/hcv coinfected patients started hcv treatment with a daas regimen. of them, had advanced liver disease (fibrosis score: f /f ) and were infected with hcv genotype . prior to starting hcv treatment, patients needed a switch in art regimen due to potential ddis with daas. simeprevir and the co-formulation ombitasvir/paritaprevir/ritonavir were the daas most frequently implicated in ddi with protease inhibitors or non-nucleoside reverse transcriptase inhibitors: / and / , respectively. also, we observed some changes of art due to other causes. five switches occurred to adequate the regimen (discontinuation of ritonavir in candidates to take the co-formulation ombitasvir/paritaprevir/ ritonavir or art improvement to decrease pill burden). as for hcv treatment effectiveness, / ( %) patients achieved sustained viral response weeks after therapy completion. conclusion: a large proportion of patients with hiv/hcv co-infection who initiate treatment with daas for hcv need to switch art due to potential interactions that may impact on effectiveness and safety of both treatments. additionally, some changes in art treatment are made to facilitate therapeutic adherence. these results highlight the need of a multidisciplinary approach in which interactions between art and hcv treatments should be carefully assessed. please specify your abstract type: descriptive abstract (for projects) background and objective: the potential impact of polymedication, iatrogenic events and medication error is a serious concern in hospitalized patients. clinical pharmacists can limit these risks by identify high risk. the aim of this study are to identify in six medical units high risk patients by using three predictive scores of rehospitalisation ( ps) , early mortality (charlson) and drug related problems (drp) . design: clinical and therapeutic variables in patients were collected through medical records and prescriptions by clinical pharmacists. scores were calculated during months in six units (internal medicine, n = ; nephrology, n = ; geriatrics, n = ; rheumatology, n = ; cardiology, n = and endocrinology, n = ). the data were analysed by mann and whitney test for the continuous variables and chi square test for the qualitative variables. the coefficient of correlation between the three scores were calculated by a pearson test for normal distribution and by a spearman test for non normal distribution. patients were considered at a high risk for re-hospitalization ( ps [ ) , early mortality (charlson [ ) and iatrogenic events (drp c ) . results: in the general population, the average age was . ± . years old and the sex ratio was . . the average treatment used was . ± . charlson scores were higher in geriatric unit ( . ± . ) follow by medical interne unit ( . ± . ). the ps and drp scores were higher in nephrology unit respectively . ± . and . ± . follow by internal medecine unit . ± . and . ± . . on contrary the rheumatology unit presented the lower level for the three scores. patients were considered at high risk for three scores, % (n = ) in nephrology unit (almost % of unit), % (n = ) in geriatric unit, % (n = ) in internal medicine unit, % (n = ) in cardiology unit, % (n = ) in endocrinology unit and % (n = ) in rheumatology unit. conclusion: knowledge of the variables associated with these predictor scores could help clinical pharmacists to prioritise various medicine units and target those at risk. we identified especially three units at risk: nephrology, geriatric and internal medicine. thanks to these results, clinical pharmacists can rapidly and efficiently target patients who present iatrogenic and/or re-hospitalization risks. design: a retrospective observational analysis was conducted in our hospital, based on medical records of patients presenting atrial fibrillation (af) and treated by doacs from january to may . to identify patients hospitalized due to severe bleeding, we analysed prothrombin complex concentrates (pccs) and activated pccs prescriptions, as well as pharmacovigilance declarations. results: patients were treated with doacs: with rivaroxaban ( . %), with dabigatran ( . %) and with apixaban ( %). fifty-nine ( . %) patients experienced at least one bleeding leading to hospitalization: with rivaroxaban ( . %), with dagibatran ( . %) and with apixaban ( . %). thirty-eight severe bleeding were identified ( . %): occurred with rivaroxaban ( . %), with dabigatran ( . %) and with apixaban ( . %). they included intracranial bleeding ( %) and gastro-intestinal bleeding ( %). seven haemorrhages resulted in hypovolemic shock (dabigatran: , rivaroxaban: , apixaban: ) and of them were fatal (dabigatran: ). rates of bleeding (p = . , v test) and of severe bleeding (p = . , v test) were not statistically different for the three molecules. in case of major haemorrhage, the recommended factor concentrate in our protocols differs between the anticoagulant. with dabigatran, the antidote idarucizumab ( g, intravenously) should be administered, without waiting for plasma concentration results. with rivaroxaban, apixaban or unknown doacs, pcc ( - units/kg) is indicated. in case of pcc failure, activated pcc ( - units/kg) is suggested. pcc, activated pcc or idarucizumab ( ) were used in / patients ( %). in rivaroxaban and apixaban-related haemorrhages, patients received activated pcc: two had a ui/kg dose and one had a ui/kg dose. regarding dabigatran-related bleeding, one patient received pcc instead of idarucizumab. compliance with local recommendations was % ( , p [ . , v test) . pharmacovigilance reports were issued. conclusion: management of doacs-associated severe bleeding in our hospital respects local protocols. it should also be pointed out that patients with life threatening bleeding may benefit from pcc. however, the risk of thrombosis associated with pcc must be weighed against the risk of haemorrhage. since specific antidotes are emerging, like idarucizumab or andexanet alpha, new guidelines for doacs-related haemorrhage are expected. please specify your abstract type: descriptive abstract (for projects) background and objective: this project is part of a prospective quasi experimental proof-of-concept investigation of a clinical pharmacist intervention to reduce drug-related problems among people admitted to a ward in a rural hospital in northern sweden. the aim of this particular study is to explore doctors' and nurses' expectations of having a ward-based pharmacist providing clinical pharmacy services in a rural hospital. design: eighteen face to face semi-structured interviews were conducted with a purposive sample of doctors and nurses working on the ward were the clinical pharmacy service was going to be implemented. semi-structured interviews were digitally recorded, transcribed and analysed using thematic analysis. results: the majority of participants had limited experience or a vague idea of what pharmacists are able to do in a ward. most participants described traditional roles such as inventory, drug distribution and dispensing. most respondents were unaware of the pharmacists' knowledge, skills and competences. for some it was unclear how having a clinical pharmacist in the ward was going to impact on their workload this was particularly important for the nurses. some doctors (mainly experienced) were concerned that having a pharmacist may mean losing or not gaining competence on drugs. for others it was unclear how the pharmacists' will work with patients or what clinical skills they have. however most participants were positive about the implementation of the new service. conclusion: this study provided a rare opportunity to explore the doctors' and nurses expectations of the role of clinical pharmacists before a clinical pharmacy service was implemented. the results showed that the participants' expectations of the clinical pharmacist role were unclear. to successfully implement clinical pharmacy services in a clinical pharmacy ''naïve'' setting; roles, clinical competence and responsibilities should be clearly described. furthermore, it is important to focus on inter professional collaborations between doctors, nurses and pharmacists. practical for the local hospital setting. seven out of experts agreed with pharmacist prescribing for the conditions identified. pharmacists (n = ) were more willing to prescribe antihypertensive and antidiabetic medication ( . %) when compared to oral anticoagulants ( . %). these values are higher than those obtained by vella in . the majority of pharmacists ( . %) recommended that pharmacists should take up further studies to a master or doctorate level degree in a clinical aspect in order to be authorised to prescribe. conclusion: the developed framework for pharmacist prescribing and the guidelines developed for pharmacist prescribing of oral anticoagulants and pharmacotherapy of hypertension and diabetes mellitus were shown to be reliable and were accepted by pharmacists and physicians. please specify your abstract type: research abstract background and objective: valproic acid (vpa) and its derivates and mycophenolate mofetil (mmf) and mycophenolic acid used during pregnancy increase risk of congenital malformation and cognitive impairment. thus, the french national agency for medicines and health products safety (ansm) decided to establish new conditions of prescription and dispensation of drugs containing vpa (may ) and mmf (april ). a signed care agreement and a co-prescription of contraceptives are now mandatory in the drug dispensation for reproductive-age adolescent girls and adult women. this study will describe the impact of these new guidelines on our practice. our objective is to compare the vpa and mmf media coverage and the impact on the prescriptions. setting and method: we compared the mass communication between vpa and mmf on social media, webpages and journal article (public and professional journal) on google and googletrends in the first months around these new rules. we combined different keywords such as ''accord de soins'' and the drug name. in the same time, we collected and analysed vpa and mmf prescribing and dispensing data and compared it to the data for the first months. main outcome measures: results: just before the vpa rule, the vpa was presented in the general press as the new health scandal after benfluorex mediator°w ith google searches in march compared to searches usually per month. simple research combining keywords reveal always more than twice more webpages concerning vpa than mmf. at the same time, a patients association (renaloo for renal failure) wrote to the ansm to contest the new rule with the double contraception and without any consultation of patients association. in our daily practice we also faced some physician reluctant to sign this prescription agreement with patient (too many agreements already asked, decision of ansm without any consultation of learned societies). the care agreements are kept in the patient records, a statement ''care agreement signed'' is reported in the electronic prescription of vpa and mmf. the overall consumption of mmf and vpa increase for respectively the first and months after rule implementation (from + to + %) except for the micropakin mg. the months mmf data will be presented for the final communication. conclusion: the media pressure and the new regulation have an impact on prescription trends. these new prescription and dispensing rules concerned two different contexts: pathology, media coverage, possible drug alternatives. we were faced to some difficulty in implementing the new guidelines, which reveals a certain reluctance of the prescribers or the patients represented by associations. tdmp : vancomycin trough serum concentrations are frequently subtherapeutic in a population of critically ill patients: a prospective observational study please specify your abstract type: descriptive abstract (for projects) background and objective: to design and characterise a framework of international pharmacy standards for pharmaceutical care application on oral anticoagulation for prevention of atrial fibrillation (af) related strokes. design: literature review (including existing international guidelines and quality measures) was conducted to characterise the standards and design an international framework for pharmaceutical practice application on oral anticoagulation for prevention of af-related strokes. expert opinions were sought through a delphi method to reach consensus on the framework domains and standards. results: the framework consisted of twelve overarching standards, which were defined and grouped into four domains as follows; ([personal care package]:-communication with patients, support decision making process, education and counselling, adherence. [medicines optimisation]:-clinical review and therapy optimisation, initiation and control, maintenance, supply and transfer between care settings. [workforce]:-workforce planning, training and development, analysing information; and [governance]:-assurance of service provision) specific to oral anticoagulation in prevention of af-related stroke. each standard was also categorised within dimensions and supporting statements to describe what a quality pharmacy service should deliver. a total of forty-five dimensions and twelve statements were incorporated into the framework. conclusion: a clearly defined framework of international standards was developed as a clinical tool and quality assurance to optimise the delivery of care for oral anticoagulation in prevention of af-related strokes. it will support pharmacists and their teams to develop their professional practice, improve services, and deliver safe and high quality patient care across all pharmacy settings. poster discussion forum i: community pharmacy and public health cp-pc : nurses' and pharmacists' learning experiences from participating in inter professional medication reviews in primary health care: a qualitative study hege t. bell *, , anne gerd granås , ragnhild omli , ingela enmarker , aslak steinsbekk nord university/ntnu, trondheim, hioa, oslo, nord university, namsos, norway, department of nursing, Østersund, sweden, ntnu, trondheim, norway please specify your abstract type: research abstract background and objective: traditionally, drug prescription and follow up have been the sole responsibility of physicians. however, interprofessional medication reviews (imrs) have been developed to prevent drug discrepancies and patient harm. what participating nurses and pharmacists learn from each other during imr is poorly studied. the aim of this study was to investigate nurses' and pharmacists' perceived learning experience after participating in imrs in primary health care for up to years. setting and method: a qualitative study with semi-structured focus group interviews and telephone interviews with nurses and pharmacists with experience from imrs in nursing homes and home based services. the data was analysed thematically by using systematic text condensation. main outcome measures: a qualitative method is useful when looking at objects from the perspective of how they are experienced. results: sixteen nurses and four pharmacists were interviewed. the nurses' perception of the pharmacist changed from being a controller of drug management routines towards being a source of pharmacotherapy knowledge and a discussant partner of appropriate drug therapy in the elderly. the pharmacists became more aware of the nurses' crucial role of providing clinical information about the patient to enable individual advice. increasingly the nurses learned to link the patient's symptoms of effect and side effect to the drugs prescribed. with time both professions jointly spoke of an increased awareness of the benefit of working as a team and the perception of contributing to better and more individual care. conclusion: imrs in primary health care meet some challenges especially concerning how to ensure participation of all three professions and how to get thorough information about the patient. possible solutions might be to use shared communication tools like internet based communication programs and to introduce the patient as a participant at the imrs. please specify your abstract type: research abstract background and objective: international good pharmacy practice guidelines describe how pharmacists should counsel the patients about their medicines, offer additional services where needed, and intervene at drug related problems. daily practice often differs from theory. this study aimed at illustrating the whole process of prescribed medicines dispensing in daily community pharmacy practice. part b of the project focuses on pharmacists' opinions. setting and method: community pharmacies in basel, switzerland, were invited in random order for study participation. one master student in pharmacy performed non-participant observations during day at each included community pharmacy. at dispensing of prescribed medicines, patient data, content of counselling, communication style, and provision of further services (e.g. follow-up offer) were documented on a checklist with predefined themes. interventions were documented systematically. a semi-structured interview on the pharmacists' opinions about the counselling, triggers, facilitators and barriers, and the documentation of interventions was conducted at each community pharmacy. main outcome measures: counselling content at prescription dispensing by numbers and by pharmacists' opinions; barriers, facilitators, and triggers for counselling at prescription dispensing. results: in march and april , of invited community pharmacies participated in the study. out of documented observation periods, encounters were analysed (first prescription: /refill prescription: ). counselling was provided to ( . %) clients with an average of . (± . ) themes per encounter. a total of clients refused counselling. themes most counselled at first and refill prescription dispensing were: drug intake ( / ), dosage ( / ) , and administration ( / ). for the pharmacists (n = ), most important themes to be discussed at first prescription dispensing were indication ( ), administration ( ), and anamnesis ( ); for refill prescription dispensing they were adherence ( ), therapy benefits ( ), and adverse effects ( ). the majority of pharmacists ( ) felt that it was their obligation to ask questions about patients' health during the dispensing of prescription medicines and named trigger (e.g. patient knowledge gap, patient motivation, interactions), but one-third reported difficulties with it. barriers were refusal by patients ( ), communication problems (language, ), lack of medical data ( ) , and lack of time ( ) . conclusion: a discrepancy in counselling content by observation compared to pharmacists' opinions was revealed. this might indicate that pharmacists are aware but hindered by barriers to practice according to good pharmacy practice guidelines. please specify your abstract type: research abstract background and objective: the workforce vision in scotland envisages 'making more and better use of technology …to increase access to services and improve efficiency' across the healthcare interface. services offered by community pharmacy remain limited by lack of shared access to patients' clinical information. in scotland, every patient has a unique identifier, their chi (community health index), which facilitates identification of/searching for patient records. the aim of this research was to explore the experiences of community pharmacists granted clinical portal access to patients' records. setting and method: from april , community pharmacists across nhs tayside (n = ) who had completed technical and information governance training were invited to maintain a portal log of their experiences of using the clinical portal to access patient records. each was asked to record when/why they considered accessing a patient's record and whether their information needs were met. portal logs were subject to independent summative content analysis by two researchers. this study gained ethical approval from robert gordon university. main outcome measures: not applicable. results: clinical portal logs were received from most participating pharmacists (n = / ). two were unavailable (moved to hospital setting; maternity leave). a third had not had occasion to access the clinical portal which he speculated was due to not working at weekends but also raised concerns about gaining patient consent. frequency of seven identified themes provided a partial indication of balance of reasons for usage. firstly (# ), to confirm a patient's prescription (n = ), secondly (# ), for additional information (n = ). less frequently (# ), portal access was to check repeat medications (n = ). other reasons for access were (# ) to check hospital discharge (n = ) followed by (# ) check on multi-compartment appliance aid status (n = ) or (# ) check the emergency care summary (n = ). there were also instances (# ) when portal access was not found to be helpful (n = ) so traditional offline routes were followed. conclusion: preliminary findings indicate mainly positive experiences with no technical issues raised and community pharmacists' information needs largely met. although limited to a small number of pharmacists in only one health board, findings support scottish policy aims, including 'prescription for excellence.' further work is underway around patients' perspectives of community pharmacist access. please specify your abstract type: research abstract background and objective: multicompartment compliance aids (mcas) such as the multidrug punch cards pharmis Ò are used to support patients in the daily management of their medication. solid oral medicines are unpacked from their original packaging and repacked in mcas although controversy exists about the stability of repackaged medicines. different countries published contradictory lists of medicines not recommended for repackaging. we aimed to define and apply criteria able to assess visual alteration of medicines repackaged in mcas. setting and method: eight criteria describing physical alteration of tablets/capsules were retrieved from the who international pharmacopoeia : chipping, swelling, capping, rough surface, cracking, crushing under pressure, mottling, and discoloration. absence of one criteria gives point. a maximum score of points can be obtained. twenty-two critical medicines and three half tablets were repackaged in the multidrug punch cards pharmis Ò and stored at accelerated conditions ( °c/ % rh) for weeks. original blisters of medicines were stored at room temperature as control. each tablet/capsule was visually inspected after , , and days. main outcome measures: score according to eight criteria. results: after weeks, tablets/capsules including of the half tablets showed no visual alteration and were identical to controls. a reduced score ( - points) was given to seven repackaged medicines and one half tablet within weeks: madopar Ò ( ), pravastatin sandoz Ò ( ), carvedilol mepha Ò ( ), plavix Ò ( ), pantoprazol nycomed Ò ( ), adalat Ò cr ( ). swelling and rough surface were the most frequent. chipping and capping were not observed. conclusion: our eight visual criteria are able to detect physical alteration of repackaged solid oral medicines under stress conditions. in absence of reliable data we suggest to apply this simple quality control for repackaged medicines in pharmacy practice. because chemical stability testing is not feasible in practice, pragmatic solutions are sought. further studies are needed for storage at room temperature. cp-pc : drug-related problems and symptom burden in nursing home residents kerstin bitter *, , ulrich jaehde , christina pehe , gabriela heuer , manfred krü ger clinical pharmacy, university of bonn, bonn, aok rheinland/ hamburg, pharmacists' association north rhine, düsseldorf, germany please specify your abstract type: research abstract background and objective: drug-related problems (drp) are common in the elderly due to polymedication. community pharmacies supplying drugs to nursing homes may play an important role in detecting and solving drp in nursing home residents. this project aims to evaluate whether community pharmacists can enhance the medication safety of nursing home residents by solving drp by means of a simple medication review (mr). furthermore, the applicability of a new tool to detect symptoms as potential adverse drug reactions in elderly patients should be tested. setting and method: nursing home residents at the minimum age of years insured by aok rheinland/hamburg and regularly taking at least five drugs per day were invited to participate. pharmacists performed a mr based solely on the patients' medication data, including dosage regimens of the nursing home and self-medication data. the detected and solved drp were counted. additionally, a simple questionnaire (sympel) was distributed to the patients periodically in order to assess their symptom burden. main outcome measures: frequency and type of the patients' drp as well as their symptom burden before and after the mr. results: after testing the feasibility of this intervention in a pilot study, patients were included in the main study so far. in average, the pharmacists identified two drp per patient and reported to the responsible general practitioner (gp) . as in the pilot study, most frequent drp documented by pharmacists were drugdrug-interactions ( %). % of the pharmacists' recommendations were accepted by the gp. % of the patients took at least one drug considered as potentially inadequate in the elderly. out of six different symptoms, patients reported dizziness and bruises most frequently. conclusion: pharmacists can detect many drp in nursing home residents by means of a simple mr. however, the full potential of this service to solve drp can only be exploited if the cooperation with the gps is improved. please specify your abstract type: research abstract background and objective: medicines for rare diseases (rd) are costly and have limited efficacy evidence but they represent around % of all innovative medicines. therefore, countries are facing challenges in providing patient access to them. the purpose of the study was to assess the patient access for slovenia and compare it with other european countries in the last decade. setting and method: the medicines for rd that obtained marketing approval between and via centralised procedure were included in the study based on the orphanet list from january . using the quarterly ims health database, sales data were analysed for slovenia and other european union countries, norway and switzerland. patient access was assessed for each country and comparisons between the countries were made using the three main outcome measures. main outcome measures: the number of medicines for rd available; time to first continuous use after marketing approval; total pharmaceutical expenditure for medicines for rd in euros. results: altogether, medicines for rd were approved between and . complete sales data were available for medicines which were included in the comparison. for germany and the united kingdom the continuous use of ( %) and ( %) was observed, respectively. the following italy, france, denmark and sweden use - % of all the medicines. in slovenia, ( %) medicines for rd were introduced. germany and the united kingdom times to first continuous use were the shortest (median time - months after marketing approval). median times below months were observed for norway, sweden, austria, the netherlands, france and switzerland. other countries were slower in enabling first continuous use (median time from to months). germany, france, switzerland had the largest pharmaceutical expenditure per inhabitant in ( . , . and . euros/inhabitant) while slovenia amounted to . euros/inhabitant. in slovenia more than a half of the medicines for rd approved in europe are used which ranks it in the middle of all the countries in comparison. comparing to the important european pharmaceutical markets like germany, united kingdom, france and italy, slovenia's median time to first continuous use is longer and pharmaceutical expenditure per inhabitant for these medicines is lower. pec : mapping of the dlqi scores to eq- d utility values using ordinal logistic regression and monte carlo simulations: is it plausible? please specify your abstract type: research abstract background and objective: converting dermatology life quality index (dlqi) scores to generic measure data would allow utility calculations and enable cost-effective analysis. this would meet the needs of health technology assessment agencies (htas) such as nice, who preferentially use the general health measure eq- d. the dlqi is a specialty-specific measure unlike the eq- d, a generic measure from which utility values can be derived. often several measures are implemented in studies with increased cost and patient burden. ordinal logistic regression (olr) was used to develop a model to convert dlqi scores to eq- d based utility values for use in economic appraisal of medicines. setting and method: data from patients were randomly divided into estimation and validation sets to fit and test the model. a series of ordinal logistic regressions were fitted in spss v for the five eq- d dimensions based on age, sex and all individual items of the dlqi as predictors. the model produced three estimated probabilities per subject per eq- d domain. using these estimated probabilities, a series of monte carlo (mc) simulations were run for each subject resulting in predicted domain responses. from these, utility values were calculated and compared to actual patient values. main outcome measures: conversion of dlqi scores to eq- d domain data results: there are conceptual overlaps between items of the dlqi and eq- d. the validation data set (which was not included in the creation of the model), demonstrated that the models were highly predictive compared to actual responses, except for minor differences for the pain/discomfort domain. for example, for the eq- d ' mobility' domain, patients answered 'no' (predicted and patients answered 'some or extreme' (predicted ) . we examined the model's latent variables, which also demonstrated high predictability at individual level. for example for the 'usual activities' domain the mean latent variable scores were - . , - . and - . for those responding 'no', 'some' and 'extreme' respectively, showing a clear increase in the scores with response. after excluding subjects with missing variable data there were patients in the estimation set and in the validation set. the model was shown to be highly predictive and repeated simulations demonstrated a stable model. the average predicted utility value for the entire validation set ranged from . to . across the mc simulations compared to the actual average utility value of . . conclusion: using olr, we have developed a method of mapping the disease-specific dlqi onto the eq- d: utility values may then be derived for population data sets, and possibly for individuals. the olr technique could be used to convert data from other disease-specific quality-of-life measures to generic utility data for incorporation into cost-effective analyses, greatly enhancing the potential value of such information. tomi laptoš *, , tanja kersnik levart hospital pharmacy, the division of paediatrics, department of nephrology, university medical centre ljubljana, ljubljana, slovenia please specify your abstract type: descriptive abstract (for projects) background and objective: having to take medication during time in school may present certain discomfort for some children. suboptimal dosing regimens (i.e. dosing more frequent than determined by drug's trough:peak ratio) can be prevented by a clinical pharmacist's overview and therapy optimization. the aim of the study was to review and evaluate dosage regiments in paediatric patients on antihypertensive therapy. dosage regiments are defined as schedule of doses of a therapeutic agent per unit of time and the amount of a medicine to be given at specific time. design: electronic health records (ehr) review, evaluation of actual dosage regiments against regiments recommended in smpcs and clinical database (uptodate Ò ), a consecutive case series study. results: ehrs of patients, admitted to or discharged from the department of nephrology of the division of paediatrics, umcl in with suspected icd- diagnoses from i to i were reviewed. patients were excluded (diagnosis not confirmed or lifestyle-change disease management only). the remaining patients (average age . years (range - )) received daily on average . medications (range - ) in . individual doses (range [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . most frequently used drugs were perindopril (n = ), ramipril (n = ), amlodipine (n = ), bisoprolol (n = ) and doxazosin (n = ). patients received ex tempore oral suspensions. dosage regimen was not optimized in % (n = ) of the patients, among those % (n = ) receiving one medication only and % (n = ) receiving more than one medication where at least one was not optimized. furthermore, % (n = ) patients on stabledosage therapy (no dosage change of either medication in last months with satisfactory clinical outcomes) were eligible for fixeddose combination medication. with optimized dosage regimens patients would receive daily on average . medication (range - ) (- %) in . individual doses (range - ) (- %). the difference was statistically significant ( % ci, p \ . ) in both cases. conclusion: our data show that the majority of the paediatric patients on antihypertensive therapy ( %) received their medication in optimal dosage regimens. however, with an estimated every fifth patient not being on optimal dosage regimen, a multidisciplinary approach is crucial to assure that the individual patient achieves the best clinical, humanistic and economic therapy outcomes. ph : polypharmacy management programmes in the elderly: a case study in greece dimitra gennimata *, , christos kampolis , aggelos vontetsianos , jennifer mcintosh , alpana mair , on behalf of simpathy consortium please specify your abstract type: descriptive abstract (for projects) background and objective: polypharmacy management and medication adherence in the elderly are significant public health issues throughout the european union (eu). simpathy (stimulating innovation management of polypharmacy and adherence in the elderly) is a consortium of organizations representing eight european countries, aiming at stimulating innovation around management of appropriate polypharmacy and adherence, ultimately providing tools for eu policy makers to develop and/or improve, implement and evaluate programs addressing these issues. design: a mixed-methods case study was carried out in greece, to identify policies on the management of polypharmacy and adherence issues in the elderly. a desk review of the polypharmacy and adherence policies at the government, regional and institutional level has been completed. key informant interviews were conducted with policymakers and health professionals responsible for developing and implementing strategies. focus groups consisting of policymakers, clinicians and patients validated the research findings. results: although e-prescription implementation is widespread (& % coverage nationwide) and disease-specific guidelines have been developed, polypharmacy management is only associated with direct economic indicators. no formal policies or programmes are identified. significant contributions are coming from different health professional organizations that have chosen to provide expanded services to their patients, aiming at optimising drug therapy and thus polypharmacy management and medication adherence in the elderly. community pharmacists offer pharmaceutical care to patients, which includes management of prescribed medication, otc remedies, vitamins and supplements and food-drug interactions. hospital pharmacists in some state (public) hospitals review medication for inpatients and out-patients, communicate with prescribers and confirm the ''benefit-no harm'' principle in the prescribed medication (e.g. incompatibilities, side effects, -rights of medication). medical doctors, mostly general practitioners and some specialized ones, usually in primary healthcare settings, keep health records of their patients and have an overview of all administered medication. however, key barriers still remain the lack of coordination of institutions and authorities and overlap of their responsibilities, healthcare workforce and infrastructure shortages and several cultural issues. conclusion: all initiatives to medication management and medicines optimisation are provided without directive from national policies or guidelines. therefore, these activities rely on the goodwill of the health professionals to address pharmacotherapy and therefore polypharmacy management but they are not necessarily representative of what is happening nationwide. a national policy to implement the management of polypharmacy nationwide could mobilise the willingness of health professionals and ensure consistency of care. development and implementation of this policy should build on the grassroots efforts currently underway in this area. ph : clinical profile and treatment discontinuation in a tuberculosis control state programme in brazil: preliminary results from sinan database simone s. bezerra , mara guerreiro *, , , nathany pessoa , maria paula athayde , rodrigo auad , joão josé gomes , josé lamartine soares sobrinho post graduation program in therapeutic innovation, federal university of pernambuco, recife, pernambuco, brazil, centro de investigação interdisciplinar (ciiem), instituto superior de ciências da saúde egas moniz, monte de caparica, please specify your abstract type: research abstract background and objective: challenges remain in tuberculosis (tb) control. discontinuing treatment can leave patients infectious and contributes to the emergence of resistance. this study aimed to describe the clinical profile and cure and discontinuation rates of tb patients enrolled in the pernambuco tuberculosis control programme (pect). setting and method: the study was conducted in three sites in recife, brazil, designated a (one polyclinic plus eight general practice units), b (one hospital for medium-complexity patients) and c (one hospital for high-complexity patients). data were extracted from the notifiable diseases information system (sinan) for all pect outpatients, from / to / (n = ). analysis was performed with the aid of action for excel; there is on-going analysis to further explore differences across sites. ethical approval was granted. main outcome measures: clinical form of the disease, hiv testing, new cases, cure and treatment discontinuation. results: sociodemographic data were available for sites a and b only. most patients were male ( %, n = ), with age raging from to years old ( . %, n = ); most had a low education level ( %, n = ) and low socioeconomic status ( %, n = ). the most common clinical presentation was pulmonary tb. most cases were new ( . %, n = ); recurrence and enrolment after discontinuation were respectively . and . % (n = and n = ). with respect to hiv, . % of patients were seronegative (n = ); about a third ( %; n = ) had not performed hiv test. rates for cure were respectively . % (n = ), . % (n = ) and . % (n = ) in the sites a, b and c. correspondent rates for treatment discontinuation were . %(n = ), . % (n = ), . % (n = ), respectively. tb-related mortality ranged from in site c to . % in site b. conclusion: missed hiv tests may represent undetected tuberculosis/ hiv coinfection. site b presented the highest rates of intrapulmonary plus mixed tb forms, discontinuation of treatment and tb-related mortality; additionally, it had the lowest rate of enrolment after treatment discontinuation. patients co-infected with tb and hiv are firstly referred to this site, which may explain this finding. our findings may help managers allocating resources and assist clinical pharmacists in planning their interventions. findings also suggest the need of more intensive interventions in tb patients, such as pharmaceutical care programmes. please specify your abstract type: research abstract background and objective: pharmacists working in primary health clinics have various roles. pharmaceutical care is one of them. how to provide this service varies across countries and settings. the most optimal way to provide pharmaceutical care is important to define when developing clinical pharmacy services in a new setting such as primary care practices. general practitioners are key stakeholders in this endeavour. the aim of this study was to find the most optimal approach to providing pharmacist-led pharmaceutical care in primary health care clinics in iceland in collaboration with general practitioners. setting and method: action research provided the framework for this research. data was collected from pharmaceutical care interventions with patients, field observations, field notes, and interviews with general practitioners over the period of the study. the study ran from september to june . three separate semi-structured in-depth interviews were conducted with five general practitioners from one primary health care clinic in iceland at different time points throughout the study. pharmacist-led pharmaceutical care was provided to patients (n = ) before and between general practitioners' interviews. the study settings was a primary health care clinic in reykjavik area and the patients' homes. main outcome measures: how to provide pharmaceutical care in collaboration with general practitioners in the icelandic health care environment. results: direct contact between pharmacists and general practitioners over short distances are essential to providing optimal pharmaceutical care services. pharmacist's access to medical records is necessary even though face-to-face communication between pharmacist and patients are most effective in providing pharmaceutical care. pharmacist-led clinical service was deemed most needed in dose dispensing polypharmacy patients. patients require more information about drugs prescribed to them coupled with an accurate drug list with greater detail. conclusion: the most efficient collaboration when pharmacist and general practitioner is obtained when they work side by side at the primary health care clinic. when new services are developed it is vital to identify different requirements of the primary health care clinics to optimize the running of a clinical pharmacist service. ph : accompaniment of patients treated with oral chemotherapy: a survey on patients' experience laure napoly *, , pascal paubel , , sylvie burnel oncorif -regional cancer network, health law and health economics deparment, health law institute, inserm, umr s , paris descartes university, sorbonne paris cité, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: antineoplastic agents taken orally are more and more used in cancer care. these medicines confer autonomy to patients. although, they may cause adverse effects that can lead to treatment adherence issue, unjustified hospitalizations, or premature treatment interruption. proper accompaniment of patient can prevent these issues. in order to define how to organize this accompaniment, we conducted a survey on patients' experience. the objective is to describe patients care pathway and identify their needs. design: a descriptive, qualitative, prospective, survey was conducted using self-administered questionnaires, in hospitals of ile-de-france region. inclusion criteria were: having being treated with oral neoplastic agents during minimum months, age [ , solid tumor, no concomitant iv chemotherapy. collected data were: patients' sociodemographic and clinical profile, their insight about different steps of care pathway (information, treatment delivery, follow up), their behaviours and interactions with healthcare professionals, and their overall opinion. results: patients were recruited in six hospitals. their sociodemographic and clinical characteristics were variable. % were treated with targeted therapy, % with cytotoxic agent, and % with endocrine therapy. patients showed high satisfaction for given information at the beginning of the treatment. principal source of information identified was the oncologist ( %). while the delivery of treatment, % of patients beneficiated of advices from the pharmacist. accompaniment of patients during treatment seemed unequal, with: a frequency of visits with the oncologist ranging from less than - months; % of patients not knowing any telephone number they can call in case of worries or questions about their condition or treatment; % not remembering any particular accompaniment treatment (visits or calls from nurses or doctors for example). for adverse effects management, three principals actors were identified: oncologist ( %), general practitioner ( %) and pharmacist ( %). regarding the use of oral chemotherapy, patient are satisfied with: it's comfort ( %); being more actively involved in their cancer care ( %); and state not having any anxiety taking chemotherapy home ( %) . asked openly about their concerned and needs three major concepts were identified: high concern about adverse effects, positive feedback about maintaining contact with health professionals between cures and difficulties of communication between health professionals. conclusion: there is a high satisfaction regarding oral chemotherapy and health professionals. the oncologist has a primordial place in patient pathway, whereas implication of pharmacist and general practitioner stays variable. adverse effects are a major concern that needs proactive accompaniment. the variability of our results suggests that accompaniment must be flexible and adapted to the needs of each patients. the key to flexibility could be good coordination and communication between healthcare professionals. ph : general beliefs about medicines among independent elderly adults in sweden: data from an rct lina hellström *, , , victoria throfast the pharmaceutical department, kalmar county council, ehealth institute, linnaeus university, kalmar, sweden please specify your abstract type: research abstract background and objective: there is a need to improve prescription and use of medications by the elderly. the objective of a recent rct was to investigate the effects of e-learning about medicines among elderly adults. a positive impact on the primary outcome measure, knowledge about medicines, is reported elsewhere. a secondary outcome measure, general beliefs about medicines, is reported below. setting and method: the study was a randomized controlled trial in elderly people (aged c years). participants were recruited from patient associations and pensionerś associations. participants were randomized to either an intervention group that participated in the e-learning, i.e. internet modules with video and audio, or a control group that did not take part in the e-learning. post-intervention data was collected using paper-based questionnaires, completed within two weeks after agreeing to participate in the study. the general beliefs about medicines questionnaire (bmqgeneral), comprising the subscales necessity, harm and overuse, was used to elicit beliefs. higher scores indicate stronger endorsement of scale constructs (range [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . main outcome measures: bmq-general subscale scores. results: a total of elderly people were included in the study, in the intervention and in the control group. the mean age in the total population was . years and % were women. eleven percent did not use any prescribed drugs while % used more than five prescribed drugs. the patients' scores were very similar in the two groups for all three bmq subscales. the median ''overuse'' score was in the intervention group versus in the control group, the median ''harm'' score was (iqr - ) in both groups and the median ''necessity'' score was in both groups. in the total population the most commonly expressed negative beliefs referred to overuse of drugs. . % of respondents agreed with the statement ''if doctors had more time they would prescribe fewer medicines'', . % stated ''doctors prescribe too many medicines'', and . % stated ''doctors place too much trust in medicines''. a majority of the respondents agreed with the four items on the ''necessity'' scale. for example ''medicines help people to live a better life ( . % agreed)''. conclusion: the studied e-learning intervention was not shown to have any impact on general beliefs about medicines. beliefs about medicines have been associated with a number of background variables which might explain why increased knowledge about medicines alone cannot change such beliefs. in general, respondents in the study had highly positive beliefs about the necessity of medicines. nevertheless, the results indicate that overuse of medicines is regarded as a problem. ph : maf-plus: pharmacists' contributions to provision of financial assistance for medications ian wee * , charlene ong, niron naganathar changi general hospital, singapore, singapore please specify your abstract type: descriptive abstract (for projects) background and objective: the medication assistance fund plus (maf-plus) is a government scheme introduced in singapore in to provide financial assistance to needy patients who meet pre-set criteria based on means testing. unlike previous schemes, maf-plus provides broader discretion to institutions when providing financial assistance. in our institution, pharmacists reviewed patients' case and medication histories, and filed recommendations to a multidisciplinary committee tasked with approving deserving maf-plus applications. the pharmacists' contributions to the committee, and the outcomes of the applications, are presented in this study. design: all maf-plus applications received between st october and st december were reviewed. pharmacists' comments for each application, where provided, were noted, as well as the range of medicines applied for, review approval rates, and cumulative percentage of available funds utilised. the effect of a recent widening of the scheme's scope to include notable high-cost items was also evaluated. results: between and , maf-plus applications were reviewed, of which ( . %) were approved. of the rejected applications, ( . %) were channelled to alternative financial assistance schemes on the recommendation of the pharmacists. the medicines most commonly applied for were intended for the treatment of cardiac ( . %), respiratory ( . %), and psychiatric ( . %) conditions. pharmacists' recommendations also led to a gradual expansion of our institution's list of pre-approved medicines-from in - to by end- . from onwards, pharmacists previewed increasing numbers of applications for high-cost medicines, particularly those for treatment of retroviral disease, hepatitis c, and rare diseases. cumulative utilisation of maf-plus funds (inclusive of annual replenishment) rose from . % in - to . % by end- , representing an average year-on-year growth of . %. conclusion: using a process of judicious previewing of maf-plus applications, and recommendations to the maf-plus committee, pharmacists contributed to a high percentage of patients receiving financial assistance for medications. despite a steep growth in the number of applications received between and , this approach helped to prevent over-extension in fund utilisation. pharmacists will likely be increasingly relied upon due to an anticipated rise in the number of applications for high-cost medicines. please specify your abstract type: research abstract background and objective: adolescents often treat themselves and take medications without parental supervision. lack of experience and knowledge of medicines in this age group frequently leads to inappropriate use of medicines and adverse drug reactions. data about the use of medicines among slovak adolescents and their knowledge of medicines have not been studied yet. setting and method: for our study we used the questionnaire method. the questionnaire contained multidimensional items with closed-ended and open-ended questions, which focused on the characteristics of the adolescentś health status, use of medicines, also in relation to parents and adolescentś knowledge and perception of medicineś risk. we distributed validated questionnaires for adolescents aged from to at secondary schools in all regions of slovakia. response rate was . %. questionnaires were finally analysed. the differences in the distribution of categorical variables between groups were evaluated using the chi square test. sas . . was used as statistical software. main outcome measures: to determine adolescentś knowledge of medicines in terms of efficacy, self-medication, safety of therapy and analyse which medicines are the most frequently used by adolescents. to compare adolescents with chronic disease and healthy ones from the perception of pharmacotherapy point of view. results: in the analysed group . % (n = ) of adolescents are treated for chronic disease. mostly they suffer from allergy ( . %, n = ) and skin diseases ( . %, n = ). adolescents with chronic disease use regularly prescription medicines ( . %, n = , p \ . ) and over the counter medicines ( . %, n = , p \ . ). this group of adolescents better accept the pharmacotherapy with parental supervision ( . %, n = , p = . vs. healthy adolescents) and they believe in effectiveness of prescription medicines ( . %, n = , p = . vs. healthy adolescents). most frequently prescribed medicines were azithromycin, levocetirizine, ofloxacin and over the counter medicines were ibuprofen, paracetamol, ascorbic acid. we found out in all group of adolescents that . % (n = ) prefer self-medication without check-ups, . % (n = ) used drugs in the last months without a prescription, . % (n = ) take over the counter medications independently without the supervision of parents, . % (n = ) buy medicines themselves in the pharmacy, . % (n = ) do not take medications as recommended, . % (n = ) believe that they have enough knowledge of medicines which they take, . % (n = ) resp. . %, (n = ) believe that prescription medicines resp. over the counter medicines are safe. conclusion: questionnaire analysis pointed out that slovak adolescents have not enough knowledge of medicines. the study provides new information in the field of risk perception and adolescentś knowledge of medicines in the slovak republic and highlights the areas that need to be studied in the future in terms of adolescentś education. federal university of pernambuco, state technical school prof. agamenon magalhães, recife, pernambuco, brazil please specify your abstract type: research abstract background and objective: the effectiveness of tuberculosis (tb) control programmes depends critically on patients completing appropriate treatment. this study aimed to outline the cure and discontinuation rates of patients enrolled in the pernambuco tuberculosis control program (pect), based on dispensing data. setting and method: the study was carried out in three sites in recife public health system, brazil, designated a (one polyclinic plus eight general practice units), b (one hospital for medium-complexity patients) and c (one hospital for high-complexity patients). data were collected between - / , through reports from the stock management software for public pharmacies (horus) for pect outpatients. reports corresponded to a total of patients ( , and in sites a, b and c, respectively). horus defines ''cure'' as medicines collection for three, six or nine consecutive months without interruption, depending on the treatment scheme; discontinuation is defined as non-sequential collection of medicines or treatment interruption for two consecutive months or more. patients were assigned an ''undetermined'' status if treatment was ongoing. data were inputted onto an excel spreadsheet and checked for accuracy. quisquared test, fisher's exact test and bootstrap analysis were performed with r statistical computing. ethical approval was granted. main outcome measures: cure and discontinuation rates for pect outpatients. results: demographic data are not available for the sample. rates for cure were respectively . % ( ), . % ( ) and % ( ) in the sites a, b and c, while rates for treatment discontinuation were . % ( ), . % ( ) and % ( ), respectively. discontinuation rates were significantly different among the sites a, b and c (p \ . ). bootstrap analysis showed that overall the proportion of patients with an ''undetermined'' status in each site did not significantly change these differences. conclusion: only site a had an acceptable discontinuation rate, in light of the world health organization recommendations. this deserves attention as default treatment leaves patients infectious for longer, increases the risk of poor outcomes and fosters resistance to antibiotics. pharmacists could use dispensing data to signal tb patients at-risk of discontinuation, and subsequently tailor interventions addressing its causes. site b had the greater number of patients which discontinued treatment. patients co-infected with tb and hiv are firstly referred to this site, which may explain this finding. our findings suggest the need of more intensive interventions in patients co-infected with tb and hiv, such as pharmaceutical care programmes. please specify your abstract type: research abstract background and objective: many efforts are done to organise good quality and safe pharmaceutical care. in general, the involvement of a hospital pharmacist or hospital pharmacy personnel in the process of medication reconciliation results in a reduction of the number of medication discrepancies. however, in case of emergency admissions this topic is still insufficiently studied. the introduction of good medication reconciliation on the emergency department (er) requires firm logistical and organisational efforts. we investigated the effects of a drug reconciliation intervention by pharmacy personnel during emergency admissions in order to identify discrepancies between medication lists taken by er physicians and by pharmacy personnel. setting and method: this observational, comparative, non-randomised intervention study was performed in . we calculated that a population size of patients was sufficient to perform reliable measurements. inclusion criteria: all patients presented at the er and admitted to a hospital ward \ after presentation with usage of one or more prescription drugs. exclusion criteria: age \ years, residency outside the region delftland, inability to undergo an oral interview, absence of a medication list of the public pharmacy (ozislist), decease of the patient during er-stay and patients undergoing surgical procedures. discrepancies between both medication lists taken by an er physician or pharmacy technician were classified in four categories of increasing severity ( = no discrepancy to = clinical relevant discrepancy) using the index of the national coordinating council for medication error reporting and prevention (www.nccmerp.org). discrepancies were categorised by a panel consisting of a pharmacy technician, a (senior) hospital pharmacist and a th year pharmacy student. statistical analysis was carried out with a statistical software package (spss ) using the mann-whitney u test and chi squares test. main outcome measures: during the intervention measurement we analysed the reconciliated medication by comparing the er's physician's list with the list of the pharmacy technician after a medication verification interview. the number of discrepancies were measured and judged by the panel. discrepancies were given a category , , or as defined. results: during the intervention measurement patients were admitted to the er. sixty-five ( ) patients ( . %) met the in-and exclusion criteria. the number of medication discrepancies decreased significantly after intervention of the pharmacy technician by %, from to discrepancies. the average number of discrepancies per patient after intervention decreased by . %, from average . to . discrepancies per patient. conclusion: medication verification by pharmacy personnel in the er reduces the number of medication discrepancies by half. medication lists generated with a standard interview by pharmacy technicians in combination with an ozis-list on admission of patients at the er is more complete and accurate than the current method. hp-pc : discharged patients: a problem for community pharmacists? information transfer, as well as the role, needs, and objectives of pharmacists when they care for recently discharged patients. setting and method: a focus group was conducted with a sample of six community pharmacists from personal contacts to represent different characteristics. the focus group consisted of different questions and the recording was transcribed, fragmented and categorised. based on these results, a nationwide online-survey was created with the following questions: a) responder's characteristics, b) number and origin of prescriptions, c) role fulfilment of the joint-who/fip-guideline on good pharmacy practice, rated with a -point likertscale, d) information items derived from the focus group discussion grouped into four categories and evaluated for their availability and for their usefulness by likert-scales, e) goals for discharge optimisation, f) additional comments. the questionnaire was piloted and translated forward and backward to french and italian by native speakers. it was sent to all managers of pharmacies belonging to the swiss pharmacist's association in summer (n = ). main outcome measures: conclusions from focus group discussion and responses to questions a-f from the nationwide questionnaire. results: the focus group participants ( . ± . years, % female, % employees) emphasised the importance of an expanded information transfer, especially for medication changes, unclear prescriptions, and information about a patient's medication acquisition. they were concerned about their extensive workload of discharge prescriptions, and mentioned treatment continuity as one of their goals. the questionnaire was answered by pharmacists (response rate . %, . ± . years, . % female). there were . % of responders who reported to fulfil their role (to manage a patient's therapy, function b) not satisfyingly. unavailable but essential information were allergies and the specification of off-label use prescription. unavailable although desired information were the reasons for therapy changes, indications, appointments, contact information, or compounding formulations. concerning design and transfer, information should be written in a structured way but no clear preference for a transfer method was found. goals of community pharmacists were: improved treatment continuity, patient safety, and pharmaceutical care. conclusion: swiss community pharmacists rarely receive sufficient information on discharge prescriptions. appropriate pharmaceutical care is therefore impeded. the knowledge and application of the findings enable directed optimisation of discharge. hp-pc : patients attitude for using antipsychotic medication in the norwegian early intervention in psychosis, tips study rafal yeisen *, , stein opjordsmoen , , , inge joa , , jan olav johannessen , , jone bjørnestad on behalf of centre for clinical research in psychosis, psychiatric division, stavanger university hospital, stavanger, norway background and objective: poor drug adherence in patients with psychosis leads to relapse, re-hospitalization, poor outcome and increased consumption of health services. pharmacoclinical studies have demonstrated that the treatment response decreases with each relapse. it is estimated that % of patients suffering from chronic illness are not taking medication as prescribed after months. the purpose of this study is to investigate which experiential factors that potentially might affect adherence with medication in adults with psychotic disorders. setting and method: in a descriptive qualitative sub-study in the ongoing norwegian early intervention in psychosis, tips study, where twenty-first episode patients ( male, female) participated in semi-structured interviews years after inclusion. they were still using or had used antipsychotics during the last years. data were analysed using interpretative phenomenological analysis. main outcome measures: adherence to antipsychotics. results: the data suggested four main themes, reflecting the patients' subjective experiences and their impact on the desire to adhere to antipsychotics: ( ) admission experience as a psychotic's patient; ( ) information from healthcare staff; ( ) limited involvement in decision-making; ( ) attitude to antipsychotics. conclusion: a number of factors had a positive influence on adherence to antipsychotics. pleasant admission/stay experiences, feeling that antipsychotics had therapeutic effects, mild or no side effects, and believing that antipsychotics are necessary and useful, were typical statements. please specify your abstract type: research abstract background and objective: the hospital-to-home transition is a vulnerable stage in a patient's care. patients can experience problems with medication supply, which possibly lead to therapy interruptions. the objectives of this study were to investigate medication supply after discharge, and patients' and physicians' opinions about the current discharge process and possible optimisations. setting and method: a telephone interview was conducted with discharged patients from the surgical and internal medical wards from the cantonal hospital in baden (switzerland). inclusion criteria were: patients c years old, discharged home with a discharge prescription. patients were called between the nd and th day after discharge and a piloted, structured interview was performed, consisting of questions on experiences and optimisations. afterwards, semi-structured interviews were conducted with five physicians from the study hospital. results from patient interviews and the general discharge process were discussed. main outcome measures: proportion of filled prescription, frequency and type of supply problems including therapy interruptions. opinions of physicians and patients on current discharge process and possible optimisations. results: discharged patients were . ± . years old, % female, % from internal medicine, and % regularly visit the same pharmacy. of the interviewed patients, have not filled their prescriptions yet and had their prescription filled when they were called. of these, % of them visited the pharmacy on the day of discharge, but it took up to the th day until all of them received their medication. supply problems were encountered by patients ( %), mainly because of the medication not being in stock in the community pharmacy. only four patients experienced therapy interruptions, which took up to the rd day post-discharge. patients discharged from internal medical wards had more supply problems compared to surgical wards (relative risk = . , p = . ). patients experiencing supply problems had statistically significant more medicines on a daily basis ( . ± . vs. . ± . , p = . ). physicians were surprised about the late prescription filling and worried about the disease outcomes. however, interruptions were interpreted as unfrequent. when asked if, in future, hospitals should transfer prescription to the community pharmacy prior to discharge, % of patients refused and physicians were undecided, mainly because of a questionable benefit. but both groups indicated that giving some bridging supply would be welcome. conclusion: this study showed that patients discharged from a swiss hospital encounter supply problems, but therapy interruptions are seldom. giving some bridging supply was preferred over an early information transfer by patients and physicians. interventions should consider these opinions and focus on internal medicine patients with high number of medication. please specify your abstract type: research abstract background and objective: adherence to secondary prevention evidence-based medical (ebm) therapies for patients with st-segment elevation myocardial infarction (stemi) is essential to reduce long-term rates of major adverse cardiovascular events. current guidelines recommend the long-term use of low-dose aspirin, highintensity statins, angiotensin-converting enzyme inhibitors (acei)/ angiotensin receptor blockers (arb) and beta-blockers (bb), in addition to p y inhibitors for year. we aimed to assess the adherence to secondary prevention ebm therapies from discharge to one-year follow-up among patients with stemi undergoing primary percutaneous coronary intervention (pci) in contemporary practice. setting and method: observational single-centre study including consecutive patients with stemi undergoing primary pci in a tertiary hospital in switzerland over a one-year period. secondary prevention ebm therapies were assessed at discharge and at one-year follow-up. main outcome measures: prescription of key secondary prevention ebm therapies (aspirin, p y inhibitors, statins, acei/arb and bb) from discharge to one-year follow-up after stemi. bb was recommended only for patients with heart failure or left ventricular ejection fraction (lvef) \ %. results: a total of patients were included. ebm drug prescription at discharge was . % for aspirin (n = ), . % for p y receptor inhibitor (n = ), . % for statin (n = ), . % for acei/arb (n = ) and . % for bb (n = , among patients with lvef \ %). ticagrelor ( . %) was the major p y inhibitor prescribed. overall, ebm drugs were missing at discharge, with of these missing drugs having no justification for no-prescription (contraindications, allergy or intolerance). at one-year follow-up (median . months, n = ), aspirin, statins and acei/ arb prescription rates were . % (n = ), . % (n = ) and . % (n = ) respectively. out of patients ( . %) with lvef \ % received a bb. among patients treated with ticagrelor at discharge, ( . %) were receiving ticagrelor at follow-up, whereas ( . %) were switched to another p y inhibitor. among patients who discontinued ticagrelor (n = , . %), duration of dual antiplatelet therapy was months for % (n = ) and discontinued prematurely (\ year) for % (n = ) patients. reasons for ticagrelor early discontinuation or switch were not specified. conclusion: in a real-world cohort of patients with stemi undergoing primary pci, prescription of recommended secondary prevention medications at discharge is excellent. adherence to ebm therapies at year remains high with more than % of patients receiving all ebm drugs. early discontinuation of dual antiplatelet therapy was observed in % of patients, whereas ticagrelor was switched for another p y inhibitor in . % of patients. these observations highlight key opportunities to improve longitudinal use of secondary prevention therapies after stemi in routine clinical practice. although side effects are less common than traditional chemotherapies, certain ones such as pain, fatigue, nausea and vomiting can still be bothersome. in oncology outpatient clinics, side effects are monitored by oncology nurses; however due to high patient turnover and limited numbers of nurses, the assessment of side effects might not be performed adequately. therefore, aim of this study was to determine side effects of immunotherapy and targeted therapy and to compare the severity assessment of side effects by clinical pharmacist and nurses. setting and method: the study was conducted in the hacettepe university oncology hospital outpatient clinic. the patients who have been taking ipilimumab, nivolumab, pembrolizumab, bevacizumab, panitimumab or cetuximab during october -march were included. the assessment of side effects were undertaken by a clinical pharmacist and nurses separately on each visit using the common terminology criteria for adverse events version- toxicity assessment scale. an independent clinical pharmacist compared the side effects' assessments by pharmacist and nurses for analysis. ethical approval was obtained from hacettepe university ethics committee. main outcome measures: to compare the severity of side effects of targeted drug therapies which were assessed by a clinical pharmacist and nurses. results: during the study period visits of patients were evaluated. a total of side effects assessments were recorded. among those assessments ( . %) was assessed in different ranking by nurses and pharmacist. the differences in the number of assessments were mainly seen in criteria related to pain (n = ; ), sensory loss (n = ; ), fatigue (n = ; ), stress (n = ; ), insomnia (n = ; ) which was performed by nurses and pharmacist respectively. other side effects detected only by clinical pharmacist were oedema, cough, gastrointestinal complaints (heartburn, cramp) and sensitivity of odour which require close monitoring and in-depth counselling by clinical pharmacist. conclusion: this study explores the differences in assessment of side effects by pharmacist and nurses in targeted therapies. routine assessment of side effects between chemotherapy cycles might yield to misinterpretation or inadequate assessment due to workload of outpatient clinic. therefore, inter-professional interactions in outpatient clinics might close the communicational gaps and improve patient care. hp-pc : implementation of clinical pharmacy in the acute psychiatric wards: improving quality of medical treatment across health care sectors amila zekovic *, , signe kristensen , lisbeth lund pedersen clinical pharmaceutical services, capital regional pharmacy, head of clinic, mental health services, copenhagen, denmark please specify your abstract type: descriptive abstract (for projects) background and objective: a study from shows that people with a mental disorder had a two-to threefold mortality compared with the general population in denmark. life style diseases are the major reason for the excess mortality, partly due to undertreatment of physical disease and well known side effects from medicines such as obesity, diabetes, and heart disease. in may , a clinical pharmacy service (cps) was implemented in all acute psychiatric wards (apw) in the capital region as a part of a three-year project funded by the danish health authorities. the objective is to illustrate how the implementation of clinical pharmacy in the apw in copenhagen increases the focus on drug related problems, rational pharmacotherapy and side effects, increasing the quality of medical treatment and patient safety across health care sectors. design: data was collected at the apw in copenhagen which consists of three wards and has a total capacity of beds. inclusion criteria were patients to which two or more of the following apply: • c years of age • c drugs • high risk drugs (clozapine, sertindole and opioids) • combination of antipsychotics and benzodiazepines • diagnosed with liver/kidney disease the secondary inclusion criteria were all patients receiving c drugs as a single criterion. to obtain a valid medication history and secure medication reconciliation, the pharmacist interviewed included patients. the patients were also asked about side effects, compliance, and perceived effects of treatment. a medication review was conducted based on the patient interview, screening for interactions in an interaction database, and consideration of biomedical data in order to evaluate if treatments should be adjusted, initiated or discontinued. the pharmacist's input was discussed with the doctor, as inputs are more likely to be considered if they are communicated orally. finally, all inputs were documented in the patient's journal as a pharmacist note. the model for improvement was used as a tool for implementing the cps and is being used continuously for improving the service. results: between may th and june th , patients were screened at admission to the apw, of which . % met the inclusion criteria ( patients). in this period the pharmacist conducted notes, indicating that . % of the included patients were seen by the pharmacist. in april , patients were in average admitted with . drugs and . inconsistencies between the hospital's medication orders and the medications that the patient had been taking. regarding patients who are discharged to community care shortly after admission, the pharmacist note is sent to their general practitioner for follow up. conclusion: overall, the implementation of a cps in the apw has been successful. medication reconciliation ensures that the patient is provided with correct medicine at admission, transfer or discharge. by performing a thorough medication review based on a consultation with the patient, the service contributes to an increase in quality of medical treatment. please specify your abstract type: descriptive abstract (for projects) background and objective: the importance of the role of a clinical pharmacist resident in the operating room during months, in a private hospital belonging to a group devoted to healthcare for over years. the hospital is recognized as a reference centre of excellence of hospital care in portugal. it has inpatient beds, two surgical blocks with rooms and beds in the intensive care unit. the aim of the clinical pharmacist in the operating room is to ensure compliance with good clinical practice, safety and pharmacotherapeutic effectiveness, as well as optimization of drug costs. design: . logistics restructuring of pharmaceutical services and the need of the physical presence of the pharmacist in the operating room. . furthermore, the workstation of the pharmacist is moved to the operating room and the in-depth study of all medicines used in the operating room. . in compliance with the joint commission, definition, optimization and adjustment of drug stocks to the needs of the service itself. in close collaboration with the nursing staff, consumer kits were created for registration of drugs by type of surgery in order to facilitate registration and ensure billing efficiency. control of the analgesic drug's dispensation circuit in hospitalized surgical patients that stay less than h in the hospital. ensure compliance with the project through which the health regulatory authority evaluates several hospitals in the country, creating a national ranking among hospital specialties. . clinical phase: creation of prescription protocols by type of surgical intervention based on national clinical guidelines. validate prescriptions in the intra-surgical block in compliance with antibiotic prophylaxis, antiemetic and thromboembolic, checking deviations in therapy according to good practice. identify pharmacologic hypersensitivities of patients by consulting the clinical process and anaesthesiology records. provide information on drugs, drug efficacy monitoring and adverse drug reactions in risk management platform. check off label use of drugs. results: of a total of interventions, relate to revenue optimization and relate to clinical interventions. there was an increase of approximately % in billing. on what regards to clinical interventions, the majority of them showed deviations from good clinical practice. the physical presence of a clinical pharmacist in the surgical block is essential as the prescription and administration of drugs is carried out simultaneously, allowing immediate therapy validation, in order to increase the safety and efficacy. the pharmacist has the ability to interact with the multidisciplinary team, as well as monitoring the patient's clinical process, the pharmacotherapeutic profile and drug allergies, allowing the detection of any adverse drug reaction on-time. all these interventions are possible in the pre, intra and postoperative phases. results: counselling (av.(±sd) duration: ± min) was performed in patients ( . % female; av.(±sd) age: . (± ) years; av.(±sd) medicines at discharge: . (± . )). in % of patients mrps were intercepted. the five most common mrps (%) were: need for organisational support ( . , e.g. proper prescriptions' writing), therapy-related discussions ( . ), untreated indications ( . ), errors in documentation ( . ), and medicines without an indication ( . ). patients ( . %) classified for study inclusion, of whom ( . %) consented to be followed-up and ( %) provided data. roughly % of patients report having received information about medicines at discharge, of which three-fourths remember being informed by the pharmacist. more then every second patient ( . %) reported having received valuable new information. changes in chronic-use medicines occurred in . , . , and . % of patients at -, -, and -month, respectively. at -month, in . % of patients chronic-use medicines were newly prescribed, in . % discontinued. medical specialists initiated these changes in . % of patients. one out of five patients couldn't recall the reasons for changes in medication. nearly % of patients showed moderate to little medication adherence at -month. it did not significantly change during the follow-up period. conclusion: clinical pharmacists' counselling prevents mrps at the transition from hospital to home. follow-up data show that changes occur in one out of three patients. medication adherence remains stable, but generally needs to be improved. please specify your abstract type: research abstract background and objective: until , prescription analysis was based in our hospital pharmacy. clinical pharmacy has been deployed in care units since . many clinical pharmacy services were developed: medication reconciliation, patient's therapeutic education and counselling, and prescription analysis unit based. the purpose of this study is to assess the impact of the clinical pharmacist as a direct patient-care team member on prescription analysis. setting and method: we collected pharmaceutical interventions (pis) of the first months of and at the same period of the year in the neurology unit when the pharmacist was unit based. we studied and compared type of pis (medication, drug related problem-drp), rate of pis acceptance and clinical impact. focus was made on high alert risk medications and potentially inappropriate medications. . % in versus none in . when prescription analysis was based in the pharmacy unit, % of drp detected by the pharmacist had a potential clinical impact versus % when the pharmacist performed prescription analysis in the care unit (p \ . ). three drp detected in had serious potential harm. results: ward-based prescription analysis allowed detecting five more times drp with a significant more important clinical impact than pharmacy unit based prescription analysis. the clinical pharmacist as a direct patient-care team member is more efficient in detecting serious potential harm. indeed, the pharmacist has a greater knowledge of the patient's clinical condition. nevertheless the global rate of acceptance of pis was greater when the prescription analysis was based in the pharmacy unit even if the difference is not significant. but prescription analysis is more complex when performed in the care unit, taking account adherence of the patient, and potentially inappropriate medications resulting in much higher risk-taking by the ward-based pharmacist. conclusion: this study showed that unit based prescription analysis is the best way to detect drug related problem. it must be competed by medication reconciliation and medication review to improve medication safety process. hp-pc : qt-prolongation in an acute psychiatric setting: fact or fiction? eva jacxsens *, , hans van den ameele , jü rgen de fruyt , yves vandekerckhove , frank vancoillie , veerle grootaert pharmacy, psychiatry, cardiology, az sint-jan brugge-oostende av, bruges, belgium please specify your abstract type: research abstract background and objective: several psychotropic drugs can induce qt-prolongation, which is a well-known risk factor for developing torsade de pointes (tdp) and sudden death. the clinical relevance of this side effect of psychotropic medication remains unclear, especially in patients hospitalized in an acute hospital. to interpret the clinical importance of psychotropic drug induced qt-prolongation, we investigated the prevalence of these electrocardiographic changes. setting and method: a prospective study was conducted on four psychiatric wards in a general hospital: two acute, short-term psychiatric units (asp and asp ), one addiction service unit (asu) and one geriatric-psychiatric ward (gpw). all adult patients admitted between october st and march th on a psychiatric ward were eligible for inclusion. at admission, an ecg (ecg ) was performed and creatinine and potassium levels were measured. a second ecg (ecg ) was performed at least days after the start of a psychotropic drug associated with a risk of qt-prolongation. qtcprolongation was defined as ms for males and ms for females. clinically relevant qtc-prolongation was defined as c ms. statistical analysis (r software) was done as appropriate. main outcome measures: prevalence of psychotropic drug induced qtc-changes and correlating factors. results: patients (mean age years, %female) were enrolled in the analysis. in patients, an ecg was performed. qtc + were prolonged in . %( / ) of females and . %( / ) of males. no clinical relevant prolongation (c ms) was registered. higher qtc intervals were measured in the geriatric population. . %( / ) of all measured qtc were situated between [ c qtc + b ms] in gpw versus . %( / ) in the other units. significant difference in qtc-changes was associated with sex (p = . ). there was no correlation assessed between qtc-prolongation and age, number of psychotropic drugs or a specific single psychotropic drug (p [ . ). conclusion: in this study qtc-prolongation due to psychotropic drugs is less common than previously described. ecg monitoring may be unnecessary in the follow up of patients without risk factors and could reduce hospital and community costs. however, considering the potential harm associated with tdp, qt-prolongation should be avoided. we recommend recording an ecg before the start of a qt-prolonging psychotropic drug in risk patients: patients with a chronic alcohol or drug addiction, a cardiac history, on concomitant therapy with at least two qt-prolonging psychotropic drugs, or geriatric patients ([ years). hp-pc : implementation of medication reconciliation aase m. raddum *, , anne-lise sagen major sykehusapotekene i midt-norge, sjukehusapoteket i Å lesund, avd. volda sjukehus, volda, sykehusapotekene i midt-norge, sjukehusapoteket i Å lesund, Å lesund, norway please specify your abstract type: descriptive abstract (for projects) background and objective: a correct and accurate medication list should accompany patients at transitions in care from one setting to another, including admission to hospital. complete information on drug use is a prerequisite for all hospital treatment, whereas incomplete information represents a potential patient safety risk. medication reconciliation is defined by the world health organization (who) as ''…the formal process in which health care professionals partner with patients to ensure accurate and complete medication information transfer at interfaces of care.'' the objective of this study was to investigate the quality of the medication history obtained for admitted patients. furthermore, measures to improve the quality of medication histories, i.e. implementation of medication reconciliation, were initiated. design: the study included patients admitted to the internal medicine ward. a comprehensive medication history was determined by performing a standardized patient interview and/or by using relevant sources of information. the primary endpoint was discrepancies between the medication history obtained on admission and the one determined prospectively by a clinical pharmacist. the clinical relevance of the discrepancies was not determined, but sorted according to six major categories, such as: medication not in chart, but patient reports using (omission) and medication in chart, but patient reports not using (commission). further on, in order to minimize the risk of discrepancies, it was focused on implementation of medication reconciliation. a campaign was initiated, where a clinical pharmacist held information meetings regarding the medication reconciliation procedure. for the next weeks, the degree of medication reconciliation was recorded. to spur the degree of medication reconciliation, each ward's weekly numbers were published and the ward with the highest degree of medication reconciliation won a prize. results: among the patients included, a total of discrepancies were revealed. in summary, patients had at least one discrepancy in their medication history, resulting in discrepancies in the medication lists of % of the included patients. at the start of the study, the level of medication reconciliation varied among the wards ( - %), while at the end of the study the levels were increased ( - %). conclusion: all the included wards improved their level of medication reconciliation during the study period. however, these new combinations have potential drug-drug and herb-drug interactions which can affect the safety and effectiveness of the treatment. in our clinical practice, the clinical pharmacist provides patient education about direct acting antiviral drugs (daa) based-regimens, promotes medication adherence and manages potential interactions with hcv treatment. the aim of the present study was to determine the prevalence of use of herbal products in the patients on hcv treatment, and to describe the potential hepatotoxicity of the herbal products and their interactions with hcv treatment. design: we included all adult patients on daa treatment for hcv who were dispensed drugs from / / to / / . we retrospectively recorded demographic data (age and gender), clinical data related to hcv infection (hcv genotype, fibrosis stage, daa regimen and treatment outcomes) and type of herbal products consumed. we then assessed the presence of herb-drug interactions and the potential hepatotoxicity of herbal products. results: we obtained data from patients on daa-based treatment for hcv. the prevalence of consumption of herbal products prior to starting the treatment was . % ( / ). the most consumed herbal products were (prevalence [ % among herbal products users): milk thistle, green tea, chamomile, valerian, pennyroyal, boldo and artichoke. we detected four herbal products with potential hepatotoxic effects according to the literature: milk thistle, green tea, pennyroyal and aloe vera. the prevalence of consumption of these hepatotoxic plants among herbal products consumers were, respectively: . , . , . and . %. we detected herb-drug interactions or potential for hepatotoxicity in out of patients who consumed herbal products. the management of these potential interactions consisted of stopping the herbal product before starting the hcv treatment. conclusion: the consumption of herbal products in our hcv patients was frequent. the management of potential interactions was conservative, recommending to stop herbal products. clinical pharmacists have an important role in the counselling, detection and management of potential herb-drug interactions and herbal products-related hepatotoxicity. poster discussion forum iii: hospital pharmacy and pharmaceutical care please specify your abstract type: research abstract background and objective: anaemia is a common comorbidity of chronic kidney disease. intravenous (iv) iron is used when oral iron formulation became insufficient or to reduce the use of erythropoiesis-stimulating agents (esas) in haemodialysis (hd) patients. the lack of generic group for iv iron sucrose (is) preparations leads to a controversial issue about their clinical effectiveness. in this study, we evaluated the effectiveness of original is compared to is similar (iss) in hd patients. setting and method: a retrospective monocentric observational cohort study was conducted from / / to / / , in a stable hd population to compare is and iss. the follow-up periods lasted weeks and were separated by a one-month wash-out period. original is and iss were administered respectively during the first (p ) and the second (p ) periods. the comparisons were performed using the paired student's t test or the paired wilcoxon test for continuous data and the fisher's exact test for categorical data. main outcome measures: the main endpoint was the difference in haemoglobin (hb) levels between p and p per patient. anaemia parameters (serum iron, serum ferritin, transferrin saturation ratio), the number of transfused patients, the doses of iv is and the doses of erythropoiesis stimulating agents (esas) were compared before and after the switch from is to iss, as secondary endpoints. results: a total of patients were included. there was no significant difference in mean hb value between p and p ( . ± . mmol/l versus . ± . mmol/l p = . ). anaemia parameters were significantly different between p and p (mean serum ferritin, serum transferrin and transferrin saturation ratio) with p \ . , except to the mean serum iron. the mean monthly dose of iv iron per patient and the mean dose of esas were respectively in p and in p : . ± . mg versus . ± . mg (p = . ) and . ± . ui/kg/week versus . ± . ui/kg/ week (p = . ). transfusions occurred less frequently in p than in p (p = . ). conclusion: this study showed that iss was as effective as original is regarding hb levels. however anaemia parameters appeared to be in favour of is; the mean dose of esas seemed to be higher after switching from is to iss. these outcomes should be further explored using prospective comparative clinical studies. please specify your abstract type: research abstract background and objective: the pharmacy residents are sometimes up to deliver chemotherapy when they are on night or week-end duty at the hospital. a dispensation's error (delivery of metoject Ò (methotrexate) for intrathecal (it) injection whereas it doesn't have the indication for this use), led us to test the pharmacy residents' knowledges about the it access in order to underscore the points to be improved. the final aim of this work is to secure the pharmaceutical care of the patient h a day, days a week. setting and method: an online and anonymous survey of questions was sent to the residents of our area. it was composed of three parts: specific general information, questions about the chemotherapy specifically (indication, maximum dose and volumes, molecules used), illustrated questions about real situation for the dispensation on duty. the answers were collected over a two weeks period. main outcome measures: we studied the rate of good answers in global and by respondent. results: twenty-five residents answered the survey, among them % never achieved any internship in a centralized unit of reconstitution of chemotherapy (urc). all the levels of internship are represented: st year (n = ), nd year (n = ), rd year (n = ) and th year (n = ). only % know where a medicine is injected intrathecally on the spinal column, % know on which level of the meninges. three residents think that a nurse can inject intrathecally. they also had to select the molecules which can be injected by this access: % answered vincristine, % vinblastine, % bortezomib; despite these three molecules are mortals if they are injected intrathecally. the majority know the indication of the it chemotherapy: prevention and treatment of cancers' meningeal localizations. sixty percent do not know that several molecules can be injected for the same patient in the same time. the maximum dose of methotrexate is known for half of the respondents, but only % for the cytarabine'one. only residents out of know that ml is the maximum volume allowed to be injected for an adult. six residents would have delivered metoject Ò mg in pre syringe filled if a doctor had asked for an it during a night. lastly, there are only two people who know that aracytine Ò (cytarabine) mg must be reconstituted with sodium chloride for it use and not with the provided solvent containing benzylic alcohol. the score of the residents having already done an internship in an urc is . / compared with . / for those who never did. the respective scores per year of internship are . ( st year), . ( nd year), . ( rd year) and . ( th year). conclusion: results and answers have been presented in a meeting and sent to the residents. we initially note many gaps in knowledge. the residents who already worked in an urc and the elders got better results. all the residents could be on duty at the hospital and all must be formed. a second session will be organized in a month to evaluate the formation's impact. it also has been presented to the assistants during an interactive lesson. this formation is essential to guarantee the dispensation of the adequate product and a secured medical care of the patient. please specify your abstract type: descriptive abstract (for projects) background and objective: patient adherence to prescribed medications is crucial for reaching metabolic control goal. to better understand the impact of polypharmacy on medication adherence, we undertook a detailed survey of medication use among patients with endocrinologic diseases. the aim of this study was to determine medication adherence in a cohort of patients with endocrinologic diseases and to test the hypothesis that adherence decreases with increased number of medicines prescribed. design: we conducted structured interviews to determine self-reported adherence of patient on a scale of (high) to (low observance) (srap- ) and a measurement using morisky medication adherence scales . demographic and medication information were collected from medical record. for statistical analysis, mann-whiney u-test for continuous variables, with chi square for categorical variables and kendall test for correlation were used. results: our cohort included patients, % were women and % were diabetic ( % suffering from type diabetes). the mean age was ± years, the average number of medication was . ± . . ( %) patients were not able to estimate their adherence. patients reported srap- scale with an average of . ± . , this estimation was significantly higher than mmas- with an average of . ± . (p \ . ). the proportion of adherence level were identical between srap- and mmas- with respectively and % of high, and % of medium and and % of low adherence. a significand correlation between srap- and mmas- scales (r = . , p \ . ) was found. however no correlation between adherence scale and number of treatment (r = . for mmas- and . for srap- scale) nor number of daily doses (r = . for mmas- and . for srap- scale). on the medications, % presented difficulties with observance. cardiovascular ( . %), diabetes ( . %) and psychiatric ( . %) treatment are the three most involved drug classes in nonadherence. conclusion: in this cohort, patients reported high medication adherence. we highlighted a correlation between srap- and mmas- scales. surprisingly, we didn't find correlation between adherence scales and number of treatment or dose by day. the next step of this work will be the identification of risk factor of nonadherence using logistic regression analysis. hp-pc : the office of access to healthcare: how to optimize secured access to treatments? claire chatron, adeline flatres, claudine hecquard, guillaume saint-lorant * , alexandra muzard pharmacy, chu caen, caen, france please specify your abstract type: research abstract background and objective: office of access to healthcare (oah) is an organization which offers a medical and social coverage to people who can't access to care and to medication because of the absence of social welfare, living conditions, or financial difficulties. medications are free dispensed thanks to retrocession activity in hospitals pharmacies. the aim of this study is to analyse this activity and to improve communication with patients and access to treatments by an adapted pharmaceutical interview. setting and method: this study includes all dispensations of year . in order to get medications in our hospital, a social worker and the patient come at the hospital's pharmacy. one people of retrocession team (four assistants, two externs, two residents and two pharmacists) dispenses necessary drugs to the patient according to hospital drug formulary and operating protocol. a switch or a special order can be purposed if the drug is not available. then, we give the patient a medication management plan (mmp) to explain him how to take his treatment at home. retrocession team filled a quiz about this activity and ways to improve it. main outcome measures: the main topics included in the quiz and evaluated were: dispensation organization, english talking, feeling during the interview and evaluation of the mmp. results: three hundreds and ten patients were admitted in oah . these patients come mainly from the eastern europe and do not speak french in most of cases. social workers, who can help for communication, are not always present because these patients can come during on-call duty. quiz results showed that weak points occurred during the interview: explanation of the mmp, languages barriers, mention '' if needed '' not understood by the patient. explanation of the order for a particular drug was difficult to operate too. mmp were only drafted in french which was not convenient for foreign people. however, modalities of dispensation were well understood by the retrocession team. following quiz results, mmp was translated into english by the retrocession team. mentions '' if needed '', '' number of maximum tablet a day: … '', ''your medication is in order, thank you for coming to look for this treatment back to the hospital on … '', '' … is the same as …, prescribed by your doctor on your medication list '' have been added and translated. results of the study and new mmp will be presented to the pharmaceutical team and to social workers in staff. an index card for ''communication in english with a patient'' has also been drafted. it contains sentences meadow drafted in english. conclusion: access quality health care service is important to achieve health equity and to increase the quality of everyone's life. these documents improve communication with patients and by the way their understanding about their treatment. the use and the impact of these documents on well understanding will be soon evaluated with social workers and patients. hp-pc : improve the medication in an associated to general hospital nursing home luisa alonso * , marta vidal iglesias, lucia gómez carrasco, guillermo goda, laura garcia, laura marin, ana hernandez, alvaro moreno please specify your abstract type: descriptive abstract (for projects) background and objective: in order to improve the medication reconciliation and to implement training programs for the medical team in an associated to general hospital nursing (asnh) home we measured the discrepancies between pharmacy registered treatments (prt) and medical prescriptions (mp), and we analysed potentially inappropriate prescriptions according to ''american geriatrics society beers criteria'' and ''stopp-start criteria. design: retrospective observational study that included patients admitted in the asnh. the ''consensus document on terminology and classification in medication reconciliation'' was considered for discrepancy classification. data collected: discrepancies between mp and prt. in patients from the original group of , we reviewed potentially severe drug interactions, potentially inappropriate mp and drug classes to avoid in older adults and medications to be used with caution in older adults (according to stopp-start and beers ) . all data were registered, measured and analysed in excel Ò . results: patients and a total of mp were reviewed. discrepancies ( . %) were found between the medical order and the prt, those discrepancies included errors of omission in prt ( . %), absence of discontinuation of medication ( . %), incorrect dosage ( . %). potentially moderate to severe interactions: the most frequent drug groups were proton pump inhibitors (ppis) ( . %), benzodiazepines (bzds) ( . %), oral hypoglycemiants ( . %), other groups with frequency over %, oral antihistaminic, statines, low molecular weight heparine (lmwh), laxatives, calcium salts and iron salts. stopp criteria were identified that affected to mp and the distribution was as follows: laxative combinations ( . %), long term ppis ( . %), cns depressants combinations ( . %), long half life bzds combinations ( . %), aspirin incorrect drug strength ( . %) and other groups with lower frequencies, nsaids and prokinetics. start criteria: being all of them by omission of the drug at the time of admission. beers criteria: prescriptions in the ''avoid prescription in adults'' group of which corresponded largely to concomitantly cns depressants and long term use of ppis in no risk patients. conclusion: the difficult working conditions, the excessive workload and the high staff turnover, where doctors have a patient ratio over / , make difficult to update treatments according to patient daily needs. a clear communication problem between the hospital pharmacy and the asnh prescribers exists due to lack of infrastructures, and it has been demonstrated with the high percentage of discrepancy, that implies an important logistic problem (not a safety problem) since the nurse team works directly with the original medical orders. the analysis of prescriptions showed the need for updating the medical knowledge. the high volume of stop and beers criteria and lack of doctors time made impossible the individual acting upon each patient, so short summaries of continuous training related to most frequent problems have been designed. please specify your abstract type: descriptive abstract (for projects) background and objective: our french university hospital is one of the most active centre for liver transplant ( transplants annually). various professionals are involved in the graft patient care and education. much information and education sessions are exempted before and after the transplant. the objective of this work was to realize a short movie for patients ( ) to get them ready for transplant ( ) to give the key messages to support their transplant ( ) to make family understanding the process and to promote the life behaviour changes. design: three members of the pharmaceutical team with nurses-led care coordination and a surgeon wrote the scenario. we requested two directors for days of shooting. we defined the key points for the patient and places to film, and fixed the duration ( - min). the scenario was validated by the chief of the liver transplant unit and nurses-led care coordination. after the days of film shooting, we selected sequences. results: the movie was a succession of six parts. ( ) the movie has been burned onto cds, put on flash-drives and will be uploaded on the internet. because of the international origin of our patients, the video will be subtitled at least in english. the video will be broadcast to hospitals which do not transplant patients and refer them to our hospital. since the medical team was involved in a collaborative project, the making of the video has permitted to strengthen the cohesion. indeed, this work would not succeed if everybody did not express himself. patients understood the interest to testify about their lived experience with the liver transplant, because they wished to have such information when they were waiting for the graft. conclusion: this movie is very useful for patients and families who are looking for information before and after liver transplant. it is a tool to get them into condition patients. this video presents the advantage of being personalized (local and caregivers that the patient will encounter are filmed). furthermore, it maintains a dynamic involvement of the pharmacy (already well established with clinical pharmacy, patient education and medication reconciliation) in the liver transplant unit. the making of the film has been an opportunity to bind the members of the team together, by valuing the work of everyone. the film could be screened if this abstract is selected for an oral communication. please specify your abstract type: descriptive abstract (for projects) background and objective: numerous procedures on medication management at oslo university hospital aim to minimize the risk of medication-related errors. error reports and observations show great variation in the use of these procedures, primarily due to difficulties in their implementation and maintenance. our aim was to assess the effect of a novel teaching strategy, the impala project, on doctors and nurses compliance with the medication management procedures. design: the project was carried out at general medicine wards at oslo university hospital for a period of weeks at each ward. assessment of medication-related error reports yielded the following areas of focus: (i) correct medication prescription, (ii) specification of doses for medications given on an ''as required''-basis, (iii) double control of medication dosing, (iv) correct and documented generic substitution. weekly presentations by pharmacist(s), lasting for a maximum of min, were given to doctors and nurses as part of daily ward routines. this was repeated over weeks. data on medicationmanagement procedure compliance were recorded before the start of the intervention, during and after each intervention period. the results were presented and made available to both leaders and employees throughout the project period both as an incentive to improvement and as a motivation factor for continued effort. results: there was a marked increase in medication-management procedure compliance among the nurses, especially after the second week of intervention. the most marked increase was shown for double control. increase in medication-management procedure compliance was also present among the doctors, but was less prominent. the data presented gave an extra motivational kick according to the participants. the leaders and the employees stated that the impala strategy was easy to follow and gave results without much organizational effort. conclusion: fifteen minutes presentations given by a pharmacist(s) as part of daily ward routines, combined with presentation of results demonstrated considerable improvement in medication-management procedure compliance. please specify your abstract type: research abstract background and objective: high unexpected serum vancomycin concentrations (svcs) were observed in patients without impaired renal function during the therapeutic drug monitoring (tdm) in our pharmacokinetic service. the aim of this study was to analyse the evolution of the svcs and its relationship with the markers of renal function. setting and method: retrospective study conducted at a university hospital with a follow-up period of months. only adult patients having at least two tdm were selected. trough svcs were measured by cmia (architect i- analyser, abbott Ò ) and fitted to a two-compartment model by using bayesian analysis (pks Ò , abbott). clinical and demographic data and daily dose, as well as timings of vancomycin administration and of blood sample collection were accurately recorded. spss Ò , version . was used to compare data from both tdm by student t-test (parametric data) and wilcoxon (nonparametric data). main outcome measures: concentration-to-dose ratio (cdr: trough concentration * /daily dose); glomerular filtration rates (gfr) estimated by cockroft-gault formula; measured and predicted svcs levels. results: adult patients were included (females: %); median age [ - ] years).the first and the second tdm were carried out after . [ . - . ] and [ . - . ] days from the beginning of the treatment, respectively. in the first tdm, no difference was found between the measured concentrations ( . ( . ) lg/ml) and those predicted ( . ( . ) mg/l. however, predictions were less accurate in the second tdm and predicted concentrations were significantly higher svcs ( . ( . ) mg/l vs. . ( . ) mg/ml, p \ . ). the median cdr in the second tdm was significantly higher than that calculated in the first one ( . [ . - . ] l - vs. . [ . - . ] l- ; p \ . ), indicating a lower clearance and a drug accumulation. however, no statistically significant differences in the glomerular filtration rates were found ( [ - ] ml/min vs. ml/min) in the first and second tdm, respectively. conclusion: although the markers of renal function did not change during the treatment, a decrease in vancomycin clearance was observed. the pharmacokinetic model does not accurately predict evolution of the svcs over the treatment. the introduction of covariates such as the length of treatment or the cumulative dose in the pharmacokinetic model could improve its predictive performance. please specify your abstract type: descriptive abstract (for projects) background and objective: genetic polymorphism or major physiological changes have to be considered in patient therapeutic management. clinical pharmacists have a role to evaluate and optimize the appropriateness and effectiveness of patient's medications. we report here the impact of the clinical pharmacist and his collaboration with the clinical pharmacologist in the therapeutic management of a patient suffered from anorexia nervosa, a psychiatric disorder leading to body composition change that may influence drug pharmacokinetics and efficacy. design: case report. results: the patient was a -year old woman hospitalized for chronic pulmonary aspergillosis previously treated by voriconazole, posaconazole and itraconazole. her medical history included anorexia nervosa since with a body mass index of . kg m - , pulmonary tuberculosis in with relapse in , and chronic pulmonary aspergillosis since . at admission, a treatment by oral voriconazole at mg/ h was introduced. the trough concentration of voriconazole at steady state was . mg/l (therapeutic range - mg/l) despite taking drug on empty stomach. although the voriconazole dosage increased in mg/ h, the trough concentration did not increase significantly ( . mg/l). we hypothesized anorexia led to a significant mucosal atrophy and accordingly, a significant decrease in intestinal absorption surface which is a major determinant of the level of drug absorbed. thus, a switch from oral to intravenous route was performed (voriconazole mg/ h). according to subtherapeutic voriconazole concentrations (trough concentration: . mg/l) despite the use of intravenous route, we decided to perform genotyping to look for mutations of cytochromes p a * , c * and c * , particularly implicated in voriconazole metabolism. the presence of an ultrarapid metabolizer genotype ( * allelic variant of the c isoenzyme) in our patient should lead to increase drug dosage from to %. finally, the patient was treated by intravenous voriconazole at mg/kg/ h (i.e., increase by %). the maximum concentration performed h after iv route initiation was at . mg/l, suggesting a better efficacy. conclusion: this case report highlights the potential complexity of therapeutic management in some patients given anatomical and functional changes or genetic polymorphism, which can affect drug efficacy. clinical pharmacists in collaboration with clinical pharmacologists have to be able to help physicians in this type of situations. please specify your abstract type: research abstract background and objective: posaconazole (pcz) is widely used for invasive fungal infections as prophylactic, pre-emptive or curative therapy in lung transplantation. recently, a new formulation of pcz has been available in enteric-coated tablets. this new formulation improves pcz bioavailability, as compared to the oral suspension, which leads to increase pcz plasma trough concentrations (c min ) in haematological patients. no data related to pcz exposure and its effects on tacrolimus (tac), an immunosuppressant with narrow therapeutic index widely used, exists in lung transplantation. we aimed to assess the consequences of the treatment by pcz entericcoated tablets on pcz and tac exposure in lung transplant patients. setting and method: a single-centre retrospective study was conducted among lung transplant patients receiving tac and either enteric-coated pcz or both galenic forms. main outcome measures: pcz and tac exposure were estimated by the measurement of c min . to overcome the influence of dose (d), c min were adjusted on dose (c min /d) for both pcz and tac. a spearman test (nonparametric distribution) was performed to assess the correlation between pcz c min /d and tac c min /d. results: eighteen lung transplant patients (median age [q ; q ] = . [ . ; . ] years; % female) were included between june and march . eight patients received only pcz entericcoated tablets. pcz enteric-coated tablets were associated to an increase in pcz c min /d as compared to oral suspension ( . ± . l - vs. . ± . l - , p \ . ). overall, pcz therapy initiation led to an increase in tac c min /d ( . ± . l - before initiation vs. . ± . l - after initiation, p = . ). tac c min /d was significantly higher with pcz enteric-coated tablets, as compared to pcz oral suspension ( . ± . l - vs. . ± . l - , p \ . ). a weak correlation was observed between pcz c min /d and tac c min /d, independently to pcz galenic form (r = . , p = . with pcz enteric-coated tablets and r = . , p = . with pcz oral suspension). conclusion: this pilot study in lung transplantation confirms the better bioavailability of pcz enteric-coated tablets as compared to oral suspension. our results show a more important increase in tac exposure with pcz enteric-coated tablets compared to pcz oral suspension, suggesting a concentration-dependent cyp a inhibitor effect of pcz. these findings are of interest in clinical practice to monitor transplant patients treated by the new formulation of pcz. further analyses, including the consideration of confounders, will be conducted. please specify your abstract type: descriptive abstract (for projects) background and objective: within months, two patients receiving apixaban developed agranulocytosis. based on temporal and clinical plausibility as well as published literature, the objective was to determine the causal relationship between agranulocytosis and apixaban. design: description of two agranulocytosis cases reported in our hospital. results: first case is an years old male, admitted to the neurology unit (d ) for ischemic stroke. at admission, blood count showed no abnormalities. four days after admission, treatment administered consisted in: dextrose % infusion iv, sodic heparin iv, acetaminophen, atorvastatin, metoprolol. neutrophils count was normal ( . g/l). heparin was stopped at d and replaced with apixaban according to following dose regimen . mg twice a day. at d , patient presented with hyperleukocytosis (neutrophils count g/l) and high crp ( mg/l). thus, a cytobacteriological urine test was performed. at d , patient presented with hypothermia followed by hyperthermia related to acute sepsis. blood count showed agranulocytosis (neutrophils count . g/l). broad spectrum antibiotherapy was started (ceftriaxone and gentamycin). despite treatment, death of patient occurred at d . the suspected cause of death was septic shock added to severe febrile neutropenia. following haemocultures confirmed sepsis (e. coli) possibly originating from urinary tract infection. second patient is a years old male, admitted to the cardiology unit (d ) for bronchopneumopathy associated with tachycardia and atrial fibrillation. a treatment with heparin was immediately started in association with patient usual treatment (bisoprolol, valsartan, rosuvastatin, hydrochlorothiazide and manidipine). in addition, broad spectrum iv antibiotherapy was started with ceftriaxone and spiramycine followed at d by an oral treatment with cefixime and spiramycine until d . heparin was replaced by apixaban at d ( . mg twice daily). antihypertensive treatment was adapted throughout patient's stay. patient presented neutropenia at d (neutrophils count . g/l), followed by agranulocytosis at d (neutrophils count . g/l) when it was decided to replace treatment with apixaban by fluindione. the following day, neutrophils count was about . g/l and patient received filgrastim. a myelogram showed a possible peripheral neutropenia. in the absence of other confounding factors (hiv, hbv, hcv, cmv), an iatrogenic agranulocytosis related to apixaban was suspected. conclusion: causal association with heparin is unlikely as neutropenia is not an adverse drug reaction known included in the smpc of this drug having a well-established safety profile. since the two patients were taking their usual treatment for a significant period of time, a causal relationship is deemed unlikely. temporal and clinical plausibility seem to indicate a possible relationship between agranulocytosis and apixaban. as this medicine has been recently approved, this might explain why no case has been reported in the literature and the absence of agranulocytosis as an adverse drug reaction of apixaban. please specify your abstract type: research abstract background and objective: taste is tightly connected to children's acceptability of medicines. two ways to overcome lack of acceptability are to administer solid formulations which are easier to taste mask and change to better tasting medicines. dicloxacillin is an antibiotic known for its unpalatability, and taste studies suggest that this might jeopardize its adherence. the aim of this study was to explore if prescription data can be used to estimate acceptability of antibiotics among children on a population level using dicloxacillin as an example drug. the research questions were: when comparing dicloxacillin with other antibiotics commonly used in children, ( ) is there a difference in the age of conversion from liquid to solid formulation and ( ) is there a difference in re-prescription rates on day and after the initial prescription? setting and method: we included all initial prescriptions of oral dicloxacillin, phenoxymethylpenicillin, amoxicillin and erythromycin for children - years registered in the norwegian prescription database (norpd) - due to dicloxacillin mixture being discontinued from the norwegian market in . the age of conversion was defined as the age where half of the children were prescribed liquids and the other half prescribed solid formulations. re-prescription rates were defined as re-prescriptions of a different antibiotic or formulation on day and after the initial prescription, divided by the total number of prescriptions. main outcome measures: age of conversion and re-prescription rates of dicloxacillin compared with other common antibiotics. results: the age of conversion for dicloxacillin was . years, compared to years for other common antibiotics. the average represcription rate for dicloxacillin was . % for children - years and . % % for children - years. the highest re-prescription rate of . % was found in -year olds. corresponding numbers were . , . and . % for common antibiotics. conclusion: the lower age of conversion from liquid to solid formulation and higher re-prescription rate of dicloxacillin mixture compared to common antibiotics indicates that prescription data can be used to identify antibiotics with low acceptability for children - years. further studies are needed to investigate if this also holds true for other antibiotics. please specify your abstract type: research abstract background and objective: attention deficit/hyperactivity disorder (adhd) or hyperkinetic disorders (hkf) is among the most common mental disorders in children, and may persist through adolescence into adulthood. pharmacotherapy used for treating the disorders also has potential for misuse/abuse. the aim was to describe the prevalence and magnitude of use of stimulant drugs and atomoxetine, and compare consumption in the nordic countries. setting and method: a descriptive pharmacoepidemiological study from the * million inhabitants of the five nordic countries in the period - . data were collected from national prescription registers, public drug reports and by correspondence with public health institutions. population data were obtained from official statistical databases or by correspondence with public health institutions. main outcome measures: trend over time, comparison between countries, type of pharmaceutical, gender, age, comparability of data. results: the annual consumption has been increasing from to , both in volume and prevalence of use. denmark had the largest increase in volume, from . to . ddd/ inhabitants/day. sweden had the highest increase in prevalence of use over the period, from . to . users/ inhabitants. iceland had the largest consumption of adhd medications in , . ddd/ inhabitants/day. prevalence data was not available for iceland but sweden was highest in prevalence of use among the other countries in : . users/ inhabitants. males aged - years had the largest volume and prevalence of use in , but females' consumption had been increasing faster both in terms of numbers of users (* . faster) and in volume (* faster) than men's consumption. conclusion: variation in consumption is considerable and cannot be explained by diagnostic and prescription guidelines, as these are similar in the five countries. consumption has been increasing fast in the period in all the countries, and faster for women than for men, although men still consume larger volumes than women, and are more frequent users. please specify your abstract type: research abstract background and objective: in and , regulatory bodies in usa (fda) and europe (ema), issued warnings on use of metoclopramide due to an increased risk for serious adverse drug reactions (adr), especially neurological adrs. ema recommended that metoclopramide only should be prescribed for up to days while fda concluded that treatment longer than weeks should be avoided. metoclopramide is commonly used to treat nausea and vomiting in pregnancy (nvp) and deficient breast milk production ( days course). ema did not make any recommendations concerning use during pregnancy and lactation. the objective of this study is to assess the disproportionality of reporting of adr from metoclopramide, with special emphasis on neurological adrs and women in reproductive age. setting and method: data from whos global adr database vigibase Ò for the time period november to may was used. the measure of disproportionality of reporting calculated was the proportional reporting ratio (prr), and % confidence intervals (ci). analyses were performed according to gender and age. time-toonset of adr was calculated. main outcome measures: proportional reporting ratio (prr) results: vigibase contains over million adr reports. metoclopramide is a suspected/interacting drug in of the reports, most common ( %) are neurological adrs. the majority ( %) of the metoclopramide adrs occurred within the first days of use. a total of % of the reports was received the last years ( ) ( ) ( ) ( ) ( ) . the reporting of neurological adrs was higher for metoclopramide than other medications in vigibase. women in reproductive age ( - years) reported higher proportion of neurological adrs (prr = . , % ci . - . , n = ) than women + years (prr = . , % ci . - . , n = ) but a similar proportion as men - years (prr = . , % ci . - . , n = ). conclusion: there is a . to three fold higher proportion of all reports regarding neurological adrs for metoclopramide than for other drugs. patients initiating treatment with metoclopramide should be informed about risks of adrs and that most adrs occur within days, and instructed to contact health care personnel and stop treatment if adrs occur. please specify your abstract type: descriptive abstract (for projects) background and objective: self-induced drug intoxications (sidi) are one of the most frequent reasons of hospitalization in emergency service ( %) with around - / inhabitants and represent around % of admissions in intensive care unit (icu). it is the most frequently used method of suicide attempts (sa) and the leading cause of hospitalization for young people under . the main objective of our study was to analyse, stratify and pharmaceutically map the different sidi identified in our icu. design: this is a prospective study over months, including all icu patients following sidi from june to january . we have collected psychiatric history and previous sa by sidi, usual treatment, state of consciousness, incriminating drugs, drug classes stratified according to the clinical severity score igsii, evolution, transfer in a specialized centre and average cost of stay. results: ninety-two cases were reported, representing % of icu admissions. the average hospital stay was days for an average cost of . €. this amount is low compared with the average cost of all stays gone through the icu for the period ( , €). ninety percent of patients had a psychiatric history and % a previous sa. the usual treatment was involved in % of sidi. half of the patients arrived conscious with an average of severity score igsii of / , being the highest found for a patient who had swallowed simultaneously pregabalin and nitrazepam. clinical severity of these patients is less than that found on average for all patients in the icu in this period ( / ). eighty-seven percent had a favorable evolution. only one death was observed after ingestion of propranolol. fifty-six and a half percent of patients were then hospitalized in a specialized centre. the great family of psychotropic is the most frequent with benzodiazepines %, neuroleptics %, antidepressants . % and antiepileptic . %. the main drugs involved are oxazepam %, alprazolam %, cyamemazine %, bromazepam % and quetiapine %. antihypertensives then arrive and represent % of sidi. the stratification of severity scores does not appear to show significant differences between drug classes, nor between mono or polydrug ingestions. conclusion: sa by drug ingestion are very common and are often linked to risky behaviours. for these epidemiological and economic findings, it is necessary to continue and develop prevention strategies avoiding the appearance of intoxication (primary), limiting the consequences (secondary), and reducing the risk of recurrence (tertiary). please specify your abstract type: research abstract background and objective: interpretation of quality of life scores to render them meaningful to aid clinical decision-making is an ongoing challenge. interventions often result in statistically significant quality of life (qol) improvement, but may not reach the threshold of clinical importance. the minimal clinically important difference (mcid) is the minimal score change of relevance clinically. the aim of this systematic review was to assess the impact on quality of life of topical, systemic and biologic treatments for psoriasis in randomised controlled trials (rcts). setting and method: prisma guidelines were followed. all available articles describing rcts of therapies for psoriasis that included qol measurements published up to november were identified. six databases were examined with search terms. abstracts of articles were reviewed independently by two assessors: a third adjudicator resolved any opinion differences. risk of bias was assessed using the jadad scale. main outcome measures: reporting of the use of qol endpoints and impact of interventions in psoriasis. results: of screened article abstracts, articles were selected for detailed review: trials met the eligibility criteria, describing research on a total of , patients. reports of psoriasis interventions that fulfilled inclusion criteria have gradually increased over time : - = , - = , and - = ( ) to evaluate the relationship between the use of different therapeutic agents and the severity of osa, and ( ) to determine the effects of commonly used medications on continuous positive airway pressure (cpap). setting and method: patient medical records (n = ) of patients, that underwent sleep studies between the years and were collected over an eight-month period from the sleep laboratory department at mater dei hospital using a random sampling technique. data collected included body mass index, gender, age, epworth sleepiness score (ess), drug history, apnoea hypopnoea index (ahi) and cpap therapy prescription. likelihood ratio chi square test, paired samples t-test and multinomial logistic regression were the statistical tests used for data analysis. main outcome measures: assessment of the drug history in response to osa control using the ess and ahi scores. results: one hundred and seventy ( . %) patients of the patients ( males, females) were diagnosed with osa. forty-five ( . %), ( . %) and ( . %) patients suffered from mild, moderate and severe osa respectively. patients had a mean age of years. angiotensin ii receptor antagonists (arbs) (p-value = . ), sulphonylureas (p-value = . ), insulin therapy (p-value = . ) and non-benzodiazepine sedating agents (p-value = . ) were found to be associated with the presence of osa. a decline in the use of the arbs (p-value = . ), angiotensin converting enzyme inhibitors (p-value = . ) and non-benzodiazepine hypnotics (pvalue = . ) was observed over the study year period. reduction in the cpap therapy benefit was detected with the use of histamine (h ) antagonists (p-value = . ), b-adrenergic blocking agents (pvalue = . ) and antiplatelets (p-value = . ). conclusion: it is confirmed that hypertension and diabetes mellitus type ii are the main co-morbidities associated with the presence of osa. reduction in the use of certain therapeutic agents is observed secondary to cpap therapy use. patients using specific drugs have been identified as being at risk of a reduced cpap therapy benefit. please specify your abstract type: research abstract background and objective: people are using increasingly more common of social networks such as facebook, twitter and youtube for different purposes. many people are using these networks with the aim of getting information and knowledge sharing. there are many groups that pharmacist is a member in social networks at turkey. the largest of these groups has , members. pharmacists are shared common problems, information and experiences in these groups. but the accuracy of the information shared on social networks are not always conclusive. the study aim to evaluate the impact of social network information sharing in the knowledge and attitude of pharmacists. setting and method: clinical pharmacy group has been created to share information on facebook. pharmacist joined this clinical pharmacy group. the group was fed by information which include new drugs, fda alerts, adverse event and case report and also drug related problems during the months. pharmacists were assigned in two major groups, group a active pharmacist who becomes a member of our clinical pharmacy group, share and discuss information through the network and group b who is not a member. a knowledge measurement survey (ams) was given to both of them. main outcome measures: acknowledge measurement survey (ams) was developed and the difference in the score was used to evaluate the difference between the two study groups. results: pharmacists participated in the study, . % of the participants were a member of our facebook group and . % of participants were not. . % of the participants have doctoral degree or student, . % have master degree or student, % have bachelor degree from year-pharmacy faculty, . % have bachelor degree from year-pharmacy faculty. the education level distribution between the two groups was not statistically significant. while . % of the ams questions were answered correctly in the member group only . % were answered correctly in the non-member group. conclusion: the study emphasizes the importance of social network in providing the accurate and fastest information for the daily use of the pharmacists, there is a significant difference in knowledge between the pharmacist who join, share and discuss information on the social network and the one who do not join. cp-ce : impacts of a community pharmacy practice experiences on student professionalism yunn-fang ho , , hung-wei lin *, , fang-ju lin , , sheng-ping chang , yen-ming huang graduate institute of clinical pharmacy, school of pharmacy, college of medicine, national taiwan university, taipei, taiwan, r.o.c please specify your abstract type: research abstract background and objective: professionalism is valued globally and pharmacy schools are expected to nurture competent practitioners to better serve the public with humanity attitudes and behaviours. the study aims: ( ) to understand possible differences in professionalism between pharmacy students and potential community pharmacist preceptors, and ( ) to evaluate student changes in professionalism upon completing the community pharmacy practice experiences (cppe) at the end of the third (p ) year. setting and method: a modified chisholm's pharmacy professionalism instrument ( -item, -point likert scale) was administered to p students, pre-cppe and hopefully post-cppe in september, and community pharmacist practitioners who participated in a two-day preceptor training workshop. participants also provided their significance ratings toward ten traits, namely altruism, accountability, excellence, duty, honor and integrity, respect for others, communication, ethics, humanism, and teamwork. main outcome measures: differences or changes in chisholm professionalism scores. results: thirty-two students and fifty pharmacists participated in the survey. honor and integrity ( . ± . ) and communication ( . ± . ) were recognized by students ( . %) and pharmacists ( . %), respectively, as the most significant trait. humanism was rated the lowest in both groups (students, . ± . ; pharmacists, . ± . ). the -item professionalism scores ranged from . ± . (''i do not expect anything in return when i help someone.'') to . ± . (''i am respectful to individuals who have different backgrounds than mine.'') in the student group; whereas . ± . (''i do not expect anything in return when i help someone.'') to . ± . (''it is wrong to cheat to achieve higher rewards (i.e., grades, money).'') in the pharmacist group. in general, pharmacists' professionalism scores were higher and, in certain items, statistically significant differences were achieved. conclusion: professionalism might grow with professional competency and practice experiences as demonstrated by potential pharmacist preceptors. upon completion of cppe, students could probably exhibit gains in professionalism. more investigations are still underway. please specify your abstract type: descriptive abstract (for projects) background and objective: in france, a significant consumption of benzodiazepines (bzd) is observed in prisons. they are widely used during incarceration to treat or prevent anxiety and insomnia. furthermore, it is known that, an important traffic exists with these drugs because of the releasing properties of bzd in case of misuse. based on these observations, the pharmacist has set up a plan to improve the use of bzd in prison. the purpose of the study was to evaluate the impact of these measures after year of implementation. design: in january , we shared with physicians in a meeting to explain our plan for a better use of bzd and to set up new rules of prescription in prison: • regularly reducing the dose to limit drug tolerance • promoting the use of long half-life molecules which allow reducing addiction and misuse • advising sedatives anti-histaminics to treat insomnia • providing information to patients about addictives risks of bzd on the tv channel please specify your abstract type: descriptive abstract (for projects) background and objective: some drug combinations (described in thesaurus of national agency of drug) are contraindicated because they appear to increase the risk of torsade de pointes. the aim of this work is to standardize our pharmacists' intervention and to propose guidelines for doctors and pharmacists, depending on the situation and drugs, to limit these combinations and to reduce this risk at our hospital centre ( beds). design: a prospective survey was realized over a period of months to identify the drug combinations prescribed in medical prescription software, from the national drug agency thesaurus, that might be inducing torsade de pointes. a multidisciplinary staff was then constituted composed of a cardiologist, a geriatrician, a paediatrician, an anaesthesiologist, a psychiatrist and pharmacists to identify the different situations and to establish guidelines. results: from the survey drug combinations were found to be contraindicated due to increased risk of inducing torsade de pointes on a list interventions realized by pharmacists. the work group identified three drugs with a therapeutic alternative: hydroxyzine, domperidone, escitalopram, the other drugs can't be switched because they are vital or have no alternative. the work group decided to maintain hydroxyzine but only on premedication and child anxiety, to eject domperidone from our therapeutic index and substitute it with metoclopramide or metopimazine, to not initialize escitalopram but to keep it if the patient has no have others risk factors associated or no contraindication. if the patient has a contraindication with a risk factor the doctor could prescribe other ssri. in addition, pharmacists alert doctors about the risk of torsade de pointes on medical prescription software if some contraindications are identified. conclusion: the contraindications identified must not be underestimated. this work allows identification of torsadogenic drugs commonly prescribed and provides guidance for doctors and pharmacists regarding drug combinations. the collective decision will be disseminated to sensitize all the doctors in the establishment. some treatments could not be substituted despite the contraindication; these must be retained but with clinical monitoring. conclusion: a substantial proportion of medication waste in the community pharmacy could have been prevented. unused medicines in the community pharmacy are generally of low economic value, making it unlikely that the costs that pharmacies will make with the redispensing of unused medicines will be covered. therefore, other actions to decrease medication waste in the community pharmacy, such as preventing that too much medicines are dispensed, should be considered. please specify your abstract type: research abstract background and objective: flaws in usage technique for inhalationmedicines is common, as much as half of the users may need some correction measures, to get the active substances down to the lungs and provide the intended effect. inadequate compliance, especially for regular-use preventive medications, is common. good guidance in pharmacies enhances correct use of medicines. the new norwegian pharmaceuticals policy (legemiddelmeldingen) from opened up for paid cognitive services, leading to the first such service being implemented in march . the service can contribute to a more correct use of the medicines and, as a consequence, lead to better control of the symptoms for patients with asthma or copd. our objective was to map the variation in pharmacies' handling of an inquiry regarding lack of effect of an inhalation-medicine. the study was done prior to the implementation of the standardized service ''inhalation-guidance'' in norwegian pharmacies. setting and method: simulated patient (mystery shopper) visits in pharmacies in oslo, akershus and buskerud in november/december . the mystery shopper expressed just having started to use an inhaler because of her asthma, but not experiencing effect. structured data collection sheets were used to register the handling immediately after the visit. main outcome measures: scoring of the quality and contents of the information based on the products' patient information leaflets. results: the issue of inhalation-technique was mentioned in of the pharmacies, whereof asked the ''patient'' to show their inhalationtechnique, in order to correct and advice and used an inhaler or demo-inhaler as an aid in the guidance. going through the instructions or watching a video-demonstration with the simulated patient also occurred, or referring the patient to read the instructions and/or watch the video-demonstration on his own. half of the pharmacies discussed the difference between use for preventive treatment of asthma and inhaler that is being used for treatment of attacks. sixty-five pharmacies gave no information about the importance of regular use of the preventive treatment. conclusion: there was considerable variation in how the pharmacies guided, which indicates a potential for improvement. the new guidance-service, implemented in norwegian pharmacies in march , will contribute to better guidance. please specify your abstract type: research abstract background and objective: in portugal, tobacco addiction was responsible for over , deaths in ( % of the total deaths). the community pharmacist's contribution to control this public health problem is insufficiently documented. the aim of this study is to assess the contribution of the community pharmacist for smoking cessation. setting and method: a retrospective and longitudinal study of a convenience sample of patients integrating quit tobacco consultations, as part of a pharmaceutical care programme implemented by an outsourced pharmacist was performed at several community pharmacies. the smokers, aged or over, were invited to join the programme. patients signed an informed consent and were submitted to a comprehensive approach by face-to-face consultations and telephone contacts. richmond and fagerström tests were used to evaluate motivation and nicotine dependence, respectively. the therapeutic plan (pharmacotherapy and behavioural counselling) was personalised to each smoker. the quit rates were evaluated by patient selfreport and confirmed by carbon monoxide measurements. the continuous variables are expressed as mean ± standard error of the mean. main outcome measures: quit rates at , , and months. results: between january and june , smokers joined the programme, dropouts ( . %). the remaining smokers, ( . %) were male, with mean age of . ± . years. on average, each smoker consumed . ± . cigarettes per day. the mean age of initial tobacco use was . ± . years with . ± . years of consumption. about % reported moderate or high motivation and % medium or high dependence. a total of consultations were held and, on average, each patient received . ± . interventions. all smokers received non-pharmacological interventions (e.g. motivational approach) and ( . %) also accepted pharmacological interventions, usually nicotine replacement products. the quit day was achieved by patients ( . %). a month after quit date, patients were abstinent ( . %). the number reduced to after months ( . %), to after months ( . %) and to after year ( . %). these data upgrade and are consistent with our previously published results ( ). the smoking cessation consultation in the scope of a pharmaceutical care programme in community pharmacy seems to effectively contribute to the reduction of tobacco addiction in portugal. cp-pc : patient counselling at dispensing of oral anticancer drugs in european countries from the pharmacists' perspective andreja eberl * , on behalf of epic working group pharmacy, institute of oncology ljubljana, ljubljana, slovenia please specify your abstract type: research abstract background and objective: the number of oral anticancer drugs (oads) available on the market grows constantly. consequently the number of patients, which have to manage the complex treatment with oads at home is increasing. the pharmacists present an important member of healthcare team, since they are dispensing oads to the patients, which need a high quality information at that crucial moment. therefore, our aim was to evaluate pharmacists perceived confidence and needs for specific continuing education in connection to oads dispensing in european countries. setting and method: we used an electronic mailing approach and a standardized online survey to ask practicing pharmacists in european countries about their experience with dispensing of oads. main outcome measures: frequency of patient counselling and fields of counselling, assessment of knowledge and skills. results: the frequency of patient counselling varied widely in participating countries between ''never'' and ''more than %'' at initial fill of an oad. at following refills the frequency of counselling was generally even lower. counselling mostly encompassed directions of use, the proper use of antiemetics and side effects. however many pharmacists stated, that they do not feel comfortable counselling patients of oads ( %) and even more acknowledged that they were uncomfortable with managing patients' side effects ( %). on the other hand only % of pharmacists believed, that they have received adequate knowledge of oads through undergraduate program, continuing education (ce) events and professional practice. many of pharmacists ( %) have not attended any of ce events related to oncology in last years. pharmacists' responses differed little between the countries. conclusion: the proportion of pharmacists who regularly counsel their patients on oads is insufficient in view of importance of the patients' needs to manage their therapy at home. however the pharmacists seems to be aware of their knowledge deficits and educational needs. the field of oads needs better coverage in under-and postgraduate education. the number of ces has to be increased in order to improve the knowledge and skills in the areas of oads counselling. please specify your abstract type: research abstract background and objective: treatment guidelines for diabetes recommend that patients are well-informed about their disease, treatments and treatment goals, e.g. glycosylated haemoglobin (hba c). the objective was to describe diabetes patients' self-monitoring of blood glucose (smbg) and potential need of guidance. please specify your abstract type: descriptive abstract (for projects) background and objective: in , the international pharmaceutical federation collected data of remuneration models for community and hospital pharmacy and identified large variations between remuneration models and highlighted that the focus is largely on products and not on cognitive services. the aim of the study is to map the remuneration models of different pharmacist-led cognitive services in primary care across europe, with a special interest on medication reviews and to update a prior survey by bulajeva (bulajeva a et al. medication review practices in european countries. res social adm pharm ; : - .). the definition of terms is pivotal for such a european survey to avoid results based on pseudoconceptions. hereafter we present the development of the survey and we will present first results from pilot tests. design: pharmacist-led cognitive services were selected based on a previous study by our group and by searching the literature, official government websites, the pcne wiki and arising links. the definitions of the terms of these services were based on searches in the mesh browser, medline and google scholar. additionally, a search in grey literature and in the internet was conducted to find appropriate foundation for the formulation of the definitions. the questionnaire will consist, of a first part about the remuneration of the pharmacist-led cognitive services. the focus is on country-specific differences in remuneration and the different levels of supply across europe. the second part of the survey is about the different types of medication review services with a focus on e.g. the implementation level, addressed issues, eligibility criteria. this survey will have a cross-sectional study design with an online questionnaire specific for invited participants across europe. to achieve the best quality of answers we will send this survey to at least two researchers with references in pharmacy practice, in each european country (purposive sample). the answers from each country will be checked for discrepancies and these potential discrepancies will be solved by a discussion with the responders. results: by the end of the pre-pilot phase, different pharmacist-led cognitive services were identified and the correlating definitions of the terms were developed. conclusion: at the time of submission the pre-pilot phase has been finished and the pilot will start july . please specify your abstract type: research abstract background and objective: medication adherence is one of the key aspects in assuring optimal health outcomes in majority of chronic diseases. the aim of the study was to evaluate copd patients' medication adherence in slovenia and its association with health outcomes. setting and method: patients were recruited by community pharmacists at the time of dispensing medication for copd. medication adherence was evaluated by using morisky medication adherence scale (mmas- ). patients who scored b points, . - . points and points were regarded to have poor, moderate and good adherence, respectively. quality of life was evaluated by saint george's respiratory questionnaire (sgrq) and the impact of disease by copd assessment test (cat). the study was conducted in september and february . the association between potential predictors and copd impact or quality of life was estimated using multiple linear regression in ibm spss statistics version . main outcome measures: medication adherence rate (mmas- ), quality of life (sgrq total score) and impact of disease (cat score). results: of patients, majority were men ( %) with mean age years. in average, patients were prescribed . medicines for copd and . medicines for other diseases. good, moderate and low adherence to copd medication regimens was found in . , . and . % of patients, respectively. mean cat scores and sgrq scores were . (range - ) and . (range - ), respectively. thirtyeight percent of patients experienced an exacerbation in the past year. linear regression showed no statistically significant association between medication adherence and quality of life or copd impact on patient. factors that statistically significantly predicted patients' quality of life were exacerbation in the past year, education level and number of concomitant medicines for other diseases. the latter was found to be the only factor associated with copd impact. conclusion: the study showed half of the copd patients to be optimally adherent to their treatment and only a small proportion of patients not taking their medicines regularly. due to the nature of the disease medication adherence does not seem to play the most important role in assuring optimal health outcomes in copd patients. please specify your abstract type: descriptive abstract (for projects) background and objective: intermediate care units (imcu) are designed to serve patients in need of more advanced medical care than the ordinary nursing home units can provide. the aim of this study was to see; ( ) how medication information follows patients in and out of icmu and nursing home short-termcare units (stcu) ( ) the type and amount of drug related problems (drp), focusing inappropriate drugs, and ( ) if there are differences between the icmu and stcu in drug use and drps. design: patients c years old admitted and submitted at the imcu or stcu in the study period ( weeks) were included. transfer of medication information were evaluated and given a score. the clinical pharmacist provided medication reconciliation upon admission, medication review and monitoring, and presented identified drps and a suggestion for solving the problem, to the multidisciplinary team. inappropriate drugs, identified by screening tools (stopp/norgep), and systematic medication reviews, were recorded. results: patients from imcu and five from stcu were included. a hospital discharge summary including medical history followed mostly all patients. the score of the medication history was . points out of . by submission from either imcu or stcu, the score was . . systematic drug review identified . drp in the imcu and . in the stcu. imcu patients used . drugs, stcu patients . in the icu, % of the identified drps was inappropriate drugs, none in the stcu. the clinical pharmacist in the multidisciplinary team presented % of the identified drps. the doctors agreed in % of the suggestions for solution, and started immediate changes in %. conclusion: a hospital discharge summary followed the patients, but the medical history part needs improvement. although few patients, the results suggest that imcu patients had more complicated medication and more inappropriate drugs than stcu patients did. clinical pharmacist in a multidisciplinary team provides useful contribution to identify, solve and prevent clinical relevant drps, including inappropriate drugs. please specify your abstract type: research abstract background and objective: lack of clinical effects of medication review on health-related quality of life of older people may be due to insufficient focus on health-related complaints. goal attainment scales (gas) are an instrument to formulate specific health-related goals. the objective of this early process-evaluation of the dreamer-study (drug use reconsidered in the elderly using goal attainment scales during medication review) is to investigate if pharmacists are able to formulate gas during a medication review of older people with polypharmacy. setting and method: older patients aged years or older using or more medicines are included in this study. half of the patients were randomized into the intervention group, where they received a medication review. during the patient interview, the pharmacist formulated gas in concordance with the patient. recommendations were made to reach these goals in collaboration with the gp. main outcome measures: number of performed medication reviews, total number of formulated gas and the three most frequent types of gas. results: until now patients have been included in the drea-mer study ( % of the target). half of them ( ) were randomized into the intervention group. by now ( %) of these patients have received a patient interview. goal attainment scales were formulated yet. the number of gas ranged from to per patient. the four most frequent gas were: polypharmacy-reducing the number of medicines ( ), reducing pain ( ), increasing mobility ( ), reducing fatigue ( ). conclusion: gas seem to be a feasible approach during medication review that increased focus on patient's needs and health-related complaints. cp-pc : oral transmucosal fentanyl citrate: a regional survey of dispensing practices in community pharmacy please specify your abstract type: descriptive abstract (for projects) background and objective: oral transmucosal fentanyl citrate (otfc) is an opioid analgesic indicated for management of breakthrough cancer pain in patients with malignancies who are already receiving and who are tolerant to opioid therapy for their underlying persistent cancer pain. otfc are usually use off-label prescription, especially in noncancer patients or patients without opioid maintenance treatment. this practice can expose to iatrogenic risks, lack of efficacy, abuse and addiction. the observatory of drugs, medical devices and therapeutic innovation of upper normandy, conducted a study to assess the knowledge of pharmacists on these medications and assess dispensing practices (pharmaceutical analysis and advice to patients). design: between june and september , two quizzes were sent to the pharmacists and pharmacies in upper normandy: one included questions of knowledge and general practice, the other assess dispensing practices of otfc prescriptions received at the counter, regarding indication, dosage and associated opioid medication. results: of the pharmacists who participate in the survey, % know the all of the oftc specialties, % of them confuse transdermal and transmucosal fentanyl specialties. indication, dosage, titration methods and the main interest of oftc are known by , , and % of them. only % have dispensed oftc more than times over the past months, % never have. they already have dispensed oftc in noncancer patients ( %) or without opioid maintenance treatment ( %). they consider not know enough about these drugs to be able to provide the necessary advice to patient ( %) and would like specific training on oftc ( %). of the analyzed prescriptions, only % are consistent with the marketing authorization: otfc medicines are prescribing in noncancer patient ( %) and/or dosage is higher than four units per day ( %) and/or there is no prescribed opioid maintenance treatment ( %). only two prescriptions have been discussed with the prescriber, and all were approved and dispensed. conclusion: otfc specialties are occasionally dispensed and often misunderstanding by pharmacists. a good knowledge of otfc is necessary to achieve the pharmaceutical analysis and provided appropriate advice to patients, in order to guarantee the good use of these medicines. support tools for dispensation, recalling indication, . the most frequent interventions were drug substitution (n = ), dose adjustment (n = ), and clarification of information (n = ). common services were reconstitution of suspension (n = ), provision in advance for continuing supply (n = ), and follow up offers (n = ). conclusion: the observation of the dispensing process in community pharmacies revealed a broad range of tasks performed by the pharmacy and identified several variables likely to influence the counselling. in addition, pharmacy activities could be pictured by the documentation of pharmaceutical interventions. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is a multidisciplinary process to correct medication errors resulting from miscommunicated information at transitions of care. development of this activity is essential but it is hindered by the time required for its implementation. we must carefully choose which services can develop this activity. as it was recently introduced in cardiac surgery unit, this study aims to evaluate impact of this process to hospital admission (severity of potential harm of medication error intercepted) and to determine the relevance of this activity in this unit. design: prospective study conducted from january to april . the data is recorded in an excel table, filled after each mr. there are five items: patient's age, best possible medication histories (bpmh), implementation period of the mr, inadvertent discrepancies (ids) and clinical impact. to assess the severity of ids, a scoring method was used (doerper et al. ) with the cooperation of surgeon and pharmacist. results: eighty-two patients (mean age ± years old) were included in the study, which represents % of the patients hospitalized in this service. the mean number of drugs per patient was ± . the bpmh were obtained within h to h of admission to hospital. a total of ids were detected, with a mean of . ids per patient. the most frequent type of ids was omission ( %, n = ), error of dose ( %, n = ). the three most common classes involved in ids were hypolipaemic drug (n = ), antidiabetic drugs (n = ) and the drugs for acid related disorders (n = ). the mean of ids per patient ( . ) as well as the percentage of patients affected by a ids ( %) are less important in cardiac surgery than those observed in other services of the institution and in the literature. about clinical impact, % of patients presented with ids considered as minor, % significant and % major. among the major ids, none was evaluated as critical or catastrophic. in our study, this process remains retroactive. conclusion: one of challenge experienced when implementing mr process in hospitals is demonstrating its clinical impact. in order to address this concern, we found that the little ids with a serious clinical impact in this unit. mr is an interesting process to detect drug errors. to optimize our study we will improve our organization in order to be closer to the patient and to strengthen the doctor-pharmacist collaboration. please specify your abstract type: research abstract background and objective: special packaging like multidose drug dispensing (mdd) may optimize medication use in patients with a decreased ability to manage their own medication. however, it remains unclear how a 'decreased ability to manage medication' is defined. the objective of this study is to assess potential medication problems that contribute to a decreased ability to manage medication in patients starting with mdd compared to patients who use manually-dispensed drugs. setting and method: patients starting with mdd (cases) and patients using manually-dispensed drugs (controls) were interviewed in community pharmacies. questions to assess potential medication problems covered three domains; medication adherence ( ), practical management issues ( ) and medication knowledge ( ) . every potential medication problem was scored with one point. cognition was assessed with the mini-cog and frailty with the groningen frailty index (gfi). main outcome measures: mean scores of potential medication problems on the domains medication adherence, practical management issues and medication knowledge. results: patients starting with mdd and patients using manually-dispensed drugs were interviewed. patients starting with mdd scored more potential medication problems on all domains: adherence . versus . , practical management issues . versus . , medication knowledge . versus . . on the three domains together, patients starting with mdd scored . [ . - . ] potential medication problems compared to . [ . - . ] for patients with manuallydispensed drugs. forty-two percent of the patients starting with mdd might be cognitive impaired and % was classified as frail compared to and % respectively of the patients using manually-dispensed drugs. conclusion: patients starting with mdd reported significantly more potential problems on three domains that may contribute to a decreased ability to manage their medication. cp-pc : fifteen key questions to assess patient knowledge on new oral anticoagulants corina metaxas * , valerie wentzky, sonja luginbü hl, kurt e. hersberger, isabelle arnet please specify your abstract type: research abstract background and objective: knowledge on new oral anticoagulants (noacs) is crucial for their safe and effective use. validated tools that assess patient knowledge exist for vitamin k antagonists, but not for noacs. we aimed to identify which questions are relevant for patient knowledge on noacs. setting and method: based on a systematic literature search, questions were compiled for the assessment of noacs knowledge. key questions were selected through three rounds of ranking by an expert panel (four physicians, four pharmacists, four nurses). round (online survey; importance): the questions grouped into the nine educational topics of wofford,adapted for noac (disease, mode of action, risk-benefit, adherence, accessing healthcare professionals, diet/life-style, lab-monitoring, medication interactions, self-care) were to be rated as important/not important and educational topics were to be ranked according to decreasing importance. round (online survey; relevance): the questions were to be ranked according to decreasing relevance. round (focus group): number of questions was reduced by voting. main outcome measures: ranking of educational topics and questions ( = most important/relevant) in march/april . results: experts ranked adherence ( . ± . ) as the most important topic, followed by risk-benefit ( . ± . ), disease ( . ± . ), accessing healthcare professionals ( . ± . ), self-care ( . ± . ), lab-monitoring ( . ± . ), medication interactions ( . ± . ), diet/life-style ( . ± . ) and mode of action ( . ± . ). one question was judged as unimportant by all experts. out of the remaining questions, ( . %) were selected as relevant for basic knowledge, ( . %) were combined into four questions and one new question was generated. a total of key questions remained after the focus group discussion. conclusion: a multiprofessional expert panel was able to select key questions retrieved from literature and ensured content validity. the selected questions will be compiled into a tool to assess patient knowledge on noacs. background and objective: medicines use review (mur) was defined by the slovene chamber of pharmacies in december and an education program was set to assure pharmacists competencies. in june the first pharmacists were certified and implemented the service in the community pharmacies. additionally, an online database was established to collect mur reports and provide feedback on pharmacists' performance. the aim of the study was to evaluate identified drug related problems (drp) as well as pharmacists' interventions from mur documentation. setting and method: a preliminary retrospective analysis of documentation for mur services provided in the first year after implementation was performed. drps were classified using a slovenian drp classification system, which is based on the pcne classification v . [ ] . data were analysed with descriptive statistics measures. main outcome measures: number and type of identified drp and pharmacists' intervention. results: a preliminary analysis was performed on mur cases, performed by certified pharmacists. in total drps were identified: ( . %) manifested and ( . %) potential. patient had on average two drps, however patients had none. main risk factor for potential drps was inappropriate use of medicines. adverse drug events (ades) presented . % of manifested drps; the main risk factor was again inappropriate use. in two cases ades happened due to an allergic reaction. different medicines were the cause of ades; mainly statins resulting in muscles pain and sleeplessness. another frequently manifested drp was insufficient effectiveness of treatment. drug interactions were risk factors in cases of manifested drps, mainly in connection with antidepressants: serotonin syndrome due to escitalopram, bleedings in concurrent use of escitalopram and ginkgo, sleepiness, etc. pharmacist intervened independently in . % of cases; times recommendations were given to physicians. however, in . % of cases the outcome of intervention is unknown. the preliminary results of the first mur cases points to a high number of identified manifested drps. however, the knowledge of intervention outcomes is lacking and therefore more attention has to be put on establishing adequate follow up on this issue. official definition represented harmonisation of several similar activities that have already been performed in slovenian pharmacies and also provided an educational program to assure pharmacists competencies. in may the first pharmacists were certified and implemented the service in the community pharmacies. therefore, the aim of the research was to get an insight into the implementation of mur in slovenia from the perspective of the first community pharmacists that provide the service in practice. setting and method: a focus group with seven community pharmacists, that provide mur in practice, was run in february . guided discussion included three main themes: the development and assurance of competencies, experience with the provision of service in practice and the future of the service. the discussion was voice recorded and analysed with the nvivo . written consent from included participants was obtained. main outcome measures: views, challenges and opportunities for the medicines use review service in slovenia. results: in total themes were identified and organized in three main categories: competencies for quality provision of mur, mur's recognisability and organizational aspects of mur provision. participants emphasized broad knowledge in pharmacotherapy is pharmacists' key competence and advantage in performing mur when compared with other healthcare professions. recognisability of mur among other health care professions as well as participants' work environments is low. hence a comprehensive approach in marketing of the service is needed. positive patient's feedbacks were reported, however persuading patients to attend mur presented a challenge. another barrier was the time to perform mur, which could be overcome by suitable work organization and special time intended for mur. conclusion: participants of the focus panel had positive experience with the development of competencies and implementation of the service in the practice. several challenges were presented connected with the recognition of the service by patients, physicians and health care payer. they strongly believe that continuing professional development forms the base for quality of the service in the future. cp-pc : evaluation of rational antibiotic dispensing in the community pharmacy setting: a simulated patient study betul okuyan * , mehmet ali savan, fikret vehbi izzettin, mesut sancar please specify your abstract type: research abstract background and objective: in the present study, it is aimed to evaluate rationale antibiotic dispensing without prescription in the community pharmacy setting; this will be done by using a simulated patient methods. setting and method: this study was conducted in malatya, located in the east part of turkey. the simulated patient visited the community pharmacies to meet the pharmacist, posing as the husband of a patient with acute uncomplicated rhinosinusitis. the simulated patient was trained regarding the standard information to be provided by the researchers and informed about the privacy of all information that would be gathered during the present study. the sample size was sixty-seven pharmacies, with a confidence interval of % and error of margin of %. the study was conducted over a total of pharmacies. all the pharmacies were listed alphabetically and were randomly selected and allocated random numbers by a computerbased program. main outcome measures: after each community pharmacy was visited, the simulated patient filled the check list which had been drawn up for the purpose of the present study. due to ethical concerns, no audio or video records were used during the study. any suggested medications were not purchased from the community pharmacy. results: of the total community pharmacies that were visited . % of them had female pharmacists and . % were run by male pharmacists. the mean number of questions asked by pharmacists to the simulated patient was . ± . . only eleven pharmacists did not suggest any medication for the simulated patient. however, thirty-two ( . %) pharmacists recommended various medication regimens, including antibiotics. of them, . % referred the simulated patient to a physician. conclusion: in conclusion, it was observed that dispensing antibiotics without prescription was still high, pharmacists did not take comprehensive medical or medication history from patients, and pharmacists provided insufficient medication information to the patient regarding suggested medications at community pharmacy setting. to avoid irrational antibiotic dispensing, it is essential to educate both health care providers and the general population. although dispensing antibiotics without prescription is illegal in some countries, it is necessary to actualize new regulations to avoid antibiotic dispensing without prescription. please specify your abstract type: research abstract background and objective: the medication adherence is an important part of active (as well as passive) attitude of a patient to the disease treatment. it represents the level of keeping the treating procedure as well as the recommendations of doctors, pharmacists and other healthcare professionals. this study deals with the adherence in patients with hypertension. the hypertensive patients are a substantial part of patients, daily visiting the community pharmacy to pick their prescriptions. these patients represent group of patients with typical asymptomatic disease. this means that they do not take the medicines or use them according to their own will. the result of their non-adherence could lead to later complications. the aim of the study was to evaluate the level of adherence and its relation to the clinical outcome-the blood pressure in hypertensive patients. setting and method: the methodology was based on a single anonymous questionnaire survey combined with the blood pressure measuring in a community pharmacy in slovakia. the modified morisky -item medication adherence tool was used in this study. main outcome measures: the results of medication adherence were evaluated as follows: - points = full adherence, points = partial adherence and - = non-adherence. each participant should use at least one antihypertensive agent and fulfil the anonymous questionnaire in the community pharmacy. the pharmacist measured the blood pressure in each participant twice, within the interval of min and used the average value in data sheet. results: the research included hypertensive patients ( . % females and . % males). the results showed that almost % of the respondents were non-adherent to the prescribed pharmacotherapy ( . % of those were males and . % were females). the group of partially adherent patients consisted of . % of the respondents ( . % of those were female). only . % respondents were fully adherent according to modified morisky score ( . % of those were women). fully adherent patients reached an average blood pressure . / . mmhg; partially adherent hypertensive patients recorded an average blood pressure . / . mmhg; and in the non-adherent patients has been observed the average blood pressure . / . mmhg. the results showed an alarming situation, and confirm the published data. non-adherent patients could not goal the good clinical outcomes. this leads to adding of another medications, raising the risk of interactions and adverse drug reactions, complications of undertreated disease, and finally, to pharmacotherapy costs increasing. please specify your abstract type: research abstract background and objective: in psychology, depression is a mental state characterized by feelings of sadness, dejection, inner tension and indecision. in psychiatry, the depression is defined as a severe mental affective disorder which paralyzes clarity of thought, psychomotorics, sleep cycle and raises pessimistic and depressing emotions often lead to pathological changes of personality. during treatment of depression is often needed psychotherapy and pharmacotherapy as well. using of antidepressants requires the sufficient level of medication adherence in patients. non-adherence to antidepressant medication significantly contributes to the undertreatment of depression in primary care populations. the aim of this study was to evaluate the level of medication adherence to antidepressants to better understand the socio-behavioural factors associated with non-adherence. setting and method: the anonymous, face-to-face questionnaire survey was set in the community pharmacy in slovakia. questionnaire obtained questions on socio-behavioural factors and adherence tool-modified -item morisky score (mmmas- ). main outcome measures: respondents were patients ( males, females) using at least one antidepressant. the results were evaluated as follows: points = full adherence, - points = partial adherence and - = non-adherence. results were evaluated in relation to socio-behavioural factors. results: average level of the medication adherence in our group was , which means the line between partial and full adherence. the results showed non-significant higher medication adherence level in males ( ) compared to females ( ). the highest level of medication adherence ( ) has been shown in patients - years old, the lowest average adherence level (non-adherence) was observed in patients up to years old ( ). patient living in the city were more adherent to their medication ( ) compared to patients living in countryside ( ). the highest level of the partial medication adherence has been shown in secondary educated patients ( ). partial adherence level was higher in patients with monthly income over € ( ) compared to non-adherent patients with monthly income up to € ( ). in patients using no other medications, only antidepressant, we have observed the highest partial adherence ( ). conclusion: our survey showed the partial antidepressant medication adherence levels in our study group. poor adherence results in low stabilization of clinical state in patient, in using more types of therapy and in increasing costs. there might be very important role of the community pharmacists and other health care professionals to improve the medication adherence and persistence through counselling and education patients on importance and need of antidepressant medication. ( ) and medication regimen complexity was assessed by using the medication regimen complexity index (mrci) ( ) . five and more medication usage has been defined as polypharmacy. results: a hundred and two elderly subjects ( . ± . ; male) were included in this study. of them, . % had two and more chronic diseases. the most common chronic diseases determined in study population were cardiovascular diseases (especially hypertension), diabetes and hyperlipidaemia. the polypharmacy has been defined in . % of them. the mean of mrci per elderly patient was . ± . . one or more pims use was observed in seventy-four elderly subjects ( . %). of all elderly subjects, . % were dispensed one and more medicines with a potential for drug-disease/ syndrome interaction. pims use was more frequently determined in patients with polypharmacy ( . vs. . %, p \ . ). the total score of mrci was significantly increased with elevated number of pims (r = . , p \ . ). conclusion: this study highlights a significant association between utilization of pims and both polypharmacy and higher total score of mrci in elderly patients. pharmacists could be evaluated utilization of pims in especially elderly patients with used five or more medications and/or higher total score of mrci. please specify your abstract type: research abstract background and objective: nursing home patients with multimorbidity often use multiple drugs simultaneously, which makes these patients more susceptible to adverse drug events. several studies have pointed to a need to increase the quality of prescribing to this population. to achieve this there is a need for reliable information about patients' diagnosis, and what is recorded as the drug's indication in different electronical and handwritten health records. the aim of this study was to examine the registered diagnoses, and indications for drug use in nursing home patients. we also wanted to study the extent to which diagnoses are untreated with drugs, as well as the extent to which drugs have a registered indication for use and a suitable recorded diagnosis. setting and method: data was collected for long-term patients, on average years old, and % females from four nursing homes in tromsø municipality, norway. we retrieved information about patients' diagnoses and indication for drug use from the electronic health record and written drug charts. two pharmacists conducted the linkage between the reported diagnoses and drug use. main outcome measures: percentage of untreated diagnoses and the percentage of drugs with a registered indication for use. results: as considered by the pharmacists, % of the registered diagnoses was untreated with drugs. dementia, gout and osteoporosis were the most commonly untreated diagnoses with, , and %, respectively. in comparison, the indication for use listed on the patients' drug charts was reported for % of the drugs. the drugs with the highest percentage of recorded indications were acetylcysteine (n = ), oxycodone (n = ) and zopiclone (n = ), where , and % had a listed indication, respectively. conclusion: a high percentage of nursing home patients' diagnoses seem to be untreated. however, most drugs that patients received were listed with indication for use in the drug charts. to increase quality of drug prescribing, one should put emphasis on improving the recorded information in electronical health records. cp-pc : personal changes in drug regimen: dangerous for health system? inga urtane, raivis pastars, dace bandere please specify your abstract type: research abstract background and objective: patient compliance is a key factor for a successful treatment and lack of it is the main reason for predicting treatment failure. in multiple researches patient adherence is determined to be as low as %. therefore it is important to identify the reasons of patients not following their drug regimen. objective. to analyse the patient comprehension of their drug regimen depending on the duration of hypertension and received treatment. setting and method: during the period from december to march a quantitative survey was conducted to include respondents who have been diagnosed with arterial hypertension and whose regimen includes at least one fixed dose combination drug. main outcome measures: in an anonymous survey data was collected about their demographic information, co-morbidity, other prescribed medication, intake regime, the average blood pressure during treatment, and patient's assessment of the prescribed therapy. collected data was analysed with spss. results: the study included participants, most of whom ( . %) were women. participants average age was . ± . years and the median arterial hypertension duration was ( ; ) years. the study participants, who sometimes consciously adjusted dosing regimen, observed arterial hypertension for a longer period of time compared to the group, which follows the prescribed regimen according to their doctor's recommendation, respectively, ( ; ) vs. ( ; ); p = . . group of respondents (n = ) receiving c prescription drugs, more often deliberately adjusted treatment regimen compared to respondents (n = ) treated with b prescription drugs, respectively . versus . %; p = . . respondents who deliberately adjusted drug were more often not satisfied with the number of longterm daily use of tablets (n = ) compared to the group (n = ), which had to intake fewer tablets every day, respectively, . versus . %; p = . . conclusion: arterial hypertension duration was associated with more frequent conscious adjustment of therapy without consulting a doctor. more individual prescriptions (c ) and an increase in the number of tablets per day at the same time also increases the risk of patients deliberately changing their dosing regimen. long-term drug users should receive additional attention during pharmaceutical care process to their respective treatment schedule in order to promote proper use of medication. please specify your abstract type: research abstract background and objective: diabetes is a health issue and real burden for in belgians. better adherence to the treatment could potentially reduce complications, decrease morbidity and mortality, and have a beneficial economic impact due to fewer consultations and hospitalizations. setting and method: a one-year program was started in belgian pharmacies to accompany diabetes patients taking dpp- inhibitors and encourage them to be compliant with their treatment. this study concerns of these pharmacies, all part of the same cooperative group. all pharmacists received prior training in motivational techniques and reviewed the bases of diabetes therapy with an e-learning program. materials developed for the patients included brochures on diabetes and its treatment, nutritional advice, physical exercise, foot care and tips and tricks for diabetics. main outcome measures: the impact on pharmacological adherence was measured using mmpr and pdc. two control groups were included: a historical control group and a group of patients that were not included in the project. non-pharmacological adherence was assessed using questionnaires. results: in the subgroup of pharmacies, patients were included in the program. by the end of april , only of them had completed the program; patients came only once to the pharmacy. they either stopped their treatment after one prescription, or were occasional clients. adherence rates were found to be high in all groups ( . - . % of patients with mmpr b %). only for the pdc, a statistically significant difference was measured between the intervention and control group ( . vs. . %; p = . ). no other statistically significant impact was measured (neither pharmacological, nor non-pharmacological). conclusion: adherence was very high in all groups. the underlying reasons still need to be investigated (choice of adherence measure, healthy user effect, etc.). however, both patients and pharmacists were very pleased with this type of program. this new role of the pharmacist will definitely be more developed in the future. please specify your abstract type: research abstract background and objective: oral anticoagulants (oac) have a beneficial effect on the long term survival of patients with atrial fibrillation and venous thromboembolism. however oacs have also side effects such as bleeding, especially when used inappropriately. pharmaceutical care interventions aim to optimize medicines use and improve patient health outcomes. the literature lacks a review on the impact of pharmaceutical care interventions in patients using oac. therefore, we systematically assessed the impact of pharmaceutical care interventions on the effective and safe use of oac compared to usual care. setting and method: a systematic review was performed in pubmed and embase with synonyms/detailed specifications of the terms oral int j clin pharm ( ) . it was motivate for the need to sort the instruments for urm, including professional participation, and on the basis of the clinical management unit, and reduce variability in decisions. the p&t or ''multidisciplinary commission rational use of medicines'' is constituted by people: one hospital medical director (president), head of pharmacy (secretary), and three directors of healthcare centre, three directors of department of specialities, one epidemiology, one hospital pharmacy, one primary care pharmacy and one paediatric. because some of these members are far between them, and normally dose not have too much time, we create an online platform to work, discuss and download all the necessary documents. setting and method: we used the facilities of the andalusian agency for healthcare quality (www.acsa.junta-andalucia.es), and as a base the law of the administrative decision. we have organized a session to discuss methodology with the participation of all members. main outcome measures: number of meeting and number of internal discussion emails. drug or protocol decisions. design of the platform. results: the design platform consists of five tabs: ( ) has the member information, position, telephone and address, ( ) email forum, following a subject line, ( ) a place for meeting requests and then hang up the meeting minutes. ( ) a tool allows you to upload documents to the portal ( ) a search engine. two sessions are schedules and total of mails. we have of members who have never participated online. at this moment we have adopted two decisions. conclusion: it is an online experience of one andalusian p&t committees, the low turnout makes go slower than expected, therefore physical meetings are necessary in this moment. we are working how to get more participation and involve in the project the committee members. please specify your abstract type: research abstract background and objective: liver cirrhosis can have a major impact on drug metabolism, requiring evaluation of drug safety and dosage in individual patients. currently, there are no guidelines on safe prescribing for medications in patients with liver cirrhosis, and these patients have many questions about safety and side effects of medication. the objective of this study is to explore the patient's needs on information about medication. setting and method: qualitative, semi-structured interviews were performed in patients with a (history of) liver cirrhosis. the patients were approached through an item in the newsletter of de dutch association of liver patients. topics in the interview guide were preferences about information about medication, side effects, safety, drug dosage, and how patients preferred to receive this information. interviews were audiotaped and transcribed verbatim. interviews were analysed using thematic content analysis. main outcome measures: the experiences and needs of patients with liver cirrhosis concerning information about medication. results: patients indicated they had received sufficient information about the indication, possible drug-drug interactions and the duration of treatment. they preferred (more) information about how medications work, what adverse drug reactions could be expected and practical aspects concerning intake of medication. informational needs were related to questions 'how to act': patients with more informational needs took a more active role in responsibility for their own medication management. patients needed information to know what to do, e.g. in case of adverse drug reactions or when a dosage was forgotten. the doctor and internet were the preferred sources of information: doctors because of the personal contact and internet because of the accessibility. facilitating factors were 'taking time' in healthcare provider-patient contact and 'everyday language' for texts on the internet and in package leaflets. a combination of verbal information by the healthcare professional and written information was preferred. conclusion: patients with liver cirrhosis need information about medication to take an active role in their drug management. comission for medicines and medical devices, chu de toulouse, toulouse, france please specify your abstract type: descriptive abstract (for projects) background and objective: due to its common use, insulin is often considered as a harmless medication by lots of health professionals while an overdose can lead to dramatic consequences and death. between january and june , in our university hospital, % ( out of ) of the declared adverse drug events have involved insulin: were caused by prescription errors and by administration errors. all were discovered after the medicines have been administered but thankfully none had serious consequences. the british national health service (nhs) and the french medication safety national agency (ansm) made a list of ''never events'': avoidable events which should never happen and misadministration of insulin is among them. the objective was to increase patient safety in the hospital by setting different actions to promote and improve the appropriate use of insulin and warn health professionals about the real dangers of this medicine. design: different actors participated in the implementation of these actions: the commission for medicines and medical devices (which is composed by doctors and pharmacists) directed a group made by physicians and clinical pharmacists from the department of cardiological and metabolic diseases'. results: in addition of the usual analysis of any adverse event linked to medication declared in the hospital, several actions were set up: • a didactic document summarizing all the ''sensitive'' steps during the prescription, stocking, dispensation and administration of insulin has made the front page of the hospital's intranet and was also diffused throughout the establishment. • a chart resuming all the different insulins commercialized in france has also been diffused. it contains their types, durations and onsets of action, conditions of storage and pictures of their packaging. • the computerized protocols involving insulin are going to be reviewed in order to lower their numbers and harmonize their content. • a revision of the list of insulins available at the hospital is in progress to reduce their number and avoid any confusion between the different products. • an evaluation of insulin's computerized prescription practices will be made via a data request. : this topic about insulin shows a greater willingness to secure the medication circuit in the hospital. other action plans such as this one will be set up involving other medications among the never-events list. meanwhile, the commission for medicines and medical devices pursues its actions of promoting the appropriate use of medication. please specify your abstract type: descriptive abstract (for projects) background and objective: one of the hospital pharmacist tasks is to suggest substitutions to ensure conformity of medical prescription with the hospital formulary. indeed, when an eye drop isn't available at the hospital, there is a specific supply circuit which has to remain exceptional: it's ordered directly to the pharmaceutical wholesaler. in this context, ophthalmologists and clinical pharmacists created a table proposing therapeutic equivalencies with eye drops available at the hospital. after approval by the commission for medicines and medical devices, this tool has been diffused within the establishment via the intranet website since the beginning of to the medical and paramedical staff. the purpose of the study is to evaluate professional practices concerning the use of the eye drops' equivalence chart. design: in the study, we compared eye drops' orders made to the pharmaceutical wholesaler before and after the table's diffusion, thus between january and december . for each order, we used the table available at this time to determine if equivalencies could have been proposed or not. if so, we identified the hospital ward and the pharmaceutical specialty. market changes have also been considered. results: we noticed a decreased frequency of eye-drops ordered despite available equivalencies: % in (before the table's diffusion), % in (after its diffusion), % in and % in . prisons units are responsible of % of these orders: they have the lowest rates of substitutions. their most ordered pharmaceutical specialties are ophthalmic glaucoma agents: % ganfort Ò (bimatoprost . mg/ml/timolol . %), % xalacom Ò (latanoprost + timolol . %), % azopt Ò (brinzolamide) for which the authorized substitutions are for the first two specialties: monoprost Ò (latanoprost) + ophtim Ò (timolol . %) and for the third: dorzolamide Ò . conclusion: equivalence table diffusion throughout the hospital has facilitated and improved the prescription and substitution of eyedrops. orders of pharmaceutical specialties despite authorized equivalencies available have declined by half. probably for practical reasons regarding long-term treatments, prison units make less substitutions but an awareness campaign will be carried out to reduce these rates. please specify your abstract type: research abstract background and objective: the patient's education and information is a mean to reduce medicine misuse and it can be performed with support of a leaflet or informative material about medicines. in brazil, there is a lack in regulation about this type of informative material to compounded medicines. the aim of this study was to evaluate the quality and effectiveness of leaflets developed to compounded medicines' users through knowledge's level and medicine treatment adherence. setting and method: analytical and quantitative study; month prospective study through interviews, at time zero (t ) and after days (t ) in a university pharmacy in goiás, brasil; fisher's exact test to measure effectiveness; ethics committee number / . main outcome measures: categorization into high adherence and low adherence by morisky test; categorization into sufficient, regular or insufficient knowledge about medicine prescription; perceptions and suggestions about delivered leaflet in medicine dispensing process. results: of patients ( . % female, mean = . years), . % considered as relevant the leaflet's content, as well . % of them kept it and . % of them read it. suggestions of . % included a desire in increase font size, more emphasis on drug interactions and images. there was a predominance of regular knowledge in both analysed times ( . % e . %), however there was a decrease in high adherence to medicine treatment ( . - . %). among patients who read the leaflet, no statistically significant association was found on these two variables at t and t (p = . and p = . , respectively). knowledge about ''administration schedules'' showed a significant improvement after intervention (p = . ). . % of patients considered that there was no need to obtain more information. conclusion: this study demonstrates the evaluated leaflets had relevance to patients and demonstrate clinical relevance. however was not observed statistically significance. this highlights the need of using different ways to measure the effectiveness of an informative material to promote rational use of medicines and depth studies and stimulation of greater attention from the health professionals to the topic. di : chlormethine gel: effectiveness and tolerance to treat mycosis fungoides françois dugre *, , anne lefebure , sonia martelli , marion pin , eve maubec , philippe arnaud pharmacy, dermatology, bichat-claude bernard hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: to determine the effectiveness and tolerance of chlormethine gel in treating mycosis fungoides. design: mycosis fungoides is the most common form of cutaneous t-cell lymphoma (mf-ctcl). early stages (ia and ib) can be controlled by skin-directed therapies such as chlormethine and carmustine. these drugs which are solutions for injection are usually used for skin application. chlormethine or mechlorethamine gel is an alkylating agent representing an alternative for previously treated patients diagnosed with mf-ctcl, in case of therapeutic failure and intolerance, or in case of chlormethine and carmustine solutions supply disruption. a retrospective observational analysis was conducted based on medical records of all patients treated by chlormethine gel in our hospital from the first of july to the first of september . the following data were collected with an excel table: body surface area or bsa affected by disease, location of the lesions, therapeutic management, effectiveness and treatment tolerance. results: fourteen patients ( women, men, mean age [min ; max ]) were treated with chlormethine gel in our hospital. twelve ( %) were treated three times per week, ( %) once a day. before treatment by chlormethine gel, ( %) patients were treated by dermocorticoids, ( %) by dermocorticoids and phototherapy, and ( %) by bexarotene, all of them stopped their treatment on account of inefficacity. one ( %) patient was treated by carmustine and dermocorticoid, and ( %) by only carmustin, all of them stopped it because of supply disruption. one ( %) patient received it in first line therapy. ten ( %) patients showed a response (partial or complete), one ( %) experienced a stabilization of his disease. before treatment with topical chlormethine, seven patients ( %) had an involved bsa [ % and four of them ( . %) experienced adverse effects. seven patients ( %) had an involved bsa \ % and three of them had ( . %) side effects. a total of seven patients ( %) presented at least one adverse effect. five patients ( %) stopped the treatment on account of adverse effects; two of them ( %) interrupted it temporarily. reported side effects were: irritant dermatitis and erosive toxicity ( ), rash ( ) and telangiectasia ( ) . conclusion: our results indicate that chlormethine gel can be effective to treat mycosis fungoides. however, it involves side effects that seems to be more frequent than those observed with chlormethine solution (used for skin application). indeed, the french national authority for health reports % of adverse effects for chlormethine solution versus % in our study for chlormethine gel. moreover, telangiectasia was never documented with chlormethine. this significant number of side effects of chlormethine gel can be explained by the gel formulation which induces patients to apply more product, especially in patients with plaques affecting more than % of the bsa. it is important to explain to patients to apply a thin film of chlormethine gel to involved skin areas and allow the skin to dry completely. sophie dumas *, , capucine devaux , nathalie le guyader diaconesses croix saint-simon hospital, diaconesses croix saint-simon hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: aprepitant, a neurokinin- receptor antagonist, prevents nausea and vomiting due to high and moderate emetogenic chemotherapy in combination with other antiemetic agents. it induces cytochrome p (cyp) c and moderately inhibits cyp a . drug-drug interaction could occur with intravenous anticancer or antiemetic drugs metabolised by these isoenzymes. it may lead to adverse effects or loss in efficacy. regarding recent international antiemetic guidelines, emergence of new intravenous chemotherapy and lack of bibliographic data, a report on aprepitant interactions is performed in oncology. the aim of this study is to review pharmacokinetic interactions with aprepitant in order to prevent potential toxic effects of intravenous anticancer or antiemetic agents and provide the best patient care. design: anticancer and antiemetic agents metabolised by cyp a and c were identified. pharmacokinetic literature review was performed using medline Ò database and laboratory data. clinical assessment and non-aprepitant pharmacokinetic studies were excluded. a table was established to summarize data. results: ten intravenous anticancer agents used in oncology are identified as cyp a substrates. pharmacokinetic assessments are achieved for docetaxel, cyclophosphamide, vinorelbine, irinotecan and trabectedin. studies dealing with the five other drugs are strictly clinical assessments. among the different pharmacokinetic studies, only trabectedin showed relevant interaction with aprepitant. in this association, aprepitant dose needs to be adjusted. cyp c catalyses the cyclophosphamide activation pathway with minor contribution. however, it would have few repercussions on cyclophosphamide pharmacokinetic. corticosteroids and hydroxytryptamine type ( ht- ) receptor antagonists are also metabolised by cyp a . aprepitant significantly increases corticosteroid plasma concentrations. in this case, corticosteroid dose adjustment should be applied. furthermore, no interaction has been found with ht- receptor antagonist. conclusion: regardless of the emetogenic level of anticancer agents, all drugs have been studied because of theirs potential combinations. two relevant pharmacokinetic interactions have been demonstrated leading to dose adjustment recommendation. corticosteroids doses, in association with aprepitant, should be reduced one-fourth for intravenous form and one half for oral form. aprepitant first dose should be decreased to mg when it is co-administrated with trabectedin. these two results lead us to re-evaluate our prescription practices. please specify your abstract type: research abstract background and objective: nsaids are associated with serious adverse reactions which in turn are responsible for significant risks of morbidity and mortality. the aims of this project is to identify risks involved in nsaid administration including over-usage and significant drug interactions, and to analyse occurrence of side-effects. the trends of nsaid prescribing by physicians and pharmacists are also determined. setting and method: a pharmacy from each electoral district was chosen by stratified sampling. a sample population (n = ) was obtained from pharmacies in malta. data was collected through the completion of questionnaires carried out by the patients. the trends of nsaid prescribing were determined by another questionnaire directed to pharmacists and physicians that was available online. main outcome measures: use of nsaids by patients and prescribing trends. results: back pain (n = ), muscular pain (n = ), headache (n = ) and arthritic pain (n = ) accounted for the most frequent use of nsaids. diclofenac accounted for the most commonly administered nsaid, taken by of the patients, of which use the mg dose. chronic disorders of symptoms experienced by the patients included hypertension (n = ), heartburn (n = ), dyspepsia (n = ), asthma (n = ) and a history of helicobacter pylori infection (n = ). other disorders suffered by single individuals include epilepsy, crohn's disease and renal dysfunction. more than half of the respondents (n = ) admitted to self-prescribing regardless the fact that the majority of nsaids are prescription-only medications. epigastric pain ( . %), stomach ulcers or gi bleeding ( . %) and elevated blood pressure ( . %) were the most common sideeffects that pharmacists and physicians come across. nsaids were frequently found to be co-administered with antihypertensives ( . %) and ssris ( %) regardless of their significant risks of interacting with nsaids. . % of the pharmacists and doctors believe that nsaids are being over-used and . % state that closer monitoring of nsaid adverse effects is necessary. conclusion: the risk involved with nsaid administration due to over-usage and drug interactions is identified, and healthcare professionals are aware of this risk. pierre leduc, antoine lanneluc, christophe gellis * , sylvie poux, dominique plats, regine larnaudie corrèze, ch brive la gaillarde, brive la gaillarde, france please specify your abstract type: research abstract background and objective: proton pump inhibitors (ppi) are widely prescribed in hospital while their long-term use may be responsible of many potentially serious long-term side effects (hypomagnesemia, neutropenia, gastric cancer) and drug interactions (ppi are inhibitors of cyp c ). the objective of this study was to assess the appropriateness of ppi prescriptions in a geriatric department in order to optimize their conditions of prescriptions. setting and method: this prospective study involved patients hospitalized between january and april in a geriatric department. the accordance of the prescriptions with the marketing authorization indications and the french guidelines was analysed. data collection was done using a table excel. main outcome measures: collected information were related to patients (age, sex) and ppi prescriptions (active substance, administration route, dosage, duration of therapy, therapy indication and reassessment of ppi therapy). results: ninety-one patients were included: sr: . , mean age: . years [ ; ]. ppi therapy prevalence over the period was %. the ppi were prescribed in the geriatric department in patients (mostly esomeprazole) whereas patients had ppi therapy (mostly esomeprazole) at the admission, for more than years in patients. oral route was the most frequent one (n = ). ppi were administered once a day and only three ppi were administered in the morning. % of ppi prescriptions were considered unjustified; the indications were prevention of haemorrhage with antiplatelet therapy (n = ), prevention of haemorrhage with corticoid (n = ), prevention of haemorrhage with anti-vitamin k (n = ), dyspeptic disorders (n = ), gastralgy (n = ) and others reasons (n = ). % of ppi prescriptions were considered relevant. the reassessment of ipp therapy (n = ) lead to prescribe another dosage (n = ), to stop therapy (n = ) or no change (n = ). conclusion: the study showed that the majority of ipp prescriptions were not in accordance with french guidelines. limiting the prescription to the indications, reassessing the therapy or respecting the therapy duration should reduce the risk of long term side effects and the economic burden of ppi in a long term use. please specify your abstract type: descriptive abstract (for projects) background and objective: to evaluate the effectiveness and safety of the use of high dose of tigecycline ( mg followed by mg every h) a tertiary care hospital. design: retrospective observational study. period: january to december . inclusion criteria: episodes use of tigecycline ( mg followed by mg every h. exclusion criteria: time less than days treatment. data source: corporate program stories electronic health. results: we identified episodes in patients ( men, mean age: years ( - )). treatment was directed to multidrug-resistant organism infection in cases (seven klebsiella pneumoniae oxa- , two enterobacter cloacae, two enterococcus faecium and one methicillin resistant staphylococcus aureus. in one episode they coincided e. cloacae and e. faecium). in cases had severe sepsis or septic shock (seven abdominal focus, six respiratory focus and one unknown focus). the median number of days of treatment was ( - ). tigecycline was administered as monotherapy in three cases, bitherapy and triple combination therapy in . the antibiotics were associated were: beta-lactam ( ), aminoglycosides ( ), quinolones ( ) colistin (three, two inhaled cases), cotrimoxazole ( ) and vancomycin ( ) . in episodes produced clinical and/or microbiological resolution and antibiotics are rotated by progression picture or lack of improvement, death occurred in three cases and was suspended on suspicion of hepatotoxicity. among the seven episodes of klebsiella pneumoniae oxa- infection there were four pneumonias, three with favourable evolution and one patient died, two bacteraemia, both with resolution clinical and microbiological, and one urinary tract infection resolved. among the episodes in severe/ septic shock were five cures, six cases of antibiotic rotation progression or lack of improvement and three deaths while patients receiving therapy tigecycline. patients showed an adverse effect possibly related to therapy tigecycline: diarrhoea after days of treatment and case of liver toxicity after days of tigecycline and piperacillin-tazobactam which led to their withdrawal. int j clin pharm ( ) : - conclusion: tigecycline has been used in double dose defined in data sheet especially in situations of severe sepsis or septic shock and infection multiresistant microorganisms. the effectiveness is conditioned by the clinical situation patient, being worse in severe/septic shock sepsis. tigecycline high dose was well tolerated and there was only a case of stopping the medication for suspected damage hepatic. di : wikipedia and medicines: who edits medicine articles on the english wikipedia? kristian husvik skancke , kristian svendsen *, department of history, uit -the arctic university of norway, hospital pharmacy of tromsø, tromsø, norway please specify your abstract type: research abstract background and objective: the medical profession and pharmacists are divided on the usability of wikipedia for looking up health information. nevertheless wikipedia is widely used, more than half of us physicians and percent of all medical students use wikipedia as a source of health-related information. there is a potential for incorrect and biased information being added by the pharmaceutical industry. the aim of this project was to examine who edits wikipedia articles on medicines and to investigate whether the pharmaceutical industry edits these articles. setting and method: two different groups of articles has been examined; the top ten bestselling medicines (substances) in the world in and the ten most recently approved medicines on the european market (until december ). the top ten medicines were selected from a consultancy report by evaluatepharma/ep vantage. the ten most recently approved medicines (new substances) were found on the european medicines agency webpage. we queried the english wikipedia on january and information from the edit history and the editors' user information were extracted. unregistered editors were checked using a whois service. for the new medicines all editors were checked, while for the bestselling medicines large edits and initial edits was checked. main outcome measures: edits suspected of being made by the pharmaceutical industry. results: ten bestselling medicines: there are many users editing these articles and/or watching them, limiting the risk of misinformation from the industry. there was no indication that the pharmaceutical industry had edited any of the articles. ten most recent medicines: no article existed for dasabuvir. for the nine other substances there were relatively few editors and watchers. in four out of the nine articles we found evidence of edits from the pharmaceutical industry. these edits, were done by registered editors with very few edits except for the medicine in question and they had made large additions to the articles sometimes even before the medicine was marketed. conclusion: the pharmaceutical industry seems to edit articles about medicines on english wikipedia however we found no evidence of harmful edits and bestselling medicines have many editors monitoring the quality of articles. please specify your abstract type: research abstract background and objective: the pharmaceutical professional service of the monitored dosage systems (mds) tries to improve the adherence of the patients to the treatment. the aim was to analyse the relevance of the repackaging of the most sold medicines in our country being used by patients included in the mds professional service and to determine the information discrepancies according to the source used by the pharmacist. setting and method: cross-sectional descriptive study. community pharmacy and healthcare institutions. all the patients included in the pharmaceutical professional service of mds on june , . data source: patients' records in the professional service, database of medicines ranked by sales in units in our country to december ( medicines), information sources on medicines: ( ) vademecum of medicines and ( ) the centre of drug information of our agency of medicines. main outcome measures: number of institutionalized and ambulatory patients included in the professional service of the mds and demographic characteristics, sum of different repackaged medicines belonging to the studied patients, analysis of the repackaged medicines of major use, number of discrepancies on the repackaging of the medicines according to the information source. results: patients were included in the professional service of the mds. of them were institutionalized (average age: . years, . % men, . % polymedicated defined as using c prescribed chronic medicines) and the remaining were ambulatory (average age: . years, . % women, . % polymedicated). different medicines prescribed in the institutionalized patients were taken into account, of them included in the sales ranking in our country. according to the first source, of medicines were eligible for repackaging, medicines could be repackaged according to the laboratory manufacturer and the remaining ones could not be repackaged. according to the second source, of medicines could be repackaged, and the remaining ones could not. different medicines prescribed in the ambulatory patients were taken into account, of them included in the sales ranking in our country. according to the first source, of medicines could be repackaged, medicines would depend on the laboratory manufacturer and the remaining ones could not be repackaged. according to the second source, of medicines could be repackaged, and the remaining ones must remain in the original package. discrepancies were observed in the information for ( . %) and ( . %) medicines in institutionalized and ambulatory patients, respectively, based on the sources used. conclusion: a considerable number of discrepancies in the information on the relevance of the repackaging of medications in the mds were found between two analysed sources. these findings have already improved the quality of this professional service. it would be necessary to alert the pharmacist of the existence of the above mentioned discrepancies to be able to prevent errors from occurring at the time of repackaging the medicines in the mds and, thus, increasing patient safety. please specify your abstract type: research abstract background and objective: despite the global advances of pharmacy practice and subsequently pharmacy education, students experience insufficient opportunities to practice the activities, tasks and processes essential to deliver pharmaceutical care. objective: to describe the development, implementation, and assessment of a clinical pharmacy practice (cpp) experience course in internal medicine, cardiovascular, respiratory clinics and drug information centre that is newly integrated into pharmacy curriculum at a university in north cyprus. setting and method: a weeks structured pharmacy practice experience was designed for fifth year students. student competence was assessed using formative osces and summative written exams before and after the course, and mapped in eight main cpp competences. the course utilized a wide variety of learning and practical activities including rounds participation, morning case reports, interdisciplinary activities, carrying interventions, role-play, direct patient care, formal case presentations, journal clubs and answering drug queries. competencies tested and strengthened include: taking medication history, response to the symptoms, pharmacotherapy knowledge application, comprehensive patient assessment, data interpretation using evidence-based approach, public health counselling, drug related problems management, patient counselling and communication skills. student perceptions and experience was assessed using semi-structured group interview and a questionnaire. main outcome measures: student scores in osce; student's perceptions. results: student reported that the course met pre-set objectives with substantial learning in different areas of cpp. students scored best in communication skills ( . ± . %), public health promotion ( . ± . %) and patient counselling ( . ± . %) than in resolution of drps ( . ± . %) and pharmacotherapy application ( . ± . %), while they significantly enhanced in di manipulation ( . ± . %) compared to baseline assessment ( . ± . %)(p = . ). conclusion: the course provided a rich experiential learning environment rather than just theoretical knowledge of clinical pharmacy. students well perceived the course structure assessment and knowledge attained. this could be implemented in other faculties of pharmacy through turkey. please specify your abstract type: descriptive abstract (for projects) background and objective: clinical pharmacy and clinical pharmacology have many similar aspects. both areas present professionals who have groundings in drug therapy principles and who aim to optimize the efficacy and safety of therapies for patient's benefits. however, there are clear distinctions. clinical pharmacologists are in general doctors with an additional education in clinical pharmacology. many of these are prescribers of drugs in practice but are in usual connected to academic parts responsible for education and research. they belong to a well-recognized but small sub-specialty of medicine. in contrast, clinical pharmacists are part of a much greater group of professionals working in most hospitals in developed countries. while the former one is restricted and subordinate to distributing the drugs requested by the medical prescribers, the role of the pharmacist has increasingly developed to encircle monitoring outcomes of medicine treatment and report management, patient safety and budgetary responsibilities. pharmacists are currently capable to take on prescribing responsibilities in developed countries and have been actively involved in collaboration in practice of prescribing with doctors. they also take on a great part in education related to rational prescribing that was once thought the area of the clinical pharmacologist. given the difference in size of the two areas there is understandably increasing confusion in the minds of managers in health services as to the continuing role and identity of clinical pharmacology. this may illuminate, in part, the diminishing in numbers and visibility of clinical pharmacologists in certain countries. in fact, some might see the continuous development of clinical pharmacy as a direct danger to the viability and future existence of the specialty of clinical pharmacology. however, clinical pharmacy and clinical pharmacology working synergistically would serve for the well-being of the public. design: . results: . conclusion: . maxime apparuit *, , lea boissinot , ngauv melodie , stephanie charles weber , isabelle lopez , françois chast pharmacy, hopital cochin, pharmacy, hopital hotel-dieu, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: hereditary angioedema (hae) is a rare disease characterized by episodic attacks of swelling which can be life-threatening. treatment for hae involves prophylaxis and management of acute attacks. the objective of this study was to evaluate patients' knowledge of their disease and their treatment. design: a questionnaire about the disease and drug treatment has been implemented. it was distributed to patients through either a pharmacist during patients stay at the hospital, or the french association des malades souffrant d'angioedèmes (amsao). answers were collected by electronic or conventional mail. results: patients completed the questionnaire. the average patients age is . ± . years. all of those interviewed could name their disease. for % of patients, the crisis happened unpredictably but in most cases a triggering factor was described, such as stress ( %), fatigue ( %) or an emotional shock ( %). oedema were located mainly in extremities ( %), abdomen ( %), ent sphere ( %) or face ( %). patients ( %) reported having more than crisis each year (eligible to prophylaxis), among them, patients ( %) said they had no preventive treatment. all patients knew the difference between prophylactic and curative treatment of crisis. among the patients receiving treatment for crisis, were able to define which treatment to be used depending on the intensity and location of the crisis. the majority of patients used icatibant during a crisis, but the most frequently cited prophylaxis treatments were tranexamic acid ( %) and danazol ( %). for injectable drugs to treat acute episodes, icatibant (subcutaneous) and c esterase inhibitor (intravenous) were self-administrated respectively in and % of patients. conclusion: this study showed that patients generally knew their disease and its treatment. however, they are insufficiently informed on drugs to be used according to the clinical situation and especially intravenous self-administration. therefore, it seems necessary to increase pharmacist involvement in patient's information about therapeutic strategy and drugs routes of administration. this for a major objective: an optimal self-care in a skilled patient. please specify your abstract type: descriptive abstract (for projects) background and objective: hospital pharmaceutical educations (hpe) on patients with oral anticoagulant (oa) can improve their overall management by providing skills on proper use. an ambulatory monitoring is necessary to ensure good compliance and understanding of the treatment. our study aimed at the establishment of hpe for patients with oa, the establishment of a hospital-city link in burgundy, and an evaluation of the expectations of ambulatory health professionals (ahp). design: the development of hpe has been performed in our centre for patients with oa and assessed between may and september . in order to ensure continuity in their support, patients then received a binding document to the attending physician, pharmacist and nursing home stating the treatment and acquired skills. a satisfaction survey, with anonymous electronic questionnaire circulated by the representative boards evaluating the expectations of ahp, took place in order to improve and make the programme more attractive. results: two hundred and ninety-one patients could benefit from hpe and came out with an oa. one hundred and forty-three answers were collected: officinal pharmacists and nurses. ninety-seven percent of ahp have judged relevant the following stated security goals: the name of the drug, its use, its risks and to be able to inform all ahp. ''associated pathologies and treatments,'' ''the last coagulation test'' and ''potential factors for non-adherence'' seem necessary for the binding document. more than % of participants found that this action will facilitate the establishment of pharmaceutical anticoagulant educations in cities, the dialogue around the oa with the doctor, patient's compliance and will secure the treatment. conclusion: hpe certainly help patients. its implementation for patients with oa in our hospital has generated a real interest. the addition of an ambulatory link allows continuing at best their support. the questionnaire has also allowed us to know the opinions of ahp involved and some improvements to the binding document may have been done. participants were asked to associate the task to the profession by determining whether each profession had the main responsibility for undertaking the task, a supportive responsibility, or whether they should not be involved at all. data was analysed using spss Ò, version . the chi squared test was used to assess any significant association between categorical variables. main outcome measures: perception of the oncology pharmacist's role by healthcare professionals. results: from a total of completed questionnaires, it was found that for tasks listed as ''patient education and counselling'', % were considered as the pharmacists' main responsibility, whereas % were believed to be supportive roles. main tasks included educating the patient regarding which medication to avoid during their treatment. for tasks listed as ''drug related problems'', and % of tasks were found to include pharmacists as having main and supportive roles respectively. supportive tasks included dose calculation of anti-tumour therapy required per patient. in the ''authorisation of medication'' category pharmacists' main roles carried a total of % and supportive that of % of the total number of tasks. this included ordering anti-tumour medication. further analysis of data revealed that years of experience did not have a significant association with results obtained (p-value = . ); however physicians, pharmacists and allied healthcare professionals were found to involve the pharmacist most extensively (pvalue = . ). conclusion: tasks associated with the pharmacist were representative of the current role they possess within the oncology setting; however this association was limited to professionals having a close working relationship with pharmacists. this may be due to the lack of an established multidisciplinary team approach within this scenario thus limiting the perception of the oncology pharmacist's contribution. an implemented multidisciplinary team may improve communication between the professionals involved and optimises patient care. the aim of the study is to analyse from a qualitative and quantitative point of view the pharmacy resident's activity in pneumology service. setting and method: the study included all the daily prescriptions of three units of pneumology from january to april . pi and data were extracted from the software pharma Ò and collected in a summary excel Ò table: nature of potential errors, nature of the proposals offered by residents, way of transmitting pi, and rate of pis' acceptance. main outcome measures: potential errors are collected by following the validated and standardized criterions of french society of clinical pharmacy. results: over months, lines of prescriptions from patients aged years old (median [ - ]) were evaluated. sex ratio (m/f) was . . one hundred and two medication problems have been found: overdose ( . %), contraindication (ic) ( . %), under dosage ( . %), wrong rhythm of administration ( . %), forgotten treatment ( . %), dose unit error ( . %), antibiotic indication missing ( . %), drug not listed in the hospital formulary ( . %), potassemie unchecked ( . %), dose unadapted to renal function ( . %) or to inr ( . %), treatment not indicated ( . %), wrong administration route ( . %), antibiotic unreevaluated ( . %), redundancy ( . %). the proposals made to the doctors were: stopping treatment ( . %), posology adaptation ( . %), substitution ( . %), dose unit modification ( . %), adding information about the indication ( . %), treatment renewal ( . %), administration modalities changing ( . %), biological monitoring ( . %), therapeutical monitoring ( . %), antibiotic treatment reevaluation ( . %). all pi were made by informatical way. all medicinal classes were found in this study. hydroxyzine, cyamemazine and escitalopram were often found in contraindication errors. they are involved in cardiac disorders with qt extension. pis' acceptance rate was %. conclusion: this study shows the importance of pharmaceutical analysis on the quality of access to healthcare. the statement of pi allows us to identify the most frequent errors, warn and prevent doctors from these potentials errors by proposing solutions. the rate of acceptance is high which means that doctors agree with our proposals. pharmacists' implication in clinical pharmacy activities and their participation to medical rounds will improve this activity and by the way optimization of the management of the patient. please specify your abstract type: descriptive abstract (for projects) background and objective: ppis consumption is largely practiced in europe, because of their excellent tolerance in short time, and their misuse with regard to indications, dosage and treatment duration (in , france was the nd,ppi consumer in eu). the result is drug iatrogenic disease and unjustified expenses in health insurance. objectives: assess the ppis consumption and appreciate conformity according to the latest recommendations for relevant prescriptions of ppis. design: prospective study via an audit (model created internally), every hospitalized patients with a ppis prescription, in two hospitals, on a given day. data collected through the patient's medical record. prescriptions conformity defined, by taking account of indication level ( : approved by the ma (marketing authorization), : non-valid but certified by international publications or learned society, : nonvalid without scientific proofs, : non-indicated), dosage and treatment duration. analysed situations with no conformity (inappropriate dosage despite conform indication, treatment duration unjustified and ppis prescribed in wrong indications ( and ) . results: patients have ppis prescriptions ( male, £ years] among patients ( %). % ppis prescriptions began during the hospitalization. ( %) of the ppis prescriptions are in accordance with the experiment (indication + dosage +treatment duration), as well in community than in hospitals. details: indication level ( . %), indication level -gi bleedings-( . %). of the ppis prescription aren't in accordance. details: treatment duration ( %), dosage ( %), indication level -prevention of iatrogenic bleeding risk without nsaids prescription-( %), indication level ( %). regarding level indications, ppis are always taken with anticoagulant and/or platelet aggregation inhibitors and/or corticoid. conclusion: the part of ppis prescriptions in this study is high. the majority of non-conformity is caused by ppis prescribed with an indication level . the improvement program will involve feeding back ppis' good use, to educate physicians (junior and senior) about the relevant ppis prescription and give advice in complex situations (indication and ). in collaboration with prescribers, shutdown protocol of ppis, prescribed in long term, could be implemented in order to avoid the acid rebound effect after brutal treatment discontinuation. hp-ce : impact of a self-management program on inflammatory bowel disease patient in a university hospital caroline egon * , xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: inflammatory bowel disease (ibd) is a group of chronic inflammatory diseases that affects the colon and the small intestine. crohn's disease and ulcerative colitis are the principal types of ibd and involve severe diarrhoea, pain, fatigue and weight loss. ibd affects young adult with an increasing annual incidence ( . million concerned people in europe). patients with ibd are affected by somatic or psychosocial problems and patient education may contribute to their well-being. since september , individual educational sessions have been set up and since september , collective educational sessions. these sessions have been developed to improve patient's understanding of treatment options and medical adherence. the aim of this study was to demonstrate that a therapeutic education program (tep) could have a significant effect on ibd patient's skills with regards to their disease. design: after individual education sessions with a nurse, a group education session was introduced for outpatients with ibd. the collective session include approximately six to ten patients and is organized in a half day workshops (about disease and treatment) conducted by a multidisciplinary team. the workshops were performed by an education nurse, two hospital gastroenterologists, two hospital pharmacists and a community pharmacist. these sessions were wrapped up by a short satisfaction and knowledge questionnaires. results: in total, ibd outpatients participated to the educational program, patients with crohn's disease and patients with ulcerative colitis ( . % male; median age: ). for the individual educational sessions, two competence questionnaires were performed about anti-tumour necrosis factor alpha (tnfa) therapy: one about general knowledge, another one about self-administration subcutaneous injection. patients completed these questionnaires. for the collective educational session, the competence questionnaire developed consisting of six questions covering few items: disease, symptoms, treatment and complications. patients completed this questionnaire. after the questionnaire, each participant received a summary document about drugs, side effects, therapeutic and medical advice. conclusion: the patient education program contributes to the improvement of self-management skills when it comes to ibd. pharmacists joining medical specialists and nurses provided pharmaceutical care with a positive impact on compliance, which is a determining factor for the success of the treatment and the quality of life in patients living with an ibd. this program will be continued and a new program for teenagers is to be established as well. hp-ce : desensitization study of paclitaxel and carboplatin drug in the ovary tumor protocol in cuf descobertas hospital miguel  . freitas * , daniela brites, ana bota pharmacy, hospital cuf descobertas, lisbon, portugal please specify your abstract type: descriptive abstract (for projects) background and objective: the hospital pharmacy should be an integral part of the multidisciplinary team and implement strategies that meet the patient's needs. pharmacy, in oncological area, is in constant renewal. josé de mello saúde uses paclitaxel/carboplatin protocol as first line in ovarian tumor. although the antineoplastic agents are essential for the treatment of cancer, they can also cause hypersensitivity reactions, which may carry serious consequences. both immunoallergologist and oncologist create a desensitization protocol, which allows the reintroduction of the drug with greater security. the desensitization protocols involves the gradual administration of small quantities of the drug, resulting in a refractory period of the white blood cells (mastocytes) and a lower production of cytokines until the dose has been totally administrated. objective:to evaluate the efficacy of methods used to prevent and treat hypersensitivity reactions of carboplatin and paclitaxel, in order to carry on the treatment. design: a retrospective review of the patient files was performed in the day hospital between and . we included only patients with moderate to severe immediate hypersensitivity reactions (b h) receiving carboplatin and paclitaxel. the desensitization protocol brigham and women's hospital was applied using three solutions with increasing concentrations (dilution : , : and : ) in twelve successive steps for about h. results: in the period - were desensitized five patients with platinum group drugs, carboplatin (n = ) and paclitaxel (n = ) and the total elapsed six desensitization. almost all patients reached the scheduled daily dose, except a patient, which suspended the desensitization program for disease progression. conclusion: the desensitization protocol allowed the successful reintroduction of antineoplastic drugs in patients with a history of hypersensitivity reactions, in order to treat the disease. please specify your abstract type: research abstract background and objective: in the context of harmonization of clinical pharmacy activities within our region, a common medication reconciliation project was developed between two general hospitals. the objectives of this study were to initiate, a common medication reconciliation activity in the two hospitals, to analyse the results, and to communicate to all professionals in the area. setting and method: a working group composed of pharmacists of each hospital was formed to develop analytical documents. a -month prospective study was conducted in two general hospitals: in the first one, in an emergency department, and the other one, in a medicine department. patients included in the study were either elderly and/or had polypharmacy and/or were hospitalized for iatrogenic reason. at int j clin pharm ( ) : - the point of admission and discharge for each patient, the pharmacist has completed a conciliation record, and has detected potential discrepancy. unintentional discrepancies were reviewed and corrected by doctors. at the discharge, medication changes were sent to general practitioner and community pharmacies. a satisfaction survey about this process was sent to healthcare professionals (gp, pharmacist and nurses). a medication reconciliation's workshop was organized for a hundred healthcare professionals in the area. main outcome measures: at the point of admission, the conciliation record included the list of patient's home medication, admission medical orders, and the types of discrepancies. at the discharge, drugs prescribed were compared to admission medical orders. the satisfaction survey included seven questions to assess the process. results: during the study period, patients were included corresponding to prescription lines. reconciliation process required about min per patient. we identified at admission unintentional discrepancies. the most common unintentional discrepancy was the omission of medication ( %). % concerned alimentary tract and metabolism group. at the discharge, no discrepancies were found; the process required min per patient. % of healthcare professionals answered to our satisfaction survey to date. % are satisfied and believe that the process of medication reconciliation secures the patient medicinal treatment. healthcare professionals were present at the medication reconciliation's workshop, indicating an interest in the process. conclusion: in this experience of medication reconciliation, due to unintentional discrepancies observed, we had better implement this activity in the two general hospitals. a pharmacist devoted to this activity will be hire in each hospital. this relevant practice is well accepted by clinician. thus, we will improve communication with gp and community pharmacies. please specify your abstract type: descriptive abstract (for projects) background and objective: the sickle cell disease (scd) is a genetic, chronic disease, paroxystic in its unpredictable and polymorphic acute events. this most frequent genetic illness in the world is a major public health concern in french overseas territories. haute autorité de santé (has) recommendations for the care of scd advocate the development of therapeutic patient education (tpe). in martinique (french west indies), we consider the population of patients with scd in among which are followed in the adults sickle cell centre (ascc). one of the actions carried out by the ascc of our hospital is the tpe. the objective is to set up an original (because specific in the scd) tpe method, which enables the patient to live better with his disease on a daily basis, by teaching him and his family to recognize prematurely certain complications. design: we analysed needs from the outcomes of a national french survey has which one participated martinique and retained the following themes: the red blood cell, the genetic transmission, the main symptoms, the role of the water, the medicinal treatments and the questions of everyday life. we chose the innovative educational tools called the ''malles des savoirs Ò **'', a set of unusual experiments, accessories and models, which, by using a method of active pedagogy ''omca*: observer, manipuler, comprendre, agir ***'', value the learner by offering to him to manipulate and to experiment by himself. results: in , healthcare professionals (doctors, pharmacists, nurses) and a president of patients with scd association followed one week of formation in the omca* method for the animation of six workshops for - teenagers and adults. every ''malle des savoirs Ò **'' contains the necessary material for the animation and a guide of the organizer, including, for every tackled issue, a generic introduction, a presentation of the themes, the index cards of educational animations proposing the activities and one time of synthesis grouping the approaches concepts. the interactive manipulation allows the appropriation of the discoveries become then long-lasting experiences. a final evaluation allows to spot the problems met by learners to understand, to analyse the difficulties and to proceed to the useful adaptations during the next activity. conclusion: this tool, playful and perfectly adapted to the scd, engages, accompanies and helps patients in the construction of their own knowledges to return them actors of their disease. in , we shall estimate the impact of the development of this specific tpe programme of the patient with scd. please specify your abstract type: research abstract background and objective: to investigate the frequencies and clinical relevance of unintentional medication discrepancies, between preadmission medication lists and discharge medication lists, at discharge from hospital. a discrepancy is considered unintentional if there is no documentation explaining the intent of the medication change or if it is unintentional according to the prescribing physician. setting and method: systematic literature review. main outcome measures: frequency of unintentional medication discrepancies per patient and per medication; frequency of clinically relevant medication discrepancies. results: of the patients included - % experienced at least one unintentional medication discrepancy. of the medications used by the patients, - % were involved in unintentional medication discrepancies. of unintentional medication discrepancies found in five studies, - % were clinically relevant. conclusion: the review documented a high frequency of medication discrepancies, of which many were clinically relevant. ensuring sufficient communication of correct and complete medication information in transitions of care is a process which should be better implemented, to enhance patient safety. please specify your abstract type: research abstract background and objective: to investigate the frequency of medication changes not documented in the discharge letter, at discharge from hospital, for both regular, as needed and over-the-counter medications, supplements and herbal remedies (otc). secondary, differences between variables and patients with undocumented medication changes were investigated. setting and method: the patients included were all part of the intervention groups from an intervention study, conducted by one of the authors (tg), from april to december . the best possible discharge medication list was compared against the medication list in the discharge letter and any discrepancy between the two lists was noted, taking into account the text in the discharge summary. main outcome measures: the proportion of patients affected by at least one undocumented medication change at discharge and proportion of medications with undocumented changes. the proportion of patients was compared using a test according to gender, age, number of preadmission/discharge medications and length of hospital stay. results: two hundred patients were included in the study. the proportion of patients experiencing at least one undocumented medication change for the three subgroups: regular medications; as needed; otc, were , and % respectively. the proportion of medications involved in undocumented changes for the three subgroups were , and % respectively. the proportion of patients experiencing undocumented medication changes was significantly higher in patients with more than five regular medications at admission, (p \ . ) and at discharge (p \ . ). in both regular and as needed medications, the proportion of patients experiencing undocumented medication changes was higher in patients hospitalized longer than days (p \ . and p: \ . respectively). for otc, the rate of patients experiencing undocumented medication changes, was higher in females (p: \ . ). conclusion: a high proportion of patients are affected by at least one undocumented medication change and many medications are involved in undocumented changes. correct and complete medication information at admission and discharge may resolve many of these errors, ensuring patent safety at transitions of care. hp-ce : participation in courses at learning and mastery centre and the impact on patients' beliefs about medicines merethe nilsen *, , erik oie , kirsten k viktil diakonhjemmet hospital pharmacy, department of internal medicine, diakonhjemmet hospital, oslo, norway please specify your abstract type: descriptive abstract (for projects) background and objective: patients with chronic diseases are referred to learning and mastery centre (lmc) where the main objective is to support patients to cope with chronic diseases. education about the disease(s) (by a physician) and the medication treatment (by a clinical pharmacist) are important elements of these courses. little is known about how the participation at lmc influences the patients' beliefs about medicines. design: patients c years participating at a days course at lmc regarding acute coronary disease or atrial fibrillation were included in the period september -december . the patients filled out 'beliefs about medicines questionnaire'(bmq) before and immediately after the course, and also months after the course to evaluate their concern (bmq-concern) and necessity (bmq-necessity) of their cardiovascular medications. the bmq scores were dichotomized at scale midpoint (scale - ) to evaluate high and low concern and necessity, and these scores were combined to calculate the 'ambivalence'and 'acceptance', 'sceptical', and 'indifferent'rate to medications, and also the mean scores of the bmq were calculated. results: fifty patients were included, mean age years, % were women, using a mean of . cardiovascular drugs taken regularly. fifty-eight percent of the patients had high concern prior to the course, whereas and % had high concern immediately after and months after the course, respectively. ninety-nine percent of the patients assessed their medication as highly necessary before the course, % immediately after, and % months after the course. the mean score for bmq-necessity was . (sd . ) prior to course and . ( . ) and . ( . ) immediately after and months after the course, respectively. the corresponding scores for bmq-concern were . ( . ), . ( . ), and . ( . ), respectively. the proportions of patients classified to be 'accepting'were , , and % at the three time points, respectively, and the corresponding numbers for patients classified as 'ambivalent'were , , and %, respectively. conclusion: the lmc course had an immediate positive influence on the patients' concern about their medicines and on 'acceptance'. however, the effect seems not to persist over time. a closer follow-up could be discussed. please specify your abstract type: research abstract background and objective: the narrative-based medicine was intended primarily for health care professionals, and the use of narratives can be applied in any settings to better understand the meaning of own profession, to rediscover/strengthen the motivation to work as a team. the italian society of hospital pharmacist (sifo) promotes a qualitative study aimed at getting the real picture of pharmacist's role within the national health system (nhs), the interaction with other health professionals and patients through the narratives of under specialization pharmacists (ui) and pharmacists already working in the nhs (hp). these data can be further investigated to increase the perceived value/role of the pharmacist. setting and method: sifo hps and uis joining the national pharmacy school specialization network were invited to participate. all pharmacists participating to the study were given a semi-structure interview. the methodology was developed within the conceptual framework of the grounded theory (gt) a research methodology that arises in the context of qualitative research. gt is a systematic methodology involving the construction of theory grounded in data systematically gathered and analysed. main outcome measures: analysis of narratives. narratives were analysed according to the classifications of kleinman, frank and launer and robinson together with transitional analysis (ta). results: a total number of narratives were collected ( ups and hps). narratives from both group of participants show the need of strengthening the professional identity already in the early years of the pharmacy curriculum and more effectively during the years of specialization as well as the need of being educated to deliver patientcentred care as members of an interdisciplinary team. conclusion: this is the first step of a study that also includes patient's contribution to the definition of pharmacist's professional identity. hp-ce : impact of pharmaceutical counselling on cancer patients' information desire and treatment satisfaction stephanie wuyts *, , jacques de grève , veerle foulon , hilde collier , pieter-jan cortoos pharmacy, medical oncology, university hospital brussels, brussels, faculty of pharmaceutical sciences, catholic university of leuven, leuven, belgium please specify your abstract type: research abstract background and objective: appropriately educating onco-/haematological patients is a prerequisite to improve patient empowerment, satisfaction and outcomes. objective: to quantify patients' information need and satisfaction on cancer drug therapy and how this can be improved by clinical pharmacist's counselling. additionally, the pharmacist's impact on therapy quality and costs is assessed. setting and method: setting: prospective, randomised study in the ambulatory ( beds) and in-hospital onco-/haematology unit ( beds) in a tertiary hospital. inclusion criteria: adult patients on intravenous or oral cancer therapy, with informed consent. methods: all patients were asked to complete standardised surveys (extent of information desired, eid; patient satisfaction with cancer treatment education, ps-cate and cancer satisfaction of treatment questionnaire, ctsq) on three occasions (at the start of a new therapy, during the second cycle and after months). patients in the intervention group received additional counselling by a clinical pharmacist including medication reconciliation and review. control patients received standard of care (information on drug therapy was provided by the onco-/haematologist, followed by limited administration instructions by nursing staff). main outcome measures: patient information desire and satisfaction on cancer treatment results: patients were included over a period of months (control (n = ); intervention (n = )). no significant differences were found between contact moments or patient groups for eid, ps-cate and ctsq-scores. however, scores for ps-cate on medication side effects were positively correlated with contact moment (r s = . ; p = . ). multiple linear regression analysis showed a similar trend (b = . ; p = . ). patients receiving first-line therapy (b = . ; p \ . ) and ambulatory patients (b = . ; p = . ) were more satisfied on treatment education. the clinical pharmacist documented more drugs than were recorded in the patient file ( vs. . drugs/patient; p \ . ). on average, each patient required two pharmacist's interventions per occasion. intervention acceptance rate on drug related problems was high ( %). during the study, interventions shifted from therapy adjustments towards advice on supportive measures ( st contact: %; rd contact: %). improved medication stock control on the ward led to a savings of € , . conclusion: the clinical pharmacist can play an important role on the onco-/haematological ward, leading to improved drug reconciliation, patient counselling and cost savings. hospitalised patients and patients receiving salvage therapy appear to have higher educational needs, making them possibly overlooked target groups. finally, pharmaceutical counselling should be repeated and primarily focused on side-effect management to have a meaningful impact on patient satisfaction. please specify your abstract type: research abstract background and objective: europe is ahead of the usa and canada on approval, regulatory and marketing aspects of biosimilars. however, there is still uncertainty about interchangeability and substitution of biosimilars. the aim of the study is to assess pharmacists' perceptions about biosimilar interchangeability. setting and method: a cross-sectional study was carried out in june-july . hospital pharmacists from quebec and france were invited to respond to an online survey of nine questions (surveymonkey Ò , palo alto, ca, usa). the survey focuses on pharmacist's exposition to biosimilars (general knowledge, dispensing) and their perceptions about biosimilar interchangeability. a -item likert scale was used to answer to statements based on key issues about biosimilar interchangeability. main outcome measures: levels of agreement on biosimilar interchangeability key issues. results: a total of pharmacists responded ( % in quebec vs. % in france). the global response rate is: % ( % quebec vs. % france) (n = / ). % attended at least to one conference on biosimilars ( vs. %). % had already dispensed biosimilars ( vs. %). more than % of the pharmacists knew that: biosimilars can cause immunogenicity, clinical studies are requested for their approval, automatic substitution is not permitted. % considered that post-marketing surveillance for biosimilars should be reinforced. pharmacists considered that biosimilars are cheaper than the reference product ( vs. %). there was no difference between the level of agreement of french and quebec pharmacists for the statements. pharmacists agree that a list of biosimilar and interchangeable biologic products is necessary ( vs. %), using the international nonproprietary name to prescribe a biological product can create confusion between the reference product and its biosimilar ( vs. %), pharmacists should check if patients already experienced an immunogenic reaction before dispensing a biological product ( vs. %). pharmacists disagree that a biosimilar can be used for all the indications of the reference product ( vs. %). conclusion: perceptions of quebec and french hospital pharmacists about biosimilar interchangeability issues are very similar. this study highlights the need to deal with the lack of clarity of national guidances. clinical studies on biosimilar interchangeability must be conducted in the future to help pharmacists and physicians to take clear-headed decisions. please specify your abstract type: research abstract background and objective: analgesics are essential drugs in hospitals and especially in emergency units. medical and nurse staffs are used to the narcotic status of opioids. for some drugs, a regulatory change to narcotic status can discourage their use. for others, it could limit their access particularly in developing countries; that's why who did not recommend ketamine to be placed under international control (http://www.who.int/medicines/access/controlled-substances/ recommends_against_ick/en/). yet, the french drug agency has recently considered to register drugs containing ketamine as narcotics. the aim of this study was to assess the impact of this possible regulatory change on the pharmaceutical and medical practices in some paediatric french hospitals. setting and method: the survey was conducted in january-february in four parisian paediatric hospitals: four pharmacies, paediatric neurology and anaesthesia departments, intensive care units and pain management services. main outcome measures: pharmacists, clinicians, health managers and nurses were interviewed, using a standardized questionnaire with closed and opened questions, on the drug circuit including ordering, storage, distribution, prescription, administration and destruction. results: all the health professionals (five pharmacists, ten clinicians, five nurses) indicate that the change to narcotic status would not preclude the use of an analgesic drug. they consider that the pharmaceutical aspects (dispensation, storage and transport, etc.) are not limiting, provided that clinical usefulness is demonstrated: short action onset allowing rapid efficacy, short duration of action allowing the replacement by another drugs if needed, and moderate clinical monitoring. change to narcotic status was rather seen as advantageous since allowing better traceability, use and prescription. half of the pharmacies (n = / ) had a computerized register of narcotics and % of care units (n = / ) had a drug staffing in addition to nominative prescriptions, which was used in all care services. the drugs were kept into secured rooms. none of the emergency units (n = / ) had a computerized secured cabinet. conclusion: according to this survey, narcotic status is not a limiting factor for a drug use in paediatric hospitals, when its clinical usefulness is clearly demonstrated. to promote its use, it is important to inform medical and nurse staffs and include it into care protocols. beyond the nominative prescription, implementation staffing is a key step. please specify your abstract type: research abstract background and objective: port-a-cath is an implanted venous access device most commonly used for frequent or continuous chemotherapy administration. however, the procedure and its subsequent maintenance are not free of complications and requires additional intervention by the clinical pharmacist who can provide further patient care to make a positive impact on. to assess the effective provision of appropriate patient counselling offered by a clinical pharmacist on reducing port-a-cath relatedcomplications in cancer patients. setting and method: a controlled prospective observational study carried out on patients newly diagnosed with cancer eligible for chemotherapy administration at the oncology unit. assessment of port-a-cath related-complications were assessed at regular schedule of chemotherapeutic protocols administration. main outcome measures: to assess, reduce and solve port-a-cath related-complications. results: the most significant port-a-cath related complications were skin rash . % (p \ . ) with occurrence in males (n = ) and females (n = ), skin erythema . % with equal occurrence in both genders, followed by skin discharge . % with also equal occurrence in both genders. a high occurrence of skin rash . % occurred among diabetic cancer patients. a significant improvement in port-a-cath related complications after the provision of patient counselling by the clinical pharmacist was observed as skin rash ( . %), skin discharge ( . %), and skin erythema ( . %). conclusion: results of this study pointed out the essential role of clinical pharmacist in argumenting patient care and improving port-a-cath related-complications in cancer patients. please specify your abstract type: research abstract background and objective: polytherapy, frequently used in the elderly, is associated to an increased risk of potential drug-drug interactions (pddis) and adverse drug reactions (adrs). literature demonstrated that medication reconciliation and medication review performed by hospital pharmacists are correlated to drug related problems (drps). aim: to define a structured and feasible model where hospital pharmacists support clinicians identifying drps and promote the safe use of medicines. setting and method: prospective, feasibility study conducted in four internal medicine wards of a hospital in northern italy. inpatients (c years old, treated with c drugs) were consecutively included; the recognition/reconciliation process was performed by pharmacists in order to identify changes between prescription profile at home and during the admission (active principles, dose, administration route). these changes were classified as intentional documented discrepancies (id), not documented (ind), not intentional (ni). prescriptions during the first -hours of hospitalisation were analysed to retrieve drps (ddis, inappropriate medications for elderly, off-label, over/ under dosage, duplications, adrs) then discussed with clinicians. based on literature, referring almost drp in % of patients, a sample size of patients should allow an estimate of drp rate over % (need of intervention) with a % power and a confidence interval of % (software stata version . ). main outcome measures: rate and type of: discrepancies, drps at admission and discharge, pharmacists consultations accepted by clinicians. results: ad interim results are presented. between october/ -february/ , inpatients ( male, . mean age) were included. overall, patients were admitted with drugs used at home and prescribed during the first -hours; pharmacists retrieved discrepancies ( %id, %ind, %ni) and drps, of which % ddis, % off-label, % overdoses, % duplications, % inappropriate drugs, % not notified adrs. the % of drps was known to clinicians and % considered clinically relevant for the patients. please specify your abstract type: research abstract background and objective: hypertension is a major risk factor for cardiovascular morbidity and mortality worldwide, for which management is based on two principal, complementary approacheslifestyle modification and lifelong treatment with antihypertensive medication. adherence to hypertension therapy is a major public health challenge, despite the availability of multiple classes of antihypertensive agents. factors contributing to non-adherence are multifactorial and include intolerances to drugs at standard doses that result in therapy discontinuation. medication intolerance (mi-htn) refers to patients who experience adverse drug reactions (adrs) to at least one antihypertensive medication, without a known immunological mechanism and the need to discontinue them. we sought to determine factors associated with mi-htn and to identify patients' beliefs and concerns about their antihypertensive treatment and medication in general. setting and method: a cross sectional survey consisting of selfreported questionnaires including beliefs about medicines questionnaire (bmq), perceived sensitivity to medication (psm) and quality of life was undertaken in an unselected patients attending a hypertension centre of excellence out-patient clinic based in london. main outcome measures: to determine factors associated with mi-htn and the impact of health beliefs and self-reported perceived sensitivity to medications on mi-htn and bp control. chi squared tests for comparisons between cases/controls and multiple logistic regression analysis were used for statistical analysis. results: participants were included, of which ( %) participants had mi-htn. two-thirds were female (p = . ) with a mean age of ± years (p . ), of whom . % had uncontrolled hypertension (p = . ). calcium channel blockers were the most commonly reported intolerance by drug class followed by diuretics. being female and age [ were statistically associated with a greater likelihood of reporting medicines intolerance (p \ . ). patients who believed that medicines are harmful were [ -times more likely to report mi-htn (p = . ) and -times more likely to have uncontrolled bp ([ / mmhg) (p = . ). patients with high self-perceived sensitivity to medication was -times more prone to mi-htn (p = . ). conclusion: our findings suggests the need for greater focus on behavioural change interventions to both improve patients' perception of the necessity to persist with lifelong antihypertensive medication and allay concerns regarding harmful effects of drugs may help with long term control of hypertension. please specify your abstract type: research abstract background and objective: today, the number of medical problems in heart transplant recipients has increased due to aging and complications common to immunosuppressive drugs. the co-existence or emergence of other disease states such as renal dysfunction, infection, diabetes, obesity, hypertension, hyperlipidaemia, malignancies, and osteoporosis necessitates the use of other medications. the use of these medications in combination with immunosuppressive agents increases the risk of drug-drug interactions. the aim of this study is to identify the frequency and significance of drug-drug interactions for the patients who received cardiac transplantation. setting and method: this retrospective study was conducted at a cardiovascular specialty hospital. all patients who received cardiac transplantation from the same surgery team between and ( years) were included in the study. all data were collected from the medical records of the patients. only the most recent prescription before discharge was analysed for the presence and significance of drug-drug interactions. drug-drug interactions were checked using micromedex(r) interaction checker. main outcome measures: main outcome measures were the frequency and significance of drug-drug interactions. results: a total of patients met the inclusion criteria and prescriptions were analysed. each prescription contained an average of drugs. a total of drug-drug interactions were identified: . % was classified as moderate; . % as major and . % as contraindicated. almost half of all interactions (n = ) included immunosuppressive agents ( . % was classified as moderate; . % as major and . % as contraindicated). conclusion: cardiac transplant recipients were found to have a high number of drug-drug interactions. in order to advise on these interactions which increase with poly-pharmacy, drugs with narrow therapeutic index or drugs that require intensive monitoring, it is recommended to include a transplantation pharmacist in the transplantation team. please specify your abstract type: research abstract background and objective: the aim of our study was to assess the impact of patient education provided by the pharmacist on gylcemic control, medication knowledge level and medication adherence of patients with type diabetes. patients who were diagnosed with type diabetes for at least one-year time and were receiving at least one antidiabetic medication, attending to the outpatient diabetes clinic for the control visit were informed about the study and invited to participate in the study. patients who gave their informed consent were included in the study. setting and method: the setting is a diabetes outpatient clinic of a state hospital. the medication knowledge levels, medication adherence scores, fasting blood glucose levels, hba c levels and blood pressure of the patients were measured before pharmacist's education. after provision of standard information and individualized patient education all these parameters were measured again after monthstime and the impact of the education was assessed. main outcome measures: main outcome measures are change in the clinical parameters (hba c; fasting blood glucose; blood pressure), as well as improvements in medication knowledge and adherence levels. results: the study was conducted on patients who met the inclusion criteria; none of the patients were lost to follow-up. majority ( %) of the patients was female and the mean age was . years. pharmacist intervention resulted in positive outcomes at all clinical parameters. systolic blood pressure decreased by mmhg, while diastolic blood pressure decreased by . mmhg (p \ . ). hba c level decreased by . % (from . to . %; p \ . ) and fasting blood glucose level by . mg/dl (p [ . ). on the other hand, the number of patients reaching the blood pressure goal increased from to ; and those reaching to hba c goal increased from to (p \ . for all). similarly, the medication knowledge level [usual range - ] increased from . to . (p \ . ); and the medication adherence score [usual range - ] increased from . to . (p \ . ). conclusion: it can be concluded that pharmacist's contribution results in positive outcomes in glycaemic control and management of co-morbid conditions of type diabetic patients by improving medication knowledge and adherence levels of the patients. pharmacists should take active role in management of chronic diseases. hp-pc : impact of a pharmaceutical care program on glycemic control, medication knowledge and medication adherence levels of type diabetic patients residing at a nursing home nimet saglam *, , sule apikoglu-rabus , betul okuyan , fikret v. izzettin , nuran yildirim clinical pharmacy department, marmara university faculty of pharmacy, darulaceze nursing home, istanbul, turkey please specify your abstract type: research abstract background and objective: the aim of our study was to assess the impact of pharmaceutical care provided by the pharmacist on glycaemic control, medication knowledge level and medication adherence of patients with type diabetes residing at a nursing home. setting and method: this prospective cohort study was conducted in a state nursing home (darülacaze nursing home) in istanbul, turkey on patients who completed the whole study. all the patients received pharmaceutical care provided by the pharmacist. this pharmaceutical care program was held for months. it consisted of an initial visit, followed by ''care and control'' visits and a final control visit; each visit was held at two-week time intervals. at the initial visit, demographic and general clinical data were collected and medication knowledge and medication adherence levels of the patients were also assessed. pharmaceutical care needs were identified for each patient and recommendations addressing these issues were structured. education regarding the medications of the patients was provided in both verbal and written forms using the standard patient education leaflets prepared by the pharmacist. at each visit pharmaceutical care needs are assessed and pharmaceutical care is tailored accordingly. main outcome measures: main outcome measures are change in the clinical parameters (hba c; fasting blood glucose), as well as improvements in medication knowledge and adherence levels. results: majority ( %) of the patients was male and the mean age was . years. pharmacist intervention resulted in positive outcomes regarding hba c levels. hba c level decreased by . % (from . to . %; p \ . ) and fasting blood glucose level by mg/dl (p [ . ). similarly, the medication knowledge level [usual range - ] increased from . to . (p \ . ); and the medication adherence score [usual range - ] increased from . to . (p \ . ). conclusion: it can be concluded that pharmacist's contribution results in positive outcomes in glycaemic control of type diabetic patients by improving medication knowledge and adherence levels of the patients. pharmacists should take active role in management of type diabetes at the nursing home setting. please specify your abstract type: research abstract background and objective: haemoglobin variability is related to mortality and morbidity in haemodialysis, renal transplantation and pre-dialysis patients. some demographic, haematological and pharmacological variables may affect hb variability. but there are some controversies about the influences of different erythropoiesis stimulating agents (esa).the objective of this study is to determine the influence of different esa on haemoglobin variability in pre-dialysis patients. setting and method: we conducted a prospective observational study with chronic kidney disease patients recruited from outpatients of nephrology department of a tertiary university hospital (from january to june ). exclusion criteria were: stage i and ii, not treated with esa, haemodialysis, peritoneal dialysis, renal transplantation, thalassemia, and deficit of glucose- -phosphate dehydrogenase . main outcome measures: patients included were treated with esa in maintenance phase (stable months prior).hb variability was calculated by standard deviation (sd) and residual standard deviation (residual sd) of hb levels. statistical analysis was performed with spss . (spss inc, chicago). observation period was months and data were recorded from the clinical records. ( ) . %, sofosbuvir/daclatasvir/ribavirin ( ) . %, sofosbuvir/simeprevir ( ) . %, sofosbuvir/ledipasvir ( ) . %, sofosbuvir/ledipasvir/ribavirin ( ) . %, dasabuvir/ombitasvir/paritaprevir/ritonavir ( ) . %, dasabuvir/ombitasvir/pari taprevir/ritonavir/ribavirin ( ) % ombitasvir/paritaprevir/ritonavir/ ribavirin ( ) . %, sofosbuvir/ribavirin ( ) . %. viral load at week was \ iu/ml in patients and at the end of treatment . conclusion: the results of rapid viral response at end of treatment were similar to those obtained in studies published to date. due to its recent access to these treatments it is necessary to continue monitoring these patients to assess virologic sustained response at weeks after end of treatment. please specify your abstract type: research abstract background and objective: fragile patients are considered those vulnerable patients with a certain degree of complexity in their care (polypharmacy, multi-pathological, palliative and/or residents in social and healthcare institutions). to ensure their continuity of care and safety in the use of drugs we applied a medication reconciliation process at admission, transition of care and/or hospital discharge. objective: to analyse the results of the medication reconciliation process of a fragile patient. setting and method: we developed a list of current medication with the following sources of information: medical history, clinical databases and information provided by the patient (interview). clinical case: -year-old woman admitted through emergency department due to severe dyspnoea. no known drug allergy. background: heart failure, chronic hypertension, hypercholesterolemia, hyperthyroidism, hyperuricemia, gouty arthritis, chronic kidney disease and cognitive impairment by alzheimer disease. exploration and complementary tests: echocardiogram and analytical control. clinical judgment: acute decompensated heart failure. acute myocardial infarction. prerenal acute kidney injury. main outcome measures: medication reconciliation made at admission with the detection of discrepancies and deprescribing criteria at hospital discharge. results: fragile patient (high-risk) with medicines as home treatment. patient was hemodynamically stable during the hospital stay. discrepancies were detected between the prescribed medication and the home treatment. discrepancies justified ( ): five by omission of medication (two new clinical situation, two therapeutic exchanges to adapt to the pharmacotherapy guide and one wrong drug) and two beginning of medication. discrepancies unjustified ( ): by omission of medication. to discharge: one antiplatelet therapy was. after the comprehensive review, we made the following recommendations of deprescription: suspend one non-steroidal anti-inflammatory drug-nsaid (by risk of bleeding in association with concomitant antiplatelet and antidepressant therapy) and one benzodiazepine (central nervous system-cns side effects); modify treatment: reduce doses of diuretics (blood pressure lowering effect). pharmacotherapeutic recommendations were accepted. conclusion: detection of discrepancies in the medication reconciliation and deprescription process are effective and safe strategies that allow optimization of pharmacotherapy in fragile patients. the use of drugs such as nsaids (gastrolesive effect), the combination of drugs with cns side effects and hypotensive action (associated with falls) in elderly patients constitute situations of risk that should be reviewed in fragile patients, as an essential part of the clinical evaluation. please specify your abstract type: descriptive abstract (for projects) background and objective: there are no positions for clinical pharmacists at the hospital, so we are dependent on projects to be able to show how pharmacists can contribute in the clinical team. our aim in this project was to introduce pharmaceutical knowledge by implementing medication reconciliation and medication review in different hospital wards. we wanted to show that many patients have discrepancies in their medication lists during hospital stay and that some of the drugs or doses given can cause drug related problems for the patient. our final goal was to get the physicians to be more aware of these issues when treating their patients. design: the method used was based on the two first parts of the integrated medicines management. the pharmacist conducted a standardized drug interview with patients who prior to admission were responsible for their own drugs. for patients who could not be interviewed or were not responsible for administering their own drugs, a current medication list from relevant care level was obtained. the medication lists obtained were compared to the documentation in the patient's drug chart and discrepancies communicated to the physician. during the hospital stay, a medication review and monitoring was also conducted by the pharmacist. results were presented to the patients physician and discussed. results: a total of patients were included and of these % had c discrepancy identified by the process. the most frequent type of discrepancy was the use of a drug that was not registered on admission (omission discrepancies). other discrepancies were wrong dose, dosage or formulation and registration of a drug the patient didn't use. drug-related problems were discovered in % of the patients and the most frequent were use of anticholinergic drugs in elderly, interactions, lack of treatment and monitoring and too high doses regarding kidney function. many of the detected drug related problems results in change in medication, other times the physician addresses the problem to the gp. the physicians were surprised of the high numbers of discrepancies in medication lists and drug related problems discovered. almost all the physicians considered that the pharmacist could be an important part of the treatment team and they wanted the participation of the pharmacist to be permanent. conclusion: the project led to increased awareness of the importance of medication reconciliation and medication review and showed the importance of pharmaceutical knowledge in the treatment team. unfortunately this was not sufficient to create positions for pharmacists in our hospital. new projects will focus on pharmacists teaching interns to improve the reconciliation at admission. please specify your abstract type: descriptive abstract (for projects) background and objective: we aimed to assess the quality of fluoroquinolones (fq) prescriptions at the toulouse university hospital emergency department as part of significant increase in consumption. design: retrospective mono-centric study of fq prescriptions written to adult patients managed at the emergency department (february th, -march th, . a pair consisting of a biologist pharmacist and a clinical pharmacist has analysed them using tools provided by the centre de coordination de lutte contre les infections nosocomiales (cclin). various criteria (pertinence of prescription, choice of antibiotic, dosage, duration of treatment, method of administration…) were faced with the guidelines issued by the société de pathologie infectieuse de langue française (spilf). results: about files were examined, contained fq prescriptions for systemic use. the most frequently prescribed antibiotic was ofloxacin ( %) and the most frequent indications were urinary tract infections ( %). among the prescriptions of fq, the establishment of fq was justified in % of cases and the antibiotic chosen was always the most suitable. nonconformities of dosage and/ or treatment time were found in a quarter of cases. overall, % did comply with guidelines. the prescriptions, due to the particularity of emergency were still permormed probabilistic. however, a reassessment of them was scheduled for two-third of outpatients. conclusion: this study highlights the conformity of less than half of the prescriptions. this demonstrates that there are still actions to ensure the accuracy of fq prescriptions. and it is in this sense that this audit should be registered under the impetus of the committee on anti-infectives. it will raise awareness among doctors in the proper use of this family of antibiotics. please specify your abstract type: descriptive abstract (for projects) background and objective: the number of persons suffering from end-stage renal disease (esrd) is growing worldwide, mainly due to the aging of the population. esrd incidence has been increasing by - % per year for years. it is estimated that worldwide, more than . million patients with established renal failure are being treated with haemodialysis (hd). water for haemodialysis must meet the physicochemical and bacteriological compliance standards defined by the european pharmacopoeia. as a medicine, this water is placed under the responsibility of hospital pharmacists. addressed to hospital pharmacists, this methodology guide will enable them not only to validate controls of haemodialysis water as well as drug prescriptions for dialysis patients, but also to familiarize themselves with the best currently existing dialysis techniques and medical devices. we have tried to simplify and synthesize existing circulars and guidelines so as to render them more readily understandable for the pharmacist in charge of a haemodialysis service, and thereby help to ensure optimally safe treatment of haemodialysis patients. design: the themes developed in this guide are: • a review of the different existing dialysis techniques, • a review of the different sampling points for controls of hd water, • a review of the physicochemical and bacteriological standards of these controls according to the latest recommendations of the european pharmacopeia, and of appropriate conduct for exceeding established thresholds, • a review of the main international recommendations with regard to clinical signs of chronic kidney disease: anaemia, mineral and bone disorders (ckd-mbd), high blood pressure. • a review of the various medical devices used in haemodialysis and haemodiafiltration. results: the recommendations of good practices summarized in this guide are integrated perfectly adapted to the concept of quality assurance and its role in the accreditation process. they are focused on improving patient safety by harmonizing pharmaceutical haemodialysis practices in different dialysis centres. conclusion: these types of recommendations may be transposable to other pharmaceutical fields and/or be used as a training tool for pharmacy students or young pharmacy school graduates. the format of this guide makes it convenient, easy to use every day. it will be revised regularly to ensure the sustainability of quality plans. please specify your abstract type: descriptive abstract (for projects) background and objective: combination antiretroviral therapy (cart) has strongly improved disease control in hiv-infected patients. however, aging and comorbidities are becoming a major problem in this group of patients. most hiv-infected patients are treated with five or more medications, and harms by polypharmacy increase proportionally with number of medications. possible risks include: poor medication adherence and consequently inefficient care, increased risk of drug interactions and adverse events, with prolonged hospitalization. the problem is worsened when patients are of nonnative language and so their comprehension and adherence to drug therapy can be very poor, compromising efficacy. the hivig study is designed to evaluate the impact of the interventions promoted by the clinical pharmacist in the optimization and comprehension to personal drug therapy, favouring compliance, in a cohort of patients, hiv infected with comorbidities like cancer; the cohort includes a high number of non-native italian language individuals. design: hivig is a randomised, parallel groups clinical trial. in april the study protocol was approved by the local ethical committee, aviano. the project is scheduled to start in autumn . main objective: evaluation of the impact of a series of tools-''drug therapy setting interventions'' (dtsis) applied by the clinical pharmacist on a cohort pf hiv-infected patients with comorbidities, afferent for care at cro aviano. the treatment arm will be submitted to dtsis. dtsis interventions (treatment group) consist in: motivational interview, sharing and delivery of printed, explanatory material in the patient's native language, reconciliation of patients medications at hospital admission and at discharge; identification of potential risks due to drug-drug interactions; monitoring of compliance to drug therapy, and finally detection of adverse drug reactions (adrs) occurring in the course of care. the control group will undergo only to scheduled standard medical visits at cro. results: we expect to recruit a total patients for a -months period of follow-up. statistical analysis will be performed by intention-to-treat and by protocol. at cro aviano, the italian cooperative group on aids and tumors (gicat) has studied malignancies in hiv-positive patients since and has a leading role for studies conducted in italy (vaccher, ) conclusion: previous collected data from the previous trial performed at cro aviano (target-vig), showed a positive impact in the optimization of individual drug therapy and in the reporting of adrs. hiv has an enormous impact on life of infected patients and represents a priority issue for the entire community. we consider the method of dtsi, combined with a close monitoring of patients by means of telephonic motivational interviews, the best added value performed by the profile of the clinical pharmacist in optimizing drug therapy and personal awareness about medicines. please specify your abstract type: research abstract background and objective: the world health organization reports that ''one in four people in the world will be affected by mental or neurological disorders at some point in their lives. around million people currently suffer from such conditions, placing mental disorders among the leading causes of ill-health and disability worldwide». to review the evidences published about the roles and the impact of pharmacists in psychiatry. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, mental illness and psychiatry from january st until june th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in psychiatry. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), medication reconciliation ( ), patient care needs assessment ( ), drug therapy assessment ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ), competencies maintenance ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome measures. of these outcome indicators, ( %) were positive, neutral and negative (knowledge transfer strategy). positive impacts of pharmaceutical interventions were identified in the following areas: morbidity ( ), patient adherence ( ), patients or clinicians satisfaction ( ), side effects management ( ), medication errors prevention ( ), mortality ( ) please specify your abstract type: research abstract background and objective: the world health organization reports that . million people die each year from cancer, an estimated % of all deaths worldwide and that there is a % increase in new cases of cancer expected over the next two decades. to review the evidences published about the roles and the impact of pharmacists in cancer. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, neoplasms from january st until june th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions as well as descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in cancer. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), patient care needs assessment ( ), drug therapy assessment ( ), drug compounding/dispensing ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome measures. of these outcomes indicators, ( %) were positive, ( %) neutral and ( %) negative. positive impacts of pharmaceutical interventions were identified in the following areas: morbidity ( ), patient adherence ( ), patients or clinicians' satisfaction ( ), side effects management ( ), medication errors prevention ( ), mortality ( ), costs ( ) setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, myocardial infarction, acute coronary syndrome from january st until june th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in myocardial infarction. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), medication reconciliation ( ), patient care needs assessment ( ), drug therapy assessment ( ), drug compounding/dispensing ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome int j clin pharm ( ) : - measures. of these outcome indicators, ( %) were positive, ( %) neutral and ( %) negative. positive impacts of pharmaceutical interventions were identified in the following areas: morbidity ( ), patient adherence ( ), side effects management ( ), mortality ( ) and others ( ). conclusion: the role and the impact of pharmacists have been studied in myocardial infarction and % of outcome indicators used in these studies show a positive impact of pharmaceutical interventions. pharmacists should pay attention to these evidences to improve their practice, contribute to prevention or insure treatment of patients with potential or found myocardial infarction. hp-pc : impact of pharmaceutical care in vaccination: a review of literature please specify your abstract type: research abstract background and objective: the world health organization reports that ''immunization is the process whereby a person is made immune or resistant to an infectious disease, typically by the administration of a vaccine and a proven tool for controlling and eliminating lifethreatening infectious diseases and is estimated to avert between and million deaths each year. it is one of the most cost-effective health investments, with proven strategies that make it accessible to even the most hard-to-reach and vulnerable populations». to review the evidences published about the roles and the impact of pharmacists in vaccination. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, vaccination and immunization from january st until july th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in vaccination. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), medication reconciliation ( ), patient care needs assessment ( ), drug therapy assessment ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ), competencies maintenance ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome measures. of these outcome indicators, ( %) were positive, ( %) neutral and negative. positive impacts of pharmaceutical interventions were identified in the following areas: cost ( ), errors ( ), morbidity ( ), patient adherence ( ), patients or clinicians satisfaction ( ) please specify your abstract type: descriptive abstract (for projects) background and objective: evaluating the appropriateness and effectiveness of the patient's medications by analysing prescriptions is pharmacist side work. bedside drug administration and computerised drug administration traceability (cdat) in nursing care plan (ncp) are nurse's one. however, in order to check adherence, efficiency and tolerance of a drug, pharmacist has to ensure that the patient takes the medication appropriately. therefor ncp could be a useful tool. the aim of this study is to evaluate the effectiveness of cdat, and if not, define causes of divergences with real life situation. design: • comparison between unused drugs remained in individual patients' seven daily pill dispensers (considered as not taken) which come back from the evaluated service to the pharmacy, and their cdat status completed by nurses (taken, not taken or no status) • two recorded data, each collecting a three-week period, separated by a period of discussion with nurses: first results presentation, analysis of divergences by taking into account their feedbacks, and actions to raise their awareness about the importance of cdat. • pill dispensers' cdat is correct only if all returned drugs' status in ncp is ''not taken''. results: during the first period (n = pill dispensers), . % of pill dispensers had an incorrect cdat status. on average, . drug per pill dispenser didn't have an appropriate status in ncp (taken or no status). major causes of divergences were the lack of time and insufficient human resources, the fact that they often are interrupted in the middle of this task, a software which isn't ''user-friendly'' and a deficit of information about the issue. corrective actions were implemented, prior to the second recorded data period, targeting human factor of divergences (oral and written reminders about cdat with didactic memorandum on computers). after awareness actions, results (n = ) were . and . respectively. conclusion: efforts about cdat have been done but not enough to observe a significantly improvement in short terms. ncp's level of reliability is not optimal yet and still dependent on nurses' practices. this study allowed us to strengthen the relationship between clinical service and pharmacy, and opens the way for further works particularly through corrective actions targeting material and organizational causes of divergences. please specify your abstract type: descriptive abstract (for projects) background and objective: in , our teaching hospital has participated to a worldwide survey (global point prevalence survey (global-pps)) aimed to explore antimicrobial consumption and resistance in hospitals. because broad spectrum antibiotics have to be followed with the attention of resistance prevention, we focused our analysis on these antibiotics in our hospital (carbapenems, piperacillin/tazobactam and amoxicillin/clavulanic acid). design: the survey was performed by pharmaceutical team (senior and resident) with help of microbiologists and referring physicians. all wards of the hospital were included. hospitalized patients treated with antimicrobial agent (j , j , j a, p ab, a , j ah, p b from the atc classification) prescribed at a.m. on the day of the survey, were involved. from those who were treated by carbapenems, pip/taz or amx ac, following data were collected: age, gender, weight, doses, indications (probabilistic? documented? and if documented microbiological data), and mention of stop/review date of prescription. results: the survey was carried out from april to june in wards. among the patients included, patients ( . %) were treated with antimicrobial agents. patients ( . %) were treated with broad spectrum antibiotics: ( . %) with amx-ac, with pip-tz, with imipenem and with meropenem. the mean age of patients was . ± . and the weight was . ± . kg. their prescriptions were concentrated in three types of wards: ( . %) in icu, ( . %) in medicine, ( . %) in surgery. moreover, we observe that bsa were used to treat ( . %) community acquired infections, ( . %) nosocomial infections, ( . %) used as medical prophylaxis, or surgical prophylaxis (n = , . %). in relation to the type of treatment: were empirical treatment (including prophylaxes) and were targeted treatments ( bacteraemia, joint and bones infections, cardiovascular system infection, urinary tract infections, lower respiratory tract infections, skin and soft tissues infections and others infections). finally, extended spectrum beta-lactamase (esbl) producing enterobacteriaceae and third generation cephalosporin resistant enterobacteriaceae non-esbl producing were targeted by bsa regimen. conclusion: in this survey, use of bsa is globally compliant to french guidelines and we identified no improper prescription: multidrug resistant bacteria infections, several diseases and empirical treatments with limited duration of regimen. this shows that control of the proper use of antibiotics especially those with a broad spectrum is efficient in our hospital and has to be continued. this has been made possible due to a multidisciplinary approach including physicians, bacteriologists and pharmacists. please specify your abstract type: descriptive abstract (for projects) background and objective: in , our teaching hospital has participated to a worldwide survey (global point prevalence survey (global-pps)) aimed to explore antimicrobial consumption and resistance in hospitals. from these results, we observed that sulfamethoxazole/trimethoprim (tmp/smx) was largely prescribed in our hospital. we focused then our analysis on these results with the attention of check of its proper use. design: the survey was performed by pharmaceutical team (senior and resident) with help of microbiologists and referring physicians. all wards of the hospital were included. hospitalized patients treated with antimicrobial agent (j , j , j a, p ab, a , j ah, p b from the atc classification) prescribed at a.m. on the day of the survey, were involved. from those who were treated by tmp/smx, following data were collected: age, gender, weight, doses, and indications (probabilistic? documented? and if documented microbiological data), and mention of stop/review date of prescription. please specify your abstract type: research abstract background and objective: in france, benzodiazepine (bzd) is frequently prescribed in elderly people (ep). long-term efficacy is often questioned, and treatment has to be regularly re-examined, especially in ep. in our geriatric day-hospital for assessment of frailty, a multidisciplinary team evaluates the patients and gives them preventative measures against the loss of autonomy. medication evaluation is part of these measures. the aim of our study was to evaluate the impact of a standardized intervention on the optimization of bzd treatment. setting and method: after a short interview and the delivery of an information booklet about bzd, patients were proposed an optimization of their bzd treatment (dosage reduction, occasional medication, switch to a short half-life bzd, or total discontinuation). patients were followed up monthly by a phone-interview over a -months period. main outcome measures: the main outcome measure was the prevalence of bzd optimized treatments after a months follow-up. results: patients were included. among them, % have been taking a bzd for more than years, and % were prescribed a long half-life bzd, which can be qualified as inappropriate in ep. % of the subjects were frail and % pre-frail according to the fried criteria. at the end of the study, % of the patients had their bzd treatments optimized, including % of total discontinuation. conclusion: in frail or pre-frail elderly population, a standardized intervention can be useful to improve bzd treatment. an extension to this intervention would be the creation of an organisation tasked with routinely monitoring the patients withdrawal over a month period. hba c and weight were significantly reduced by . ± . %, p \ . and . ± . kg, p \ . , respectively; systolic bp ( . ± . mmhg, p \ . ), diastolic bp ( . ± . mmhg p \ . ) and triglycerides ( . ± . mg/dl, p . ).genital-and urinary tract infections were reported by . % patients. any diabetic ketoacidosis case was reported. conclusion: sglt- inhibitors added to other oral antidiabetic drugs or insulin in patients with uncontrolled t dm significantly improved glycaemic control, reduced weight, blood pressure and triglycerides, and was generally well tolerated. in conclusion, sglt- inhibitors, appears to be an important addition to the therapeutic options for the management of type diabetes, particularly when used as add-on therapy. ( ). treatment safety takes part of the decision to undergo bariatric surgery. during multidisciplinary team meetings, the clinical pharmacist must rely on guidelines to limit drug-induced iatrogenesis. this review aims at assessing influence of bariatric surgery on the clinical impact and pk of cardiotropic drugs so as to document pharmacists' notifications. setting and method: literature review on medline- to may -with terms: cardiovascular drugs and bariatric surgery or malabsorption syndrome. related articles were reviewed. main outcome measures: pharmacokinetic or pharmacodynamic data and clinical impact of cardiotropic drugs. results: a total of titles, and abstracts when necessary, were screened for eligibility. after reviewing process, studies were included: nine concerning digoxin, five beta-blockers (bb) and one amiodarone. published studies varied in methodology: five case report, seven case control and three cohort studies. studies reported variations of digoxin plasmatic concentrations among patients versus , suggesting liquid oral form are preferred. no clinical event was notified. more the bb is liposoluble (propranolol), the higher the toxicity is, such as heart rate and blood pressure decreasing, with potential fatal outcomes. a case of amiodarone-induced hyperthyroidism is described after bariatric procedure showing an increase plasma concentration adjusted to weight. conclusion: while the impact on narrow therapeutic range drugs is documented, others cardiotropic drugs may cause serious patient injury justifying their monitoring. therefore, risk must be identified for all patients undergoing bariatric surgery to setting up closely therapeutic monitoring. further studies are still expected to lead to recommendations about posology and treatment withdrawal to improve patient safety. please specify your abstract type: research abstract background and objective: the issue of non-compliance to prescribed medical treatment has been reported to be a crucial problem in psychiatric outpatients. the aims of this study were to assess the extent of non-compliance in a cohort of psychiatric outpatients in malta and to investigate the applicability of using a -day multi-dose pill box in terms of practicality, ease of use and impact on compliance within this patient group. setting and method: the study was conducted at mount carmel hospital, a psychiatric hospital in malta. twenty outpatients were recruited by convenience sampling. the study was divided into two phases. during phase , patient compliance was assessed using the medication adherence rating scale (mars) survey and patients were administered part a of a questionnaire entitled 'assessment of the -day multi dose pill box'. this questionnaire evaluated the patients' opinion regarding the -day multi dose pill box before and after its use. in phase , the chosen patients were given a demonstration on how to use the -day multi dose pill box and the device was given to them to use at home for one week. after one week, part b of the questionnaire was completed and compliance was re-assessed using mars . main outcome measures: evaluation of adherence before and after use of the compliance aid device. results: of the patients recruited, were male and were female. the mean age was years (range - ) and the mean number of daily medications (range - ). upon initial scoring using mars, patients were adherent and patients were nonadherent. a higher adherence was observed in patients taking or more medications daily. ten patients accepted to move on to phase of the study and took the device home to use for one week. out of these patients, felt that the way they take their medication improved following use of the device and out of patients would consider buying the device since they found it practical and easy to use. statistical analysis of mars score before and after use of the device showed no significant improvement in compliance (p [ . ). there was no significant association between level of adherence and type of psychiatric condition (p [ . ). furthermore, results did not indicate increased adherence in patients who have a carer in-charge of their medication administration or in patients using a compliance aid device (p [ . ). conclusion: the use of a compliance aid device in psychiatric patients is challenging due to difficulty in establishing patient communication and motivation. the pharmacist is in a position to identify patients who would benefit from the compliance aid device. adrien borowik * , anne fratta, fabien hernandez pharmacy, ap-hp, armand trousseau paediatric hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: enoxaparin, a low molecular weight heparin, is the most prescribed anticoagulation treatment in paediatric indications. however, the marketing authorization mentions that due to the lack of data, the use of enoxaparin is not recommended to children nor anyone weighing less than kg. thus, expert recommendations described specific dosage for the paediatric use: we aimed to compare these with our hospital practices. ( ), sofosbuvir/ledipasvir ( ), ombitasvir/ paritaprevir/ritonavir ( ), ombitasvir/paritaprevir/ritonavir + dasabuvir ( ), simeprevir + ifn ( ) . twenty-six patients ( %) were treated for weeks ( week pay-back policy). twenty pharmacists' interventions were carried out with an acceptance rate of %. the interventions included treatment adjustments due to drug interactions ( ), inappropriate treatment according to genotype ( ), duration of treatment ( ) and switch to a more cost-effective therapy ( ). seven pharmacists' interventions concerning treatment switch were applied ( %) resulting in a cost saving of € , . . all assessable patients ( ) have a negative serum hcv rna weeks after the end of treatment (svr = %) while patient died during follow-up (due to the disease). conclusion: the hospital pharmacist, as an active member of the multidisciplinary team, has an essential role in guaranteeing optimal care for hcv patients at the best cost. monitoring has also shown to be fundamental to evaluate the real world effectiveness of these drugs approved with surrogate endpoints. hp-pc : are hospital pharmaceutical staff educated on the criticality of thermosensitive drugs? camille castel, guillaume saint-lorant * please specify your abstract type: research abstract background and objective: in the use of thermosensitive drugs, the safety of patient care involves compliance with allowed temperatures. having the right information at time of care is essential. the aim of this study is to assess, within a french university hospital, pharmaceutical staff knowledge on the criticality of thermosensitive drugs and to educate them accordingly, including associated patient risks. setting and method: an assessment of knowledge using a questionnaire was led in january among pharmaceutical staff in a -bed hospital ( pharmacists, pharmacy residents, pharmacy technicians). evaluation criteria were: storage temperature of refrigerated drugs and frozen drugs, thermosensitive drug retention period after removal from the refrigerator, highest risk situation for a thermosensitive drug (t [ °c or t \ °c) and action to be taken during a temperature excursion. main outcome measures: to determine shortcomings in the management of thermosensitive drugs in order to adapt appropriate tools. results: completed questionnaires were collected. collected questionnaires included % from pharmacists (n = ), % from pharmacy residents (n = ) and % from pharmacy technicians (n = ). regulatory variations in storage temperatures of refrigerated and frozen drugs are known in respectively and % of cases. % of pharmaceutical staff are aware of thermosensitive drug retention periods after removal from the refrigerator and % of the highest risk situation for a thermosensitive drug (t \ °c). the measures to adopt during a temperature excursion are understood in % of cases. conclusion: this study highlights the lack of knowledge on the management and criticality of thermosensitive drugs and the lack of information available to pharmaceutical staff. dissemination of data and questionnaire reponses have been beneficial for the pharmacy department and have reduced inequalities in available information among pharmaceutical staff. subsequent to the study, thermosensitive drug management procedures have been revised. the deployment of this questionnaire is continuing via the university hospital intranet in order to train all health professionals in good patient care. please specify your abstract type: research abstract background and objective: temocillin is a beta-lactam antibiotic exclusively active against gram-negative pathogens. its use can avoid that of broad spectrum antibiotics, such as carbapenems, for the treatment of infections due to extended-spectrum beta-lactamase producing enterobacteriaceae. however, the absence of recommendations by learned societies on temocillin use could lead to misuse and the emergence of resistance. the aim of this study is to identify the role of temocillin in a french university hospital arsenal in order to limit ecological risks. setting and method: a retrospective study was conducted in a -bed university hospital. all adult patients having received at least days of treatment between june and april were included. data collected for the study were: age, sex, treatment indication (type of infection, identified pathogen, dosage and treatment duration), previous antibiotics and therapeutic outcomes. main outcome measures: the indicators chosen were: treatment indication, prescribed dose and treatment duration. results: two patients were included. in july , temocillin was used in a year old female as first-line treatment of intraperitoneal haematoma infection due to multiresistant klebsiella pneumoniae. prescribed at a dose of g twice daily by an infectious diseases specialist, treatment was continued at the same dose for up weeks with therapeutic success. in august , temocillin was used in a year old male for the treatment of bacteraemia due to multiresistant enterobacter aerogenes. previously treated by imipenem/cilastatin, temocillin was prescribed as second-line treatment at a dose of g twice daily by an infectious diseases specialist. treatment was continued at the same dose for up weeks with therapeutic success. conclusion: the dissemination of antibiotic resistance among gramnegative enterobacteriaceae continues to be an increasing threat for healthcare worldwide. within this context, temocillin could be an interesting alternative. determining the role of temocillin in a therapeutic arsenal is essential. our hospital considers temocillin as a ''critical antibiotic'' although its use is not exclusively limited to the new drug application. therefore, temocillin prescriptions are monitored permanently by infectious diseases specialists, microbiologists and pharmacists in order to improve the good use of this antibiotic and to optimise patient safety. please specify your abstract type: research abstract background and objective: drinkable solutions are more susceptible to deterioration and can lead to a potential risk for patient care. having the right information at time of care is essential. the aim of this study is to assess nursing staff knowledge in a french university hospital on the management of drinkable solutions to elaborate tools to help health professionals and to enhance equality of information in order to optimise patient care. setting and method: an assessment of practice using a questionnaire was conducted in may among a share of the nursing staff in a int j clin pharm ( ) results: completed questionnaires were collected. % of nursing staff replied that the period-after-opening is the same for all of drinkable solutions. this period is estimated at month in % of cases, weeks in % of cases and days in % of cases. % of nursing staff do not know how to store drinkable solutions after opening. the date of opening or the date of expiry after opening are specified on the medicine bottle in respectively and % of cases. only % of nursing staff have tools pertaining to the management of drinkable solutions. these observations led the pharmacy to create and distribute appropriate tools. storage methods for the drinkable solutions available in our hospital were collected directly from pharmaceutical laboratories. this information has been made available to nursing staff via drug control software (pharma Ò , computer engineering, paris). conclusion: this study highlights the lack of knowledge on the management of drinkable solutions and the lack of information available to nursing staff. in our hospital, the dissemination of appropriate data reduced inequalities in available information between care units. data will soon be integrated within the drug prescription software (mc kesson usv Ò , crossway, san francisco) in order to homogeneously train all health professionals in good patient care. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is a process which allows prevention of iatrogenic injuries during patient's hospitalisation and transfers. since , a clinical pharmacist has been integrated into the orthopaedic surgery care. he has performed mr at patients' admission. the aim of this study was to evaluate the impact of medication reconciliation performed by a clinical pharmacist. design: a prospective monocentric study was conducted on patients admitted in an orthopaedic surgery care (elective or unplanned surgery), during months. the clinical pharmacist established the best possible medication history (bpmh) from at least three sources of information (including patient interview when possible). then, it was compared to the admission medication order (amo) (from anaesthetists when elective or orthopaedists when unplanned). unintended medication discrepancies (umd) detected were discussed with prescribers in order to be corrected. epidemiological data, number and type of umd, therapeutic classes involved and the percentage of corrected umd were collected and their potential clinical impact was assessed. results: in this study, patients were included during months. elective surgeries were concerned in % of the cases. at least one umd was identified in patients ( %) (median age: . years old; male/female ratio: . ). of these, ( %) were older than years old. finally, umd were detected, being . by patient. main therapeutic classes concerned cardiovascular system ( %), nervous system ( %) and digestive system ( %). of the umd detected by mr, there were % of omissions, % of inappropriate dosing and % of renewal prescriptions stopped by the patient. finally, % of umd were corrected. of these umd, % were major errors (i.e. causing potential harm), % were significant errors (i.e. monitoring or intervention potentially required to preclude harm) and % were minor errors (i.e. without potential harm to the patient). conclusion: medication reconciliation process performed by a clinical pharmacist allows detection and correction of umd on half of patients in surgery care particularly on elderly patients. the high proportion of umd can be explained by the multiplicity of actors involved in medication management. health information technology could help to focus mr on patients at high-risk of adverse drug events. please specify your abstract type: research abstract background and objective: darunavir plus ritonavir (drv/r) have shown optimized results in simplification strategies (monotherapy (mt) or dual therapy (dt)) for selected hiv + in randomized clinical trials and real life experience. recent introduction of one pill drv plus cobicistat co-formulation (drv/c) may be particularly suited for both mt/dt allowing once daily administration optimizing dosage and adherence. the objective of our study is to evaluate efficacy and security of drv/c in mt and dt. setting and method: all hiv + adults with antiretroviral change to drv/c in mt/dt at a reference hospital in the northwest of spain were included in this retrospective study. a statistical analysis was performed using the spss v. .software. main outcome measures: epidemiological, clinical, antiretroviral regimen, serum creatinine, lipids and inmunovirological data (rna-hiv and lymphocytes cd ) were compared previous and after change to drv/c. results: hiv treatment-experienced patients have received drv/c in dt ( ) or mt ( ). . % were men with a mean age of years. main risk factors were: . % heterosexual, . % msm, . % injection drug users, . % mother-to-child transmission, . % transfusion in haemophiliac patient and . % unknown. cdc category distribution was . % a, . % b, . % c and . % unknown. overall mean nadir cd counts were . ± . cells/mcl. mean time since drv/c prescription to discontinuation or until analysis was . days [range - ]. . % drv/c mt were prescribed to patients with prior drv/r mt in order to simplify treatment and the mean time of the duration of these prior therapies were . years. in case of dt, . % were prescribed on patients with prior drv/ r + tc with a mean duration of . years. serum creatinine increases ( . vs. . ; p \ . ) and cd decrease ( . vs. . ; p = . ) when patients move to drv/c. no significant change in the other analytical parameters and all patients maintained undetectable. patients discontinue drv/c due to intolerance and inability to swallow in each case. conclusion: this preliminary study concludes that drv/c in mt or dt is efficacy (no viral rebound) and safety. although an increase in creatinine was observed, it would not be considered clinically significant. of note, lymphocytes decreased significantly and it will be important closely monitored to check that maintain effectiveness during the follow up. hp-pc : developing clinical pharmacy in emergency department setting up a medication reconciliation process marion collignon *, , antoine gantier , florent lapacherie , hélène dewaele , laura foucault , anne-laure raso , emmanuel cirot , said laribi , xavier pourrat pharmacy, emergency department, chru tours, tours, france please specify your abstract type: descriptive abstract (for projects) background and objective: in emergency department (ed), if a drug related problem (drp) happens at the patient admission, the risk is the error remains until discharge. one part of drp may be avoided with using medication reconciliation (mr). the objective of this study was to evaluate the feasibility of setting up a medication history (mh) of patients in ed in an acceptable lap of time before they were transferred in another unit or discharged. design: a months prospective study was conducted in ed in a university hospital in france. two junior pharmacists coached by a senior pharmacist, after a months training for mr, were in charge of the data and mh collection. for all patients, we collected age, mh according to number of sources, discrepancies identified, adherence to treatment (according to the social security questionnaire), type of sources. mh were established according to community pharmacies, patients, previous electronic patient files, prescription sheets, patient's family, packs of pills and to the general practitioner (gp). for patients from long term care facilities (ltcf), the mh was established only by communication with the ltcf. then, the current prescription was compared with the home medication regimen. mh and discrepancies (omitting medication, incorrect dose, ambiguous name) were recorded in the electronic patient files to be available during hospitalization. because ed does not have a pharmaceutical review of prescriptions, only major discrepancies were transmitted to physicians. results: we collected mh ( from ltcf), with a sex ratio of . and a medium age of . years old. it represented an average of . mh per day or min per mh. among patients who did not come from ltcf, sources used by pharmacy students were patient's community pharmacy ( , % of cases), patient ( , %), previous electronic patient file ( , %), prescription sheets ( , %), call to gp ( , . %), gp mail ( , . %), patient's family ( , . %), packs of pills ( , . %), community nurse ( , . %). finally, patients ( %) had been hospitalized, others were discharged. we analysed mh for patients: at least one drp occurred for patients ( %). among patients, ( %) had an immediate pharmaceutical intervention because of the risk due to discrepancy. among patients who did not come from ltcf and who could communicate, were good adherent to treatment ( %). conclusion: this study highlights the great interest of the mh by pharmacists at ed, which avoids many drp. the presence of pharmacists in ed contributes to maintain a safe environment for medication and to assist prescribers in the continuity of treatment between home and hospital. spending min by mh, we identify one drp every min. nevertheless, it could be benefit to develop this activity because of the satisfaction of the emergency physicians. currently, mr is the first step to develop clinical pharmacy in the ed. please specify your abstract type: research abstract background and objective: emerging evidence in the literature suggests a high prevalence of suboptimal vitamin d (vitd) and an association between lower serum levels and higher mortality in cancer. the objective of this study was to quantify vitd deficiency in patients after surgery for head and neck cancer, and to determine the effect of one cholecalciferol intramuscular dose. setting and method: intervention study with a follow-up period of months (november -february ) performed on patients followed by the nutrition support unit after surgery for head and neck cancer. demographic and physiopatological data, including admission diagnosis, age, gender, calcium, magnesium and phosphate were collected. nutrition screening by conut index was carried out. a single intramuscular dose of . ui cholecalciferol (vitamine d bon Ò ) was administered to vitd-deficient patients and serum -hidroxy-vitamin d (s ohd) records after the administration, including primary carés records after discharge, were evaluated (reference range - ng/ml). main outcome measures: s ohd (\ ng/ml: deficiency; - ng/ml: insufficiency; c ng/ml: sufficiency). results: data from patients with a mean (sd) age of . ( . ) years were collected (males: %). the admission diagnosis was laryngeal squamosis cell carcinoma (n = ), glottis carcinoma (n = ) and nasopharynx, tongue and skull base cancer (n = ). at baseline, , and patients were considered have high, medium and low risk of malnutrition, respectively. the mean (sd) serum ohd was . ( . ) ng/ml (deficiency: patients; insufficiency: patient). despite the role of vitd in mineral balance, calcium, magnesium and phosphate mean (sd) serum levels were between the normal range . ( . ) mg/dl, . ( . ) mg/dl, and . ( . ) mg/dl, respectively. s ohd records were available week after the administration (mean (sd) = . ( . ) ng/ml). and patients still showed deficiency and insufficiency, respectively. primary care's records from patients were available after discharge ( . , . and . ng/ml). conclusion: poor nutritional status and high prevalence of suboptimal vitd in patients with head and neck cancer were found. a single dose of intramuscular cholecalciferol slowly raises s ohd. follow-up after discharge is essential to evaluate the achievement of the therapeutic objective. setting and method: this is a descriptive retrospective study. it took place in a teaching hospital. antifungal broad spectrum therapies (liposomal amphotericin b, caspofungin, micafungin, posaconazole, voriconazole) used between st january and st december were included. main outcome measures: indications, type of combination and patients specifications were analysed. results: only patients ( . % over all patients receiving antifungal therapy; n = / ) received an antifungal combination therapy during the study period. majority of patients presented risk factors: % of patients had an organ transplant (n = ), % suffered from malignant blood disorders (four acute myeloid leukaemia, two chronic lymphoid leukemia, one non-hodgkin's lymphoma, one hodgkin's lymphoma and two refractory anaemia with excessive blast), % suffered from solid cancer (one lung cancer and one breast cancer) and % suffered from chronic obstructive bronchopneumopathy (n = ). antifungal combination therapy was used against invasive aspergillosis in % of cases (n = ) among which complications such as brain and cardiac impairment were found in % of patients (n = ). the six remaining patients ( %) were co-infected with candidiasis for three patients and mucomycosis for three patients. voriconazole was logically the most used in combination, and just one patient received oral form. it was in majority prescribed with caspofungin ( %, n = ) and intravenous liposomal amphotericin b ( %; n = ). combination including liposomal amphotericin b and caspofungin (n = , %) or posaconazole with liposomal amphotericin b (n = ) were found in our study. five patients deceased during the hospitalization of the fungal infection ( %) which shows the gravity of these cases. majority of patients ([ %) was treated less than days with these combinations. conclusion: this retrospective study shows that patients who received antifungal combination therapy were mostly immunocompromised, co-infected or experienced a severe infection with severity factors. the antifungal combination was in majority initiated because monotherapy failed to cure the patient. all prescriptions were discussed with a mycologist who tried to shorter the combination treatment duration. this multidisciplinary approach is a major key in the process of these type of treatments. please specify your abstract type: research abstract background and objective: because of its broad spectrum and the risk of resistance mutation, delivery of posaconazole is nominative and controlled by hospital pharmacists. the aim of this work was to describe the use and pharmaceutical follow-up of posaconazole tablets over a -months period. setting and method: this is a descriptive retrospective study over a -months period from november to may in a teaching hospital. all patients who received posaconazole tablets were included. main outcome measures: indications and dosage were reported. results: patients were included in the study. posaconazole tablets were used for: fungal invasive infection prophylaxis in case of stem cell transplantation ( %; n = ), fungal invasive infection prophylaxis if a chemotherapy was started to treat a chronic myeloid leukaemia or a myelodysplasic syndrome ( %; n = ); treatment of invasive aspergillosis ( %; n = ); mycetoma ( %; n = ); zygomycosis or mucormycosis while patient had renal impairment ( %; n = ). all of these indications were approved for posaconazole (marketing authorization and local guidelines). only patients ( %) received a loading dose ( milligrams twice a day) as recommended in approval authorization. posaconazole blood levels were monitored by pharmacologists: % of patients (n = ) did not need dosage modulation which shows that variability is not so important. but three patients did not have any assay to monitor posaconazole blood concentration. patient received a loading dose and was switched to intravenous voriconazole after icu transfer. patients needed increase and/or reduction dose to obtain optimal posaconazole blood levels. conclusion: this study describes the use and the follow-up of posaconazole tablets during the first months after its approval in europe. all indications are approved for posaconazole but this analysis shows that pharmacist have to remind the necessity of a loading dose. dosage can be adjusted according to assays results. please specify your abstract type: research abstract background and objective: due to the acute, hectic environment in a fast-paced work-flow emergency department (ed) it is a challenge to verify the correct and updated medication list for the admitted patients. when performing medication reconciliation (mr) in this environment, these challenge has to be taken into account and prioritizing patients for mr could be necessary. the objective of this study was to identify risk factors correlated to clinical relevant medication discrepancies (crmds) among patients admitted to ed, and based on these revealed risk factors, develop a model for prioritizing patients for mr in the fast-paced work-flow at the ed. setting and method: patients continuously included at the ed, diakonhjemmet hospital (dh), oslo, norway. trained pharmacists and emergency nurse conducted mr. patient specific factors and revealed crmds, between hospital admission records and information about prehospital medication use, were recorded. binary linear regression was used to identify risk factors correlated to crmds. the prioritizing model was built using statics and clinical experiences. main outcome measures: what risk factors is correlated to crmds and how precisely do the prioritizing model classify the patients as high-and low-risk patients. results: % of the patients had c crmd. the following were identified as risk factors correlated to crmd and were suitable for inclusion in the prioritizing model; gender (woman), age (c ), c admission to hospital last months, admission causes; surgical, malfunction, cancer. the model correctly classified . % of the patients with crmds as high risk. further, . % of the patients with crmds were classified by the model as low-risk patients (false negatives). the model classified . % of the patients who did not have a crmd as high-risk patients (false positives). conclusion: the prioritizing model developed can be helpful in identifying what patients are at increased risk of having crmds in the fast-paced work-flow at the ed. identifying these patients will result in using the resources available in the ed in the most efficient manner and utilizing the full potential of the mr method. as a consequence of this, patient safety would be increased. hp-pc : intravenous potassium chloride: quick audit of prescribers knowledge and recommendations regarding safe practice and proper usage asmaa damou * , vincent zaugg, martine postaire please specify your abstract type: descriptive abstract (for projects) background and objective: our hospital has established methods that try to ensure the safe use of high alert medications. intravenous potassium chloride (kcl) was the subject of preventive measures: separation of different dosages (kcl . % vials reserved for paediatric services and kcl % vials reserved for adult services); creation of an advice record for doctors and nurses; specific labelling of storage areas; double-check the prescription and administration. the objective of this study was to evaluate the knowledge of the safe use of intravenous kcl by prescribers. design: multiple-choice questions were developed for prescribing recommendations established by our hospital with the collaboration of the doctor who is chairman of the central committee of vigilance and risk associated with care (cvris). a link to the online survey was sent by email to physicians practicing in departments (eight paediatric services and six adult services). the results were extracted and interpreted in excel Ò . results: % of physicians responded to the survey ( medicine residents, hospital doctors). in paediatric services, % of doctors know that only the kcl . % should be used. % know the unit of prescription to be used (mmol/kg or meq/kg), and % know that the maximum recommended infusion rate is . mmol/kg/hour (or mmol/kg/h in recovery unit). in adult services, the recommended maximum rate of infusion ( g/h) is known to all prescribers, but only % know that the concentration of kcl must be less than g/l. % of paediatric doctors say that their kcl prescriptions are checked by a second doctor, but the answers in the same service area are sometimes contradictory. in adult services, only % of physicians say that the prescriptions are double-checked. the information brochure available on the intranet of the hospital is known by % of prescribers. the response rate of physicians to the survey was satisfactory. therefore, the recommendations are rather well known by prescribers, except the value of the maximum concentration of infusion for adults. the results of this audit were returned to the doctors, accompanied by a reminder stating the need to double-check the prescription and the existence of advice records on the website of the hospital. conclusion: this audit is an approach to increase the safety of the use of high alert medications. it will be completed a second time, by an evaluation of prescriptions collected and the storage conditions of potassium chloride in the care units. please specify your abstract type: research abstract background and objective: data listed behind each unit dose of a primary packaging of a pharmaceutical product are essential for a safe identification for the patient. however, the last medical services of the lausanne university hospital where nurses remove the solid form drugs (sfd) from their blisters when they prepare in advance the week container were in the vaud's prisons. the aims of the study were: ( ), quetiapine ( ) and ibuprofen ( ) and were psychotropics ( . %). part . the four data identified as essential: brand name, dosage [mg], batch number, expiration date. the sfd unit doses were classified as green when the blister included four data, yellow with two or three and red with less than two. of the sfd in cupboards, were green ( %), yellow ( %) and red ( %); an infovigilance was sent to each manufacturers. part . potential barriers identified: trays' sizes and space in drug's cupboards; preparation time to cut versus to remove the blisters; risks of self/hetero-aggression with pre-cut blisters; drugs packaged in bulk; multidose liquid medications. using containers larger than is usual was rarely necessary; space in cupboards was sufficient. the preparation time gradually decreased during the study. ingestion or aggression with pre-cut blisters was considered as limited, based on literature and experiences of two others prisons (geneva; lyon). for bulk sfd and multidose liquid drugs: proposals to the pharmacy to store some alternatives blistered sfd; blistering expensive bulked drugs; availability of the entire package delivered to inmates. the pilot phase was initiated in may . conclusion: a majority of inmates takes a drug treatment. half of sfd unit dose is identifiable (trade name and dosage) but an effort from manufacturers would better secure the drug supply chain. the study of the barriers helped to further implement the pilot phase. since early , none of the five prisons medical wards are removing the blisters and no incident was reported. please specify your abstract type: research abstract background and objective: nefopam is a widely used antalgic in hospital. its use is contraindicated in the epileptic patient as it results in lowering the epileptogen threshold and is likely to trigger epileptic seizures. the clinical pharmacist should systematically warn the prescriber against this contraindication when analysing prescriptions. following the onset in our establishment of an epileptic condition in a patient treated with nefopam, who had not been subject to any pharmaceutical intervention (pi), we set about analysing the validation practices regarding this contraindication and possibly implementing actions designed to improve those practices. setting and method: retrospective collection over a period of months of prescriptions for patients hospitalized in hospital beds with clinical pharmacy service (associating med-reconciliation, checking prescription according to medical file and participation to medical rounds): orthopaedic surgery, hepatic-gastro-enterology, general surgery, liver transplant and chest surgery. records of patients with nefopam prescription associated to medication belonging to the therapeutic class of antiepileptics were consulted with a view to finding cases of epilepsy. the pharmaceutical alerts were extracted from the pharmaceutical software. main outcome measures: number of epileptic patients treated with nefopam, number of pharmaceutical interventions issued when prescribing nefopam in epileptic patients. the study focused on , patients. ( . %) of them were prescribed nefopam, and ( . %) of them were prescribed nefopam associated to medication belonging to the therapeutic class of antiepileptics. after analysis of the patients' records has shown that of them were really epileptic. only pi's were effected ( . % of problematic prescriptions), and ( %) of them had an immediate prescription change. . % ( / ) of the patients have a pi in medicine services compared to . ( / ) in surgery services (p \ . ). the results of this study show that % of the contraindications related to the use of nefopam in epileptic patients are not reported to the prescriber. these results will be presented to our pharmacists so they can take them into account. subsequently a new study will be conducted to measure the relevance and efficiency of this program. hélène dewaele * , anne-laure raso, emmanuel cirot, marion collignon, laura foucault, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) has been demonstrated to reduce drug-related problems in inpatients. in our university hospital, mr has been performed for beds for years at the same time as prescriptions review. the aim of this study was to assess the impact of mr on pharmaceutical interventions (pi) during prescriptions review. design: a -month prospective study in orthopaedic surgery, hepato-gastro-enterology, general surgery, liver transplant and chest surgery was conducted. during medication review all pis were collected and those related to mr (rpi) were identified. thereafter for each patient we collected age, type of hospitalization unit (med or surgery) and for pis the drug associated and its acceptance by the medical team. results: during the study patients had a daily prescription review. patients ( %) had at least one drug-related problem. lines of prescriptions were mentioned to have at least one rpi. rpi represent % of drug-related problems. ( %) discrepancies were corrected by prescribers. the age of the patient was significantly different between patients with rpi (mean age: years old) and with pi (mean age: years old; p \ . ). the type of unit did impact the percentage of prescriptions with drug-related problems (medicine: . ; surgery: . ; p \ . ), the rate of corrected pi (medicine: %, surgery: %, p \ . ), but did not impact the rate of corrected rpi (p = . ). in surgery units the rate of corrected rpi ( / ) is significantly higher than corrected pi ( / ; p \ . ). medicines belonging to the four classes of: digestive and metabolism system, blood and blood flow, cardiovascular system, neurological system represent more than % of all the medication concerned by a resolved pi or rpi. the proportion of medicines from the digestive and metabolism class is the only class among those four that is not significantly different between resolved pi and rpi. conclusion: mr highlights a large number of discrepancies in inpatients. a modification of prescriptions due to mr occurs in % of the patients. in surgery units, these rpi are more frequently taken into account than drug-related problem warned by pis. indentifying patients for whom mr has the bigger impact could help us to reinforce our actions. please specify your abstract type: research abstract background and objective: diabetes is very frequently causing cardiovascular complications, thus impairing various systems and organs. therapy for these multiple conditions has to be revised and improved constantly. the aim of this closed retrospective study lead in bucharest emergency clinical hospital was the assessment of some of the diabetes mellitus (dm) complications and the related medication. setting and method: data was collected from cardiology, neurology, gastroenterology, internal medicine wards from bucharest emergency clinical hospital. only patients diagnosed with type dm were included in the study. there were analysed records from patients aged - of whom were men, following the presence, signalling and monitoring of diabetic nephropathy and arteriopathy. main outcome measures: we investigated the relationship between diagnosis and/or biochemical signs of kidney disease (serum urea, serum creatinine levels), diagnosis of arteriopathy, and the drug therapy administered in the respective cases. we also assessed the sex and age distribution of the patients diagnosed with diabetes mellitus and facing at least one of its complications. results: kidney disease, as a dm complication, was present in % of cases, patients aged - , of whom % were men. patients received diuretic treatment, of them being given hydrochlorothiazide, contraindicated in dm because of its hyperglycaemia-inducing effect. of the patients, had high serum urea levels ([ mg/dl), had high levels of serum creatinine ([ . mg/dl), and presented risen levels for both, but only were also diagnosed with kidney disease. patients with kidney disease were given furosemide, known for altering the renal function. circulatory failure was found in % of the patients, aged - and % of subjects, aged - , had both diabetic complications. conclusion: the present study emphasizes the role of the clinical pharmacist in adapting the medication of the diabetic patient, an inappropriate pharmacotherapy worsening dm complications. this is essential especially for elders, where polypathology and polymedication lead to a significant increase of dm complications risk. hp-pc : epileptic seizure after treatment with thiocolchicoside: discussion about a case report valérie dobremez *, , adeline martin-dupray , jacqueline berlioz , pierric giraud pharmacy, neurology, centre hospitalier annecy-genevois, metz-tessy, france please specify your abstract type: descriptive abstract (for projects) background and objective: thiocolchicoside is a semisynthetic derivate of naturally occurring colchicoside, which is largely used in humans as a centrally acting muscle relaxant. this compound also has anti-inflammatory and analgesic effects. the objective of this work is to report a recent case of serious adverse effect of thiocolchicoside occurring in context post traumatic brain damage without sequalae. design: a -year-old woman suffered from headaches and neck pain since days, she was treated with thiocolchicoside. she took mg in the evening and mg the next morning. five generalized tonic-clonic seizures, without recovery of normal consciousness between seizures, have occurred suddenly - min after the second administration. the patient was admitted to intensive care unit in order to control the epileptic seizures. a status epilepticus was diagnosed requiring intravenous drugs with clonazepam, phenobarbital and propofol. the patient was controlled and transferred in neurologic unit in order to complete paraclinical investigations. its main antecedent was a severe head injury at the age of years following a public road accident. the brain scan revealed an old frontal hypodensity. rest of etiological assessment was negative (lumbar puncture, no infectious disease), numbers were normal. the definitive diagnosis was a status epilepticus on post-traumatic sequelae, sensitized by taking a proconvulsant drug. a treatment with levitiracetam was initiated at mg twice a day. outcome was favourable with no recurrence months later, a recommendation was requested to pharmacovigilance. results: the muscle relaxant activity of thiocolchicoside results of an agonist action on glycinergic receptors located primarily in the brain stem and spinal cord. however, thiocolchicoside also acts as an antagonist of the gaba-a receptor (mainly located in the cerebral cortex), this pharmacological action can cause a proconvulsant effect. epilepsy is a very rare adverse effect, only few cases have been reported in literature. the epileptogenic activity of thiocolchicoside occur mainly in patients with a history of epilepsy, acute brain injury or possible blood-brain barrier disruption. the chronology is consistent with the responsibility of the drug as a promoting factor. pharmacovigilance retains after analysing drug causality. conclusion: the case history indicates that thiocolchicoside has a powerful epileptogenic activity. thiocolchicoside can precipitate seizures in predisposed patients, and that its use should be avoided in patients with brain diseases (and therefore lower seizure thresholds) or blood-brain barrier disruption. pharmacists could warn physicians and should verify the absence of notable history before dispensing thiocolchicoside. hp-pc : acute exacerbation generalized myasthenia after red yeast rice use: a case report valérie dobremez *, , amélie serra , déborah grosset-janin , jacqueline berlioz , aymeric dopter , jean-henri ruel pharmacy, neurology, centre hospitalier annecy-genevois, metz-tessy, nutrivigilance, french agency for food, environmental and occupational health and safety, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: many drugs can induce acute exacerbations or reveal myasthenia gravis. self-medication or complementary and alternatives medicines expose patients. the objective of this work is to report a recent case of acute exacerbation of myasthenia gravis because of a dietary supplement use. design: intermittent vertical diplopia and ptosis of the left eye settled in a -year-old man. its main antecedent is hypertension treated with perindopril. the neurovascular origin was ruled out. the electromyogram (emg) found a significant decrement ( %) of a postsynaptic block in the tongue and right orbicularis muscle. acetylcholine receptor-antibodies were positive. myasthenia gravis was diagnosed (osserman score / ) and the patient was treated with pyridostigmine. the identification of carotid atheroma required a treatment with a statin that the patient refused. he preferred a cholesterol lowering dietary supplement, containing red yeast rice. six days later, he was hospitalized for an acute decompensation of myasthenia with bilateral ptosis, oculomotor paresis, drooping head, int j clin pharm ( ) : - chewing trouble and dysphagia (osserman score / ). the patient is treated with high-dose intravenous immunoglobulins then corticosteroids. the dietary supplement is stopped. an opinion was requested to the clinical pharmacist of neurology. the osserman score gradually increases to / . results: red yeast rice contains a range of compounds known as monacolins, of which monacolin k-renamed lovastatin, which was found to be an inhibitor of cholesterol synthesis and the progenitor of the statin family. a literature review has highlighted the responsibility of statins in acute exacerbations or reveal myasthenia gravis occurrences. in this case, the chronology is consistent with the responsibility of red yeast rice. the case was reported to the french system of nutrivigilance, which retained after analysing a probable intrinsic imputability score. conclusion: dietary supplement with red rice yeast are not recommended in case of myasthenia gravis. this is the first case of acute decompensation of myasthenia recorded with red yeast rice in the french system of nutrivigilance. multidisciplinary collaboration (neurologists, clinical pharmacist) has optimized the patient management. fanny durand * , camille lambert, antoine dupuis please specify your abstract type: descriptive abstract (for projects) background and objective: development of computerized prescription highlights the need to harmonize pharmaceutical analysis practices. the aim of this study is to analyse the antibiotics prescriptions in the treatment of urinary tract infections, to develop a pharmaceutical validation tool. design: a prospective observational study was conducted for one week, in care units. pharmacists, interns, and pharmacy students were trained on spilf (french society of infectious pathology) recommendations, on pharmacist's role in the management of urinary tract infections, and on the data collection. all patients with antibiotic prescription for urinary tract infection were included. some data were collected: reason for hospitalization, clinical signs, results of susceptibility testing, risk factors for complications (organic or functional abnormality of urinary tract, male, pregnancy, elderly, severe immunodeficiency, severe renal impairment) and signs of severity (severe sepsis, septic shock, interventional surgical drainage). then, the treatments prescribed to the patient, probabilistic on the one hand and documented on the other hand, were compared to spilf recommendations. finally, during a multidisciplinary meeting (pharmacist, expert in infectious diseases), we selected the relevant pharmacist interventions. results: twenty-three patients were included ( women, men), % had a urinary catheter. . % of prescriptions were concordant with spilf recommendations: probabilistic and documented treatment, and duration. among the non-conforming prescriptions, nine pharmacist interventions have been formulated: four prescriptions did not specify the duration of treatment, one antibiotic was prescribed on an insufficient period, two cases of severe acute pyelonephritis without prescription of aminoglycoside, one prescription was not reassessed according to results of susceptibility testing, one pregnant woman with urinary colonization without clinical signs, was treated before obtaining results of susceptibility testing. three cases of poor management are identified: two cases which treatment began only after results of susceptibility testing (a urinary tract infection linked to care, an acute pyelonephritis with complication risk), and a cystitis treated with nitrofurantoin while the germ was resistant. conclusion: a synthetic tool was created. there are three elements for helping pharmaceutical analysis: the questions to ask oneself facing a prescription of antibiotic for urinary tract infection, a flowchart to identify the recommendation adapted to the case, and finally a summary table showing spilf recommendations. this tool will be distributed and evaluated. hp-pc : off-label use of rituximab in refractory antisynthetase syndrome (as) through a long-time experience in a neuromuscular diesases center lise durand * , carole metz, patrick tilleul, helga junot pharmacy, gh pitié salpêtrière, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: as is an idiopathic autoimmune inflammatory myopathy, characterized by presence of antisynthetase antibodies: anti-jo , anti-pl , anti-pl . patients are usually first treated by corticosteroids (cs) or immunomodulating drugs. rituximab (rtx) has become another option for refractory as, supported by few uncontrolled studies . because of its off-label use, our hospital pharmacy has implemented a controlled drug delivery. this work assesses a -years follow-up of patients treated by rtx and the resulted drug costs. design: patients registered in our database since december who received c injections of rtx to treat as, were analysed to describe their eligibility criteria, conditions of management and the clinical and biological effects of the treatment (creatine kinase (cpk) used as biomarker). patient files were consulted to collect all individual data and pharmaceutical software was used to review deliveries. drug costs were also reckoned based on prices from french health insurance. results: for months, patients (median age (min-max): ( - ), % women) have been treated with rtx for refractory as, the majority with anti-jo antibodies ( ). all patients suffer from muscular and lung affections, particularly interstitial pneumonia. many are also living with arthropathies ( ) or cutaneous disorders ( ). cardiac involvement is seldom ( symptomatic patients). the mean age of diagnostic is . years and the mean treatment period is . years. the common treatment is g at day (d ) and d , then g all months. before rtx treatment, seven patients received c other drugs such as cs ( %), azathioprine ( %), methotrexate or mycophenolate mofetil ( %). prednisone and azathioprine are also prescribed with rtx respectively for and %. treatment is associated with cures of intravenous immunoglobulins for four patients. to date, median number of administrations per patient is ( - ), d and d included. all patients have presented positive effects on both clinical and biological markers, mainly during the first months after treatment induction. wilcoxon tests show a significant difference in cpk level between d and m , also between d and the last known result. today, three complete remissions are specified in patient file; only one hepatitis b virus reactivation is reported. since , budget impact due to drug cost amounts to €. conclusion: whereas the use of rtx is controverted for treatment of all types of myopathy, as could have one of the best response . our cohort shows real clinical results and positive effect on usual biomarker. our experience demonstrates the safe and successful use of repeated administrations in refractory as. however, there is a need for further controlled studies to assess the efficacy/safety of rtx and to define its place in the strategy in view of its cost-effectiveness ratio. the pharmaceutical controlled drug delivery has to be continued to supervise, support and document its proper off-label use. please specify your abstract type: descriptive abstract (for projects) background and objective: as a part of the national patient safety program, the northern norway regional health authority are implementing new procedures for medication reconciliation (mr) in hospitals in the region. the procedure defines that mr is the doctor's responsibility and describes how it should be performed. the aim of this study was to investigate whether the implementation of the procedure reduces medication discrepancies (mds) in the charts at bodø hospital. and . % ( / ) of the patients died before discharge. parenteral nutrition was administered an average of . days ( % ci . - . ), of which . ( % ci . - . ) were with ipn. previous spn had been administered in . % ( / ) of the patients. before beginning ipn, the average triglycerides level was . mg/ dl ( % ci . - . ) but at the end of the ipn it was . mg/ dl ( % ci . - . ), which lead to a mean reduction of . mg/dl ( % ci . - . ; p = . ). regarding to the total amount of lipids provided with parenteral nutrition, with ipn there was a mean reduction of . g ( % ci . - . ; p = . ) comparing to those administered with spn. conclusion: usage of ipn in critically ill patients with htg permits to adjust parenteral nutrition formulations to meet specific nutrition needs, enables to reduce the total amount of lipids administered and, therefore, it allows to significantly decrease triglycerides levels. jennifer a. esteban gonzález * , elisabet nogué pujadas, angels andreu crespo, xavier bonafont pujol, nuria romero pascual please specify your abstract type: descriptive abstract (for projects) background and objective: the incidents involving patient misidentification (pm), or wrong patient medical errors (wpme), are medication errors (me), near-miss or close-call situations which can pose a considerable threat to patient health. pm may be under-reported due to the unawareness of the error or the difficulty of identifying them. the aim of this study is to describe the incidence and categories of wpme in a university hospital. design: observational, retrospective analysis of the voluntary reported wpme in the pharmacy database since march until june . these were classified in prescription, transcription, dispensing, administration and drug system errors. in addition, the national coordinating council for medication error reporting and prevention (nccmerp) taxonomy was used for classifying me according to the severity of the outcome. results: of me registered, of them were wpme ( . %). . % of them were due to prescription errors, which consist on wrong labelled medical orders, intermingled patient prescriptions or patient misidentification in computerized physician order entry (cpoe). the administration errors supposed a . % of the total amount of wpme and dispensing errors were . %. % of wpme were transcription errors, which occurred previously to the implementation of cpoe, and the remaining . % were system errors after cpoe. the wpme reported took place in the hospitalization wards ( . %), pharmacy ( . %), outpatient services ( . %), intensive care unit ( . %) and day-care hospital unit ( . %). . % occurred at working days and . % at the weekends. wpme were notified by pharmacists ( . %), nurses ( . %) and physicians ( . %). referring to the classification according to nccmerp, . % of wpme didn't reach the patient (category b) whereas . % reached the patient but didn't cause harm (category c) and . % required patient monitoring (category d). the remaining wpme ( . %) caused harm to patients and required medical intervention (category e). finally, in int j clin pharm ( ) : - more than half of wpme ( . %), reporters suggested measures to prevent these errors. conclusion: wpme represents near % of total me reported in our hospital. given that more than % reached the patient, safety measures must be implemented to reduce the risk of hazardous events. additionally, further encouragement in notification is necessary in order to improve patient safety. results: two men diagnosed with rrms aggressive evolution were included in the study. age: and . both of them without any treatment by the time they started being treated with alemtuzumab (previously one of the patients had been treated with fingolimod, suspended by inefficiency). the protocol design for the elaboration and control of alemtuzumab in the pharmacy service ensures greater safety and represents a saving strategy. in addition, the development of the protocol in the electronic prescription system (silicon Ò ) facilitates the prescription, proper administration and standardization of treatment among patients. the protocol includes daily alemtuzumab infusion for days and other necessary medications including premedication (metylprednisolone, omeprazole, paracetamol and metoclopramide) and anti-infective prophylaxis (aciclovir). developed adverse effects during infusion were skin erythema, pruritus and fever. it was not necessary to stop the alemtuzumab infusion in any patient. during treatment, one patient developed a severe lymphopenia and upper respiratory tract infection (influenza a). conclusion: the role of the pharmacist is critical at various stages, from the preparation and the administration guidelines, to detection, monitoring and reporting of adverse effects. alemtuzumab is presented as an alternative for those patients who do not respond to standard therapies or who have rapidly evolving severe rrms. because of its mechanism of action it is important to closely monitor patients, with particular emphasis on prophylaxis of possible infections. hp-pc : descriptive analysis of patients receiving oral anticoagulation following acute coronary syndromes sadeer fhadil * , paul wright, sotiris antoniou please specify your abstract type: descriptive abstract (for projects) background and objective: triple therapy with concomitant anticoagulant and dual antiplatelet therapy (dapt) following acute coronary syndrome (acs) increases bleeding risk by % compared to patients on dapt. bleeding post acs increases mortality and reinfarction risk; balancing ischemic and bleeding risks is particularly challenging in this population. european society of cardiology (esc) produced a consensus document, providing guidance for patients presenting with acs requiring concomitant anticoagulation; however optimal duration of triple therapy and safety and efficacy of novel oral anticoagulants (noacs) and more potent antiplatelet agents requires further evidence. design: a registry was collated of patients presenting with acs requiring concomitant anticoagulation. baseline characteristics, bleeding and ischemic risk scores, periprocedural treatment and antiplatelet/anticoagulant choice and duration was recorded and analysed for trends in prescribing. results: patients have been included in the registry between oct and june , of which ( %) were naïve to anticoagulation prior to admission, ( %) were taking warfarin and ( %) were on noacs. atrial fibrillation (af) accounted for ( %) cases, (average chadsvasc score of , hasbled score of ), and ( %) were for lv thrombus. of those naïve to anticoagulation, ( %) were initiated on warfarin and ( %) on a noac (last patients all received noacs). of those on a noac for af, ( %) were dose reduced on triple therapy; apixaban being the most commonly prescribed ( % apixaban, % rivaroxaban, % dabigatran). background and objective: solid oral formulations are more convenient than liquids to manufacture, store and administer for most adults. given this superiority, one would think that children were prompted to use solid formulations when available in an eligible dose. there are indications, however, that the conversion from liquid to solid formulation in children is influenced by characteristics of the liquid medication, rather than the child's ability to swallow solid medications. the aim of this study was therefore to explore if the proportion of oral liquid formulations differed between antibiotics commonly used for upper respiratory tract infections (urti) in hospitalized children. setting and method: we collected the sales data for for the children's department of the five university hospitals in norway. the three most common oral antibiotics used for urti in children were included: penicillin v, amoxicillin and erythromycin. the proportion of oral liquids was calculated by dividing the number of defined daily doses (ddd) of liquids by the total oral ddds for each substance. main outcome measures: the proportion of ddds of oral liquid antibiotics. results: a total of ddds of common oral urti antibiotics were sold in , distributed as % erythromycin % amoxicillin and % penicillin v. amoxicillin had the highest proportion of liquid with %, followed closely by erythromycin at %. in contrast, only % of the ddds sold of oral penicillin v were liquids. conclusion: higher proportions of liquid amoxicillin and erythromycin compared to penicillin v were sold to children's departments in hospitals. there are several limitations regarding the quality of sales data, as we lack information of the administered doses as well as the child's age, gender, infection and specific needs. infections in hospitals often require initial intravenous treatment, and oral switch will often be based on the initial treatment. despite these limitations, the results fit well with earlier findings which indicate that children prefer liquid amoxicillin and erythromycin to penicillin v. hp-pc : proactive medication reconciliation: a preliminary study to identify barriers before its implementation in surgery departments laura foucault * , marion collignon, hélène dewaele, anne laure raso, emmanuel cirot, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: it's well known that medication reconciliation (mr) decreases drug-related problems at patient admission (pa). in surgery departments, for planned hospitalizations, mr is performed - h after the pa (pourrat x and al, ). during this period, some chronic treatments are unintentionally not prescribed to patients. the aim of proactive mr (pmr) is to anticipate the pa by collecting their medication history before their hospitalization. the objective of this study was to identify the barriers preventing pmr implementation in our hospital. design: one week prospective study in digestive and orthopaedic surgery units in a beds' university hospital. the main outcome is to identify which barriers prevent the collection of mr before pa including the evaluation of time required to collect the relevant information, reconcile any discrepancies after the pa and identify the right sources from which to perform the mr. results: eighteen patients with a median age of years old ( - ) were contacted by phone one week before their scheduled surgery. these calls were conducted by pharmacy residents mainly between and p.m. (a more practical time for patients and at the end of pharmacist's routine tasks). an average of . ( - ) calls per patient were conducted. one patient was unreachable by phone. the average duration of the calls was min ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . twelve community pharmacy (cp) were contacted. in all cases, cp have accepted to share information about the patient's prescriptions by phone and sending it by fax during the day. five pharmacists were not contacted because patients had no chronic treatment and consequently no regular cp. on lines of prescriptions, discrepancies between the patient's information and prescriptions were identified and between prescriptions and the anaesthesia records. drug history was reported in the patient's records by pharmacy students on the day of pa in order to be used immediately by prescribers. surgery was cancelled for one patient. conclusion: the first step of an mr is made by a hospital anaesthetist some weeks before hospitalization but we have demonstrated that this step is not able to avert all potential errors. our study highlights that the time necessary to perform an mpr appears to be shorter than for an mr. in fact, it's sometimes difficult to properly interview patients during hospitalization (patient in operating room, drug-induced drowsiness). additionally, a key hurdle is to obtain any necessary modification of the prescriptions by surgeons. pmr can be expected to produce time saving efficiencies given that at pa, prescribers will have their full medication history. this study also allowed us to highlight the good cooperation between patients, cp and the hospital. it is worth noting that efforts were made to accommodate the schedules of a majority of working patients. however, as we would expect pharmacy student to perform the pmr, they will most likely attempt to contact patients during standard working hours which may impact the number of patients they are able to reach. laura foucault * , hélène dewaele, marion collignon, emmanuel cirot, anne laure raso, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: the french legislation has clearly defined and integrated the therapeutic education of patient (tep) for healthcare professionals. the pharmacist is invited to get involved in tep as a caregiver around the patient. in our study, we are investigating how the pharmacist's role is viewed by patients with chronic diseases that are included in a tep program. design: prospective study on patients included in a tep program (chronic inflammatory bowel diseases, rheumatoid arthritis, ankylosing spondylitis) between september and april . in july , the participants of group sessions (gs) conducted with health professionals, including a pharmacist, were interviewed on the phone. the principal outcome of the interviews was to evaluate how their view of the involved health professional's roles evolved before and after gs; to evaluate if they would consider being followed by their pharmacist for individual sessions (is) in a community pharmacy (cp); and if the information supplied by the pharmacist during gs was understandable. to health care. however, discussions between patients appear to be essential to facilitate their acceptance of a chronic condition. some patients also questioned the cp's skills and knowledge when it comes to their particular disease. nevertheless, . % of patients have found that the vocabulary and documents used by pharmacist during gs was adapted and that the information supplied was very useful. conclusion: this study highlights that although the pharmacist is the drug's specialist, a majority of patients will more likely ask their physician about medication. their participation to the gs hasn't changed their habits even if the pharmacist intervention was relevant and understandable. the fact that the pharmacists took into account the level of health literacy of each participant was an appreciated aspect. cp should be more proactive in their relationship with the patients in order to highlight their skills and the assistance they can provide in a chronic disease. however, it's important to take in consideration that in some cases, patients have lived with their disease since childhood. the role of is is likely to be much more limited than in other situations given their key need is to interact with patients afflicted with the same condition. hp-pc : use and safety of trastuzumab emtansin in her + metastatic breast cancer in a tertiary hospital c. chaguaceda galisteo * , alba manzaneque gordon, héctor josé del río torres, natália creus baró please specify your abstract type: descriptive abstract (for projects) background and objective: novel anti her drugs have changed the management of her + metastatic breast cancer patients. the aim of this study is to describe the use of trastuzumab emtansin (tdm- ) in clinical practice in a tertiary hospital and to evaluate its safety profile. design: we performed a retrospective study of patients who started tdm- between january and december . we recorded demographic data, clinical and treatment variables, number of doses received, reasons for discontinuation, progression-free survival (pfs) and adverse effects (aes). data were obtained from the chemotherapy prescription program and medical records. aes were classified according to the common terminology criteria for adverse events version . of the national cancer institute. results: eleven female patients with a median age of . years [ . - . ] and an ecog ( / ) were included. tdm- was prescribed as a third or further line treatment in / patients and as firstline in one patient who develop disease recurrence within months of completing adjuvant therapy. median number of tdm- cycles was . . all treatments discontinuations were due to disease progression ( / ). pfs was . [ . - . months] (patients that received less than three cycles were excluded (n = )). most frequent aes were plaquetopenia, neutropenia and transaminitis but only grade in three patients (two transaminitis and one neutropenia). conclusion: the lower pfs obtained comparing to the pivotal study ( . vs. . months) could be explained by the later use of tdm- in clinical practice ( / patients received tdm- as third or further line while % in the pivotal study were first or second line). tdm- safety profile was according to the summary product characteristics. few data are currently available regarding the use of tdm- in clinical practice. further data are required to position this drug in clinical practice. please specify your abstract type: descriptive abstract (for projects) background and objective: the hospital pharmacist for their specialized training in the area of medicines, possess a greater responsibility in the detection and reporting of adverse drug reactions (adrs), as well as other problems related to treatment, which may be subject to monitoring and reporting to the regulatory authorities and the respective laboratories. thus, the pharmaceutical services of the cuf infante santo hospital has implemented a pharmacovigilance program, with two main objectives: . optimization of the detection and reporting of problems related to therapy; . implementation of corrective and/or risk minimization measures. the pharmacovigilance program is based on the following methodology: . detection of adrs/problems related to therapy/medical device: the detection can be performed by the pharmacist or other health professional that guides the process to the pharmaceutical services. . information processing by the pharmaceutical services and realization of spontaneous reporting: the notification is performed both for the portuguese regulator (infarmed) as to the appropriate laboratory (if applicable). after evaluation by both entity, the conclusions are communicated to the pharmaceutical services, which has the responsibility to share it with all the other hospital services. . report of the event in the internal risk management platform: when applicable, the pharmaceutical services internally report the adverse event to the hospital's risk management department, leading to an internal evaluation of the current process. . completion of the process and implementation of corrective measures: when the regulatory authority and/or the laboratory sends the report/technical advice about the notification, the pharmaceutical service in partnership with the risk management team perform a reassessment of the whole process. if needed, corrective and/or monitoring measures are implemented. . monitoring of implemented measures: after the implementation of corrective and/or monitoring measures there is a period of evaluation. results: the implementation of this program for the period of year, has led to a total of fourteen spontaneous reports. from all of these notifications, seven were related to quality defect of medicines, four were of adr, one was due to suspected lack of therapeutic efficacy, and lastly, one of the notifications was medication error derived. conclusion: the obtained results, over a year period, by the pharmacovigilance program were satisfactory but the aim of the pharmaceutical services is to consolidate and optimize the same program with a view to achieving better results. the pharmaceutical services will continue to take responsibility for the pharmacovigilance circuit management in this hospital, by promoting a proactive approach to monitoring the safety, quality and efficacy of medicines, which possess the primary objective to patient safety assurance. please specify your abstract type: descriptive abstract (for projects) background and objective: for prematures, parenteral nutrition (pn) is essential for medical care but is complex (specific needs, daily change of intakes…). now, the software logipren Ò , developed by the french society of neonatology, allows the prescription of pn as well as all the childish therapeutics. it is also in link with our production robot (baxa pomp) for individual pn bags. our objective was to integrate this software while optimizing our pharmaceutical validation process. design: the software implementation was lead by a physician/ pharmacist collaboration with several preliminary steps: • identification of pharmaceutical validation settings (pertinence of individual pn vs. industrial bags, parenteral approach, elements…). before the life-sized use of logipren Ò , a base test has been experimented to identify possible difficulties and to realize some correctives actions of the software or our process. results: logipren Ò leads us to a change in our pharmaceutical validation process, by introducing new elements: • the pharmaceutical validation of pn bags is done in collaboration with the physician, during the prescription step. • all the therapeutics are known, which allow the pharmacist to take into consideration all the intakes (micro-nutrients, vitamins…). • remove the transcription step of pn bags in our production software (abacus Ò ) thanks to an interface with our production robot. • less production problems because of the coverage of those pharmaceutical aspects during the prescription. since months, this reorganization helped us to propose pharmaceutical notices for prescriptions: • omissions (remove lipids, levocarnyl Ò , micro-nutrients, electrolytes, remove industrial bags…) • modification (reduce proteins according to urea level, micronutrients and electrolytes posology, duration of lipids infusion…) conclusion: the implementation of logipren Ò enabled us to reorganize of the pharmaceutical validation process with a consolidation of the role of the pharmacist during the prescription step, in the paediatric ward. it had a beneficial aspect by the reduction of the validation and production time, a decreased risk of error (suppression of job interrupts and better communication) and an improved production by the end of transcription step to abacus Ò . furthermore, during our experimentation, we could bring to the software editor new ways to improve it and make it more efficient. % ( . % in ) , difficulties in swallowing/psycho-behavioural distress in . % ( . % in ) , and rejection of oral drug in . % ( . % in ) . physicians and nurses indicate the reason in the medical record in . % of case versus % last year. this year, drug were crushed versus drugs in : % concerned nervous system group (vs. % in ), % concerned cardiovascular system group (vs. % in ) , and % concerned alimentary tract and metabolism group (vs. % in ) . nurses use guideline in % of cases versus . % last year. as the previous year, in % of cases, washing hands before preparation and after administration are met. last year, none of them was wearing mask and gloves during this operation while this year, % was wearing mask and gloves. finally, in the two assessment, for each patient, drugs are systematically crushed together and then mixed with the patient's meal. conclusion: this study shows that crushing drugs is still problematic in our units. however, best practices were observed, such as the indication of the reason of crushing in the medical record, or the consultation of guideline. a new training for nurses will be conducted to create awareness about risks of crushing drug. please specify your abstract type: research abstract background and objective: in invasive candidemia, three echinocandins are indicated: caspofungin, mycafungin and anidulafungin. the aim of this work is to establish which echinocandin to prescribe in a french university hospital, given the scarcity of available clinical data in the literature regarding obese patients. setting and method: in a french uhc with beds, a multidisciplinary working group composed of a microbiologist, an infectious disease specialist and a pharmacist has been set up to analyse the various therapeutic options. main outcome measures: analysis of the literature, pharmacoeconomic study. results: four medications have been identified as possible therapeutic options. their adverse effects are similar and their administration rhythm is the same. according to recommendations by the esmid ( ) and the idsa ( ), the level of evidence for these three echinocandins in initial treatment of candidemia is equivalent. concerning obese patients, no weight limit is mentioned, int j clin pharm ( ) : - despite recommended dosage adjustment. caspofungin must be prescribed at a dose of mg/day for patients weighing over kg. micafungin must be administered at a dose of mg/day regardless of patient weight. in the case of persistence of cultures or if clinical condition does not improve, the dose may be increased to mg/day. anidulafungin, which is not referenced in our establishment, must be prescribed at the same dose regardless of patient weight. from an economic point of view, in our hospital, micafungin at a dose mg/day remains the least costly therapy. however, if its posology is doubled as indicated, caspofungin then becomes the most economic therapy. amphothericin b, an optional treatment, is never the most economically advantageous therapy. conclusion: as a result of this study, the chosen prescribed therapy for obese patients is caspofungin at a dose of mg/day. this work has improved access to healthcare for obese patients. pharmacokinetics and survival data must be collected on the basis of various patient weights in order to predict clinical efficacy. kristin f. heier * , liv czynski please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of this study was to develop a system to prioritize patients for medication reconciliation by pharmacists in the emergency department. it also proved a useful setting for evaluating how other health care professionals perceived the role of the pharmacist performing medical reconciliations within the emergency department. design: the study was located in the Østfold municipal hospital, located in kalnes, norway. pharmacists used a prioritization model to identify ''high-risk patients'' having clinically relevant prehospital medication discrepancies between hospital admission records and the information obtained via medication histories, general physician referrals and nursing homes. pharmacists registered patient information such as age, gender and drug-related problems (drps). seventeen physicians and thirty nurses in the emergency department answered structured questionnaires anonymously. main outcome measures: • number of patients with medication reconciliation performed by a pharmacist. • number of drug-related problems denoted in the electronical journal and presented to the physician. • the overall experience physicians and the nurses had with pharmacists when located in the emergency department. results: pharmacists performed medication reconciliation for patients, identifying drps and potential drps in total. fourteen of the physicians had read the journal notes from pharmacist and found them helpful (n = , %) or greatly beneficial (n = , %). most physicians (n = , %) and nurses (n = , %) reported a good cooperation with the pharmacist in the care of the patients. some of the physicians (n = , %) and most nurses (n = , %) wanted more information about the pharmacists work in the emergency department. the majority of ed staff ( % of physicians and % of nurses) found pharmacist as a good academic resource in the emergency department. conclusion: the physicians reported an improvement regarding the quality in the medication reconciliation made by pharmacists in the emergency department and both physicians and nurses expressed a need that pharmacists work in the emergency department on a more permanent basis. more information in general and especially better communication with nurses regarding the care of the patients are important actions need to optimise collaboration with pharmacist in the emergency department. results: a total of patients were included in the study, median age was years and % were males. they used in average four drugs regularly (range - ). almost three-quarters ( %) of the patients reported high or moderate adherence to all their regularly used drugs (mmas- c (max )). of the patients using oral spasmolytics, % reported high or moderate adherence to these drugs. the majority ( % of the patients) had high perceptions of necessity to their treatment (bmq [ . (max )), and % had a high level of concern (bmq [ . (max )). logistic regression analysis showed that there was no association between adherence and pain, nor between adherence and spasticity. younger age was found to be associated with higher risk of nonadherence. conclusion: even though overall adherence was high, the patients were more concerned to take their medicines compared to other patients with other chronic conditions. further studies are required for understanding adherence and attitudes toward medication in this population, and to help the patients feel safe about their medication regime. please specify your abstract type: descriptive abstract (for projects) background and objective: errors in medication lists often emerge in transition between health care levels, and there is need for strategies to communicate medication information. therefor we aimed to describe reasons why medication discrepancies (md) occurs in the transfer of patients between hospital and primary care service. design: in conjunction to a study based on use of structured medication report at transition from hospital to primary care service, we observed different reasons to why mds occurs. our observations and experiences linked to communication between health care levels is outlined. results: we observed that many md's disclosed at discharge could most likely be attributed to lack of medicines reconciliation at admission to hospital. for instance, several medicines were prescribed in primary care service prior to admission, but not at admission to the hospital. in addition, at admission, some medicines were listed as prescribed medications although not found in the medication lists in primary care service. we also observed that newly started and discontinued medicines were documented in the hospital discharge letter, but not implemented in primary care service. according to health care personnel in primary care service, insufficient communication about the patients' medications at discharge from hospital, led to corrections in the medication lists based on their previous knowledge about the patients. in addition, justified medication changes at discharge from hospital were not always implemented in primary care service due to professional disagreement. some stated that lack of trust was one reason for not always taking changes into account, often based on earlier experience. conclusion: these observations indicated that mds occurred both with and without intent when patients from primary care service were admitted to hospital and returned back due to poor communication. medication errors during hospitalisation and unproven intentional changes may be the consequences. due to this, it is important to improve the communication and confidence between professionals in the hospital and primary care service in order to reduce the number of mds and to enhance patient safety. please specify your abstract type: descriptive abstract (for projects) background and objective: intravenous human immunoglobulins (iv igs), plasma protein products, may cause in patient to a range of adverse side effects (headache, skin rash, kidney failure, thromboembolic event). in the framework of securing medicinal care, an assessment of professional practices has been conducted within our university hospital. the overall goal of this study is to evaluate the process of intravenous administration of human immunoglobulins done by the nurse staff. design: this prospective study has been carried out in three departments of neurology. an observation grid was established on the basis of guidelines on good practices. all in all, criterions have been examined resuming: prerequisites before administration, patient setup, iv igs administration, monitoring, traceability of drug delivery and management of adverse side effects. results: during the course of this investigation, administrations were observed. only % of nurses deliver information about the treatment to their patients before administration and % question patients about previous hypersensitivity reaction. the presence of spontaneous diuresis is verified in % of cases. emergency cart is not reachable in % of all cases. % of nurses ask patients to decline their identity. the use-by date on the bottles is checked in % of cases. at the time of preparation of perfusion, labelling does not mention either patient's name ( %) or date and hour of perfusion ( %). int j clin pharm ( ) : - during perfusion, only % of nurses follow diuresis and % watch rate of administration. hydration is not always kept min after the end of perfusion ( %). patient monitoring varies between min and h after perfusion's end. in % of cases, diuresis is monitored after the end of administration. % of nurses explain to patients side effects that may occur remotely. finally, administration traceability is was conform in % of all cases and in the event of adverse side effects, statement was made in % of cases. conclusion: best compliance scores have been achieved in myology department where patients are fewer than in the two others departments ( vs. and ). a presentation of those results will be given in theses three departments in order to improve patient management and securitization of iv igs administration. this audit will be carried out soon in other departments. please specify your abstract type: descriptive abstract (for projects) background and objective: a new human polyvalent immunoglobulins dose ( g) for intravenous administration is available on our establishment since . in order to secure the administration, this new dosage was initially reserved for the healthcares using administration pumps (being four health-care). the aim of this survey was to evaluate the satisfaction of the nursing staff already user of the new g dose and to estimate the motivation of the nonuser nursing staff by the audit date. design: this satisfaction survey was carried out with the most igiv consumer services (being internal medicine, neurology, cardiology and haematology). the questionnaire was structured in two sections: the first section regarding igiv in general, the second section concerning the new g dose. the survey included multiple choice questions or questions with answers based on a four levels evaluation scale (not satisfied, mildly satisfied, satisfied, and very satisfied). results: the audit was realized on eight health-care, involving nurses. among the interviewed, ( %) have already used the g dosage. in % of cases, users were very satisfied and % were satisfied. the most positive points noted were: gain of time provided ( . % of satisfaction), less manipulation needed ( . % of satisfaction), and reducing of infectious risk ( . %). moreover, the influence of the injection technique on users' satisfaction was further reported. indeed, according to nurses interviewed, the use of an injection pump is safer and improves the job comfort of nursing staff, unlike the injection by gravity (used in % of cases), which seems to slow down the use of this new dosage. in two cases, a positive opinion given by patient was also reported. finally, negatives points noted were related to administration instruments (use of pump or not) and to less flexibility in daily dose regulation. among the % not-user of this new dose, the % showed a strong interest for the product apart from services making the igiv administration by gravity. conclusion: in light of these results, the use of g dose will be spread to other services. the general diffusion of this dosage will provide a gain of time also at the pharmacy, during the unitary delivery and the computer-based administration of every units. a second survey will be soon effected within patients involved in the switch g/ g. the capital region pharmacy, clinic of neurology, rigshospitalet, blegdamsvej, copenhagen, denmark please specify your abstract type: research abstract background and objective: the clinic of neurology, rigshospitalet, copenhagen, denmark experience continuous medicine-related patient safety incidents (psi) related to newly admitted patients and patient transfers between wards. in order to prevent drug related problems (drp), the pharmacists increased their focus on these patients and provided systematic medication reconciliation. thus, the objective of this pilot study was to investigate if the intervention would help identify drug discrepancies (dd) and prevent drp. four wards were included in this study; two neurological, one neuro-anaesthetic (icu), and one neurosurgical ward(s). three wards use electronic medication module (epm), whereas the icu uses critical information system (cis). furthermore, all patients' prescriptions are registered on shared medication record (smr), which provides an overview of prescribed medicine. prescriptions cannot be transferred from smr and epm to cis and vice versa. we suspected that psi resulted from these system incompatibilities. setting and method: patients admitted or transferred from may nd to june rd were included. medication reconciliations using smr, epm and cis were conducted by a pharmacist on weekdays. dd were presented to a physician orally and documented. only dd accepted by physicians led to drug prescribing change. main outcome measures: number of identified dd. results: the study included patients, of which ( %) were newly-admitted. patients ( %) were transferred between wards. of the transferred patients, ( %) were transferred from the icu to other wards and ( %) were transferred from other wards to the icu. of the newly-admitted patients, ( %) were admitted to the icu and ( %) were admitted to other wards. the pharmacists identified dd; dd ( %) in the transferred and dd ( %) in the newly-admitted patients. in the transferred, dd were all related to the icu. in the newly-admitted, dd ( %) was related to the icu and dd ( %) to other wards. of the dds, ( %) were accepted by the physician. an example of a severe dd identified was an omission of prednisolone to a patient admitted to the icu. conclusion: most dd were identified in patients admitted to or transferred from icu, which uses the incompatible system cis. pharmacist systematic medication reconciliation helps identify these dd and prevent drp. please specify your abstract type: research abstract background and objective: antibiotic related drug interactions are more likely in intensive care unit patients due to common polypharmacy and antibiotic usage. the aim of this study is to determine the antibiotic related drug interactions with three different online databases (micromedex-paid, medscape-free and drugs.com-free) and to evaluate these interaction information by clinical pharmacist. setting and method: a retrospective, descriptive study was set up in hacettepe university hospital's intensive care units, between november and december , . patients who use at least one antibiotic were involved in this study. all drugs were assessed by each three databases and only antibiotic drug interactions were evaluated. clinical significance of identified drug interactions were evaluated by clinical pharmacist. main outcome measures: clinical pharmacist's assessment in significance of drug interactions indicated by three online databases. please specify your abstract type: research abstract background and objective: an implementation of clinical pharmacy practice by postgraduate students in intensive care units is a new way of learning in postgraduate education which creates opportunities in multidisciplinary collaboration in clinical pharmacy research, and also has influence on clinicians' routine patient care process. this system in educational program was ongoing in the department of clinical pharmacy since . as a part of this educational program, drug related problems in intensive care units were described and analysed, an influence of clinical pharmacy postgraduate students on patient treatment process was sought. setting and method: a prospective, cross-sectional study was performed between the march-june in hacettepe university hospitals, department of internal diseases intensive care units which consists of beds. three postgraduate pharmacy students from the department of clinical pharmacy, faculty of pharmacy conducted medication reconciliation in order to identify any problems in patients' medical orders. drug related problems (drps) were identified by the students and recommendations for management were approved by a supervisor of clinical pharmacy department before they were directed to physicians for approval. the students were not authorized to undertake any action in patient care process, therefore all required interventions for drp were undertaken by physicians and the acceptance ratio of the interventions were recorded. the pharmaceutical care network europe foundation classification system (v. . ) was used to asses drps. main outcome measures: determination of drps by pharmacists and evaluation of their interventions' acceptance by physicians in intensive care units. results: during the study period, patients were admitted to the intensive care units. each patient's medication orders were evaluated and interventions were recommended by postgraduate students. the number of interventions per patient was . . the acceptability rate of interventions by physicians was . %. in addition, physicians were provided drug information on seven different occasions. recommendations regarding drug therapy were mainly related with treatment effectiveness and adverse reactions. the common causes of drps were requiring dose adjustment due to pharmacokinetic problems ( . %), no therapeutic drug monitoring ( . %), inappropriate timing of administration and/or dosing intervals ( . %), requiring dose adjustment due to deterioration/improvement of diseases ( . %), inappropriate drug selection ( %) and new indication for drug treatment presented ( %). the most common drugs responsible for drps were ranitidin, levothyroxine, allopurinol, pantoprazol, piperacillintazobactam and vancomycin. the study showed that the most common drps was dose-related, therefore close monitorisation of the intensive care unit patients by students in clinical pharmacy postgraduate program can help physicians in terms of detecting, preventing and minimizing drps in order to improve patients' health outcomes. please specify your abstract type: research abstract background and objective: antibiotic stewardship is the process of salvaging important antibiotic agents from becoming ineffective due to bacterial resistance. this is important because throughout the world antibiotics continue to be one of the most important classes of therapeutic agents due to their vital role in saving patient lives. key goals of antimicrobial stewardship are to improve clinical outcomes, prevent antibiotic resistance, promote patient safety, and reduce health care cost. pharmacist are in the frontlines because they perform antibiotic stewardship activities, such as selecting the most optimal antibiotic agent, adjusting drug-dosage, and stopping use of unnecessary antibiotics. as a result of the continuous rise in antibiotic resistance and decline in development of new antibiotics, antibiotic stewardship programs are proving to be indispensable in a health care settings. setting and method: adult and paediatric inpatients receiving antibiotic therapy in the hospital medipol university has been evaluated. patients were selected randomly in the hospital system. patients were evaluated for antibiotic susceptibility results and compliance with antibiotic management guidelines. main outcome measures: to evaluate the antibiotic therapy in patients with culture results and to determine according the treatment guidelines. results: it was observed different pathogens in blood culture results of inpatients out of patients who were treated with antibiotics in hospital. antibiotic susceptibility results for acinetobacter spp, staphylococcus spp, enterococcus spp, pseudomonas aeruginosa, klebsiella spp, e. coli spp, streptococcus spp, corynebacterium spp, streptococcus pneumonia and enterobacter spp are evaluated in the study. klebsiella spp was the most isolated pathogen at total of culture results. most frequently resistance were int j clin pharm ( ) results: a total of ( %) questionnaires were completed. of these, approximately % were answered by hospital nurses, the remaining mainly by physicians ( %) and % ''other''. on the question ''what is your general perception of the benefit of the clinical pharmacy service; for collaborating health professionals? for the patient?'' the total benefit was ranked . and . respectively (scale from (''no benefit'') to (''beneficial to a very large extent''). the open questions: ''what disadvantages/advantages have you experienced by the introduction of clinical pharmacist into multidisciplinary teams?'' received / comments respectively. physical obstacles regarding office space, interference with the decision making process, more time consuming processes and the issue of relying too much upon the advices given was reported as possible disadvantages. respondents answered ''none'' to this question. the comments regarding advantages dealt mainly with general increased patient safety and quality assurance. in addition, advantages as work-load relieve, time saved, collegial support, practical help, and learning interchange between professions, were highlighted. conclusion: health-professionals assessed the clinical pharmacy service as highly beneficial. the advantages outlined were higher patient safety and quality regarding medication, in addition to collegial support, practical help and learning interchange. please specify your abstract type: descriptive abstract (for projects) background and objective: in june , the french health authority, the «has», published an index resuming the recommendations of benzodiazépines (bzd) prescriptions and proposing an approach to stop using it. indeed, it has been established that there is a too high and too long consumption of bzd in france. a study of prescriptions' prevalence has been done in our hospital centre. the aim of this study was to know our situation regarding the use of bzd in order to set up some improvements and take part in their proper use. design: a prospective study has been done on a months period in different services: geriatric, post-op and rehabilitation facilities, endocrinology, internal medicine, pneumology and cardiology services. the data were raised on a given day in each services and recovered thanks to the prescriptions software but also through interviews with the patients and their doctors. it was examined whether there was a bzd prescription (hypnotic or anxiolytic), whether the duration was superior or not to the duration of the amm and whether the prescription was done in our hospital centre. if the prescription was already part of the patient treatment, we looked if it was possible for the patient to stop using it, according to the has criteria. on their discharge, the letters and bzd prescriptions were also analysed and some patients' general practitioner were contacted after their discharge. results: patients (median age years old) were included from november to march . . % ( / ) of the patients had at least one bzd prescription the day we collected the data. we found only one bzd in prescriptions ( . %) and among them . % ( / ) were anxiolytic bzd. among those prescriptions, . % ( / ) already existed before the hospitalization and . % ( / ) were given during the hospitalization ( were prescribed automatically). . % ( / ) of the prescriptions did not respect the legal duration of the amm ( pieces of data were not found). . % ( / ) already exceeded this duration limit. among the patients who already had a bzd treatment before going to hospital, . % ( out of ) could consider stopping their use of bzd. by the end of this study, patients were discharged from hospital, among them . % ( / ) with a prescription of bzd. . % ( / ) of the prescriptions established during the hospitalization had been renewed when the patient came out of the hospital, we managed to contact ten general practitioners (approximately . days after their discharge), nine patients carried on their bzd treatment, among them one patient had reduced his consumption. conclusion: this study is an example of the high proportion of bzd prescriptions in france which the majority doesn't respect the legal length of the amm. the prescriptions of bzd in the hospital are generally systematically renewed by the general practitioners. the patients must be informed about the risks of using those molecules. in order to ameliorate this practice in the hospital, a proper use leaflet, reminding the prescriptions of bzd, has been created and distributed in each services to make people aware. main causes of admission were infections ( %) (respiratory disease ( %) and other ( %)), hepatic disease ( %) and neoplasias complications ( . %). patients died during their admission; due to hepatic disorder, due to neoplasia, and due to infections. conclusion: last diagnosis of hiv or no art treatment are causes of admission. immunovirological situation is related with their adherence but isńt with admissions. coinfection with hcv or hbv or others infections are risk factors for admission. center for psychopharmacology, diakonhjemmet hospital, oslo, norway please specify your abstract type: research abstract background and objective: complex medical history and treatment can potentially cause problems. the objective of this study was to investigate the prevalence of drug-related problems (drps) and medication discrepancies in internal medical patients with complex treatment at hospital admission. further, to investigate to which extent drps were identified as a result of medication reconciliation, and to which extent drps could be associated to the hospitalization. setting and method: patients with at least four regular medicines from two different therapeutic groups were consecutively included at admission to an internal medicine ward at a university hospital in norway in the period . . - . . . pharmacists used the integrated medicines management (imm) model for medication reconciliation and medication reviews at admission. a medication discrepancy was defined as any discrepancy between the recorded medication list at admission and the patient's actual use of medications, as revealed by medication reconciliation. the patients' actual use of medications, medical journal and laboratory results, were used to perform a medication review at admission time and identify drps. the proportion of drps revealed due to medication reconciliation was calculated. moreover, the project group retrospectively assessed possible drp-induced hospitalizations based on clinical history, cause(s) of admission and identified drps. main outcome measures: the main outcome was the median number of drps per patient at admission. the proportion of drps revealed due to medication reconciliation, the proportion of patients with drps possibly associated to the hospitalization, and the median number of medication discrepancies, were included as secondary outcomes. results: patients were included, . % women. median patient age was (range - ) and most of the patients were home-living before admittance ( . %). in total drps were identified at admission, with a median number of (range - ) per patient. drps ( . %) were identified due to medication reconciliation. for patients ( . %) a causal relationship between the hospitalization and the drps was assessed as ''possible''. medication discrepancies were revealed in of the included patients ( . %), with a median number of (range - ) per patient. conclusion: internal medical patients with complex drug regime are frequently exposed to drps and medication discrepancies at hospitalization. medication reconciliation could be essential to identify drps, which is likely a common cause of hospitalization in the studied patient population. hp-pc : assessment of oral anticoagulant prescriptions and pharmaceutical analysis at the hospital by regional audit damien fuss *, , clélia monchablon , anaïs breteau , marie lefebvre-caussin , rémi varin , jean doucet , mikael daouphars , doreya monzat omedit normandie -chu rouen, chu rouen, please specify your abstract type: descriptive abstract (for projects) background and objective: oral anticoagulants (oa) are the most common drug class associated with preventable adverse drug events in hospitalized patients that require optimizing the pharmaceutical analysis (pa) process. in this context, a regional audit was conducted on pa of prescriptions oral of oa. the aim of this study is to provide an overview of the treatment by oa in the hospital by evaluating the consistency of the oa prescriptions compared with national and european guidelines and evaluate the pharmaceutical interventions. design: this study is based on the collection of pa data (demographics, indication, posology, drug interactions, monitoring) as well as the collection of pharmaceutical interventions and discordance int j clin pharm ( ) : - between guidelines recommendations and clinical practice. the inclusion criteria were any patient treated with oa (vitamin k antagonists (vka), non-vitamin k antagonist oral anticoagulants (noacs)). included patients were followed minimum months. the primary outcomes include description of baseline characteristics of patients, the number of inappropriate prescriptions compared to the different clinical recommendations, the number of pharmaceutical interventions, the number of adverse drug reactions (adrs) related to oa use and the assessment of patient monitoring. results: during the -months study period, patients were included in six health institutions. the average age was years ( % of patients over years old) and % of the patients were women. % of patients had renal impairment. % of patients were treated with vka, and % with noacs. it was the first prescription of oa for % of patients ( % with vka; % with noacs). the most common indication was the non-valvular atrial fibrillation ( %). in this indication, % of patients had cha ds -vasc score c , and nearly % had a high risk of bleeding (has-bled score c ). drug interactions were observed, and adrs occurred related to oa. % of patients with an adrs had a has-bled score c . . % of prescriptions were considered inappropriate, including % noacs (no monitoring renal function in % of patients over years initiating treatment, inappropriate posology in %, and % of contraindications). the rate of pharmaceutical interventions was %. nearly % of the prescriptions were already adapted when the pharmacist was starting analysis. conclusion: prescribers are sensitized of the risks on the oa prescriptions, which explained the delay upon pa and low rate of pharmaceutical interventions. however, the high number of inappropriate prescriptions shows the necessity to improve the pa process on these drugs, particularly by actions on therapy initiation and patient monitoring, especially for noacs. for this class, the impossibility of assess the level of anticoagulation by laboratory monitoring requires appropriate initiation and monitoring, especially an assessment of baseline renal function. please specify your abstract type: descriptive abstract (for projects) background and objective: the development of bacterial resistance these last years is a public health major problem in the world and needs to implement actions. in france, the national drug safety agency has defined a list of ''critical antibiotics''. this list includes antibiotics particularly generator of bacterial resistance (amoxicillinclavulanate, cephalosporine, fluroquinolone) and antibiotics called ''last resort'' (antibiotics against gram-positive cocci, car-bapenem…). at our regional level, an evaluation of prescription of these critical antibiotics was proposed to all medical centers. the aim was to evaluate the quality of prescription of these critical antibiotics. design: the regional working group (pharmacists, infectious diseases physicians and biologists) had developed a collection grid including data on patients, antibiotics and four criteria: adequate molecule, compliance with medical prescriptions, duration of antibiotic therapy and reassessment at h. this is a prospective study proposed to all health institutions (public and private), which had to be completed on a given day in all care units and had to be conducted by a team of multi-professional evaluators. the study included a quantitative part (number of patients hospitalized in the audited units, number of patients receiving antibiotics and number of critical antibiotic prescriptions) and a qualitative part (adequate to the four criteria). results: response rate was of %. the study investigated on patients hospitalized in the audited units, including patients ( %) receiving antibiotics. among the patients, % were hospitalized in medical, surgery or obstetrics units we recorded prescriptions of ''critical antibiotics particularly generator of bacterial resistance'' ( % amoxicillin-clavulanate, % ceftriaxone, % fluoroquinolone and % other third-generation cephalosporine) and prescriptions of antibiotics called ''last resort'' ( % carbapenem). the average age of the population was . years (± years). sex ratio was . . % corresponded to curative use and % to prophylactic use. the expertise of infections diseases physician was requested in only % of the cases. the antibiotics were prescribed in majority to treat bronchopulmonary infections ( %), urinary tract infections ( %) and intraabdominal infections ( %). ninety-two percent of the prescriptions had a proper indication. % of the prescriptions complied to the guidelines. the duration of antibiotic therapy was adequate in % of the cases. only % of the prescriptions were correct according to these three criteria. forty-four percent of the prescriptions were reassessed and adapted by the physician. conclusion: this study is original because of its regional dimension and antibiotic analysis. the number of analysed prescriptions was significant with an overall proper prescription in adequate with the guidelines. however, actions must be implemented on duration and reassessment and adjustment of treatment. these results were presented to the participating hospitals. these three points will be reevaluated during a new regional audit. the criterion «no more psychotropic drugs» has been met in . % of assessment. otherwise, or more psychotropic drugs are prescribed in . % of assessment from the point of admission. the criterion «no more a benzodiazepine drug» has been met in . % of assessment. otherwise, more than one benzodiazepine drug is prescribed in . % of assessment from the point of admission. no contra-indication is detected in . % otherwise, a contra-indication between two drugs causing torsade de pointes is detected from the point of admission in this department. no more anticholinergic drug is prescribed in . % of assessment. according to the french criteria, one or more inappropriate drug is prescribe in . % of assessment. the most common inappropriate drug group prescribed was alimentary tract and metabolism drug ( . %) (the hospital at home team needs these class of drug) followed by nervous system ( . %) (prescribed at the point of admission) and by cardiovascular drugs ( . %) (prescribed at the point of admission). finally, the criteria «no more one non-steroidal anti-inflammatory drug» and «no illogical association» have been met in all cases. conclusion: this analysis shows that most of criteria for «assessment of prescription among elderly in a «hospital at home» department have been met. when one has not been met, either the hospital at home team needs the drug prescribed, or this drug have been yet prescribed from the point of admission in this department. this study could be used for the next certification. hp-pc : access to health care: case of autologous serum eye drops batiste martel, fabien lindenberg, camille castel, guillaume saint-lorant * please specify your abstract type: research abstract background and objective: autologous serum eye drops (ase), prepared from patient's serum, are indicated in the treatment of severe dry eye syndrome and defective epithelial healing. its in-hospital preparation within a controlled-atmosphere zone unable it to be dispensed by non-equipped hospital pharmacies. the aim of this study was to implement security measures to allow transport towards distant hospital pharmacies and all patients even those residing far from a regional university hospital (uh). setting and method: this study was conducted in a -bed french university hospital. patient blood samples were taken within the university hospital every weeks. serum was then biologically controlled (negative tests for hiv, hbv, hcv, tpha, vdrl). preparation was conducted days after blood sampling. sterile preparations were then stored at a temperature of - °c. studies showed that eye drops were stable days after being thawed. transport of eye drops to distant hospital pharmacies requires to be conducted under controlled temperature i.e. below °c, to ensure the stability of eye drops. these pharmacies are located close to patient's homes. the entire process was examined by a pharmacy team in order to study and secure each step, transport in particular. main outcome measures: validation of each step of the autologous serum eye drop dispensing process, from sampling to receipt by different hospital pharmacies, transport in particular. results: patients benefitted from the preparation. all patients resided more than kilometres from a uh. a follow-up form was completed to qualify dispatching and to trace each step during transport. a temperature sensor was placed inside the box. the receiving agent was required to stop and control the sensor. a double retrospective control was performed by a pharmaceutical team via the recording of temperature sensors. a second follow-up form was drafted in order to track dispensation reviews, ongoing dispensation and future planning. a patient information booklet was distributed to hospital pharmacies to inform patients about good practice concerning eye drops. conclusion: technological necessities concerning autologous serum eye drop preparation and transport limit access to health care. in this study, the role of the pharmacist consisted in reducing inequalities among patients residing at a distance from the only regional uh. the role of the pharmacist is to ensure absolute quality of preparation between the uh and the patient. hp-pc : computerized medication reconciliation: overview of pharmaceutical software used and support for development of integrated modules julie mocquard, anaïs berthe, elise rochais * , nicolas prévost, jean-claude maupetit, on behalf of centre de ressources régional en conciliation médicamenteuse omedit pays de la loire, nantes, france please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation aims to improve continuity of care for patients. in , a national survey identified barriers for implementation of this activity in france, among which computerized systems were judged unsuitable for hospital practices. in the absence of appropriate hospital information systems (his), medication reconciliation remains a time consuming process implying manual transcriptions, potentially leading to a lack of traceability and medication errors. the objective of the study was to assess the current his used in a french region including the integration of medication reconciliation into the software and to define courses of action to assist this integration. design: an online survey conducted in may was addressed to head pharmacists of the health facilities in the region, giving a total of head pharmacists concerned. it included questions on the software used by the health facility, the development of medication reconciliation and its traceability, formulation of operational requirements to the editors of software and availability of a module integrating medication reconciliation provided by the software. results: seventy-eight pharmacists ( %) participated in the study, with all types of health facilities represented: public hospitals, clinics, home health agencies, haemodialysis structures and after care and rehabilitation facilities. thirty different software were identified in the region. ( %) pharmacists planned to develop medication reconciliation in their health facility and ( %) were already carrying out this activity. within these %, medication reconciliation was conducted on paper only for ( %) of them, while ( %) were using a computerized system (patient file, pharmaceutical software, other) for traceability. the most widely used software in the region contains a module enable for computerized medication reconciliation, and three other editors are currently developing one. no development is scheduled for another three editors nonetheless commonly used in the region. ( %) pharmacists had contact with the editor of the software, and had given thought to the preparation of requirement specifications to the editor to develop an integrated module of medication reconciliation. conclusion: despite the interest attributed to medication reconciliation and despite the need of a fully integrated module of medication reconciliation to his, only a few health facilities of the region possess an appropriate computerized system to develop this activity. this study underlines the approaches already made by pharmacists to editors in order to integrate medication reconciliation to the his. subsequently, retrieving these approaches and writing specifications common to all health facilities is scheduled, in order to assist them in providing a strong incentive for the editors to integrate medication reconciliation to existing his. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is an interactive and multiprofessional process that ensures the continuity of care by integrating the ongoing treatment to the new hospital prescription. this helps securing the patient's care pathway particularly at transition points. the objective is to initiate the mr process in our medical institution with a pilot study in the department of internal medicineemergency downstream to validate a methodology and adapted tools. design: the mr takes place in three steps performed by a pharmacy student: ( ) realization of best possible medication histories (bpmh), combining at least three sources of information and using sources' collection form. this research begins with a patient interview done in pairs with a medical student using an interview guide. ( ) comparison bpmh with the initial hospital prescription in the department (after passing through the emergency department) on the treatment reconciliation form. a status is assigned to each line of drugs and then the differences are identified (stopped, changed or added). these two steps are validated by a pharmacist. ( ) discussion and characterization of observed differences (intentional/unintentional and documented/undocumented) with the senior physician. results: twenty-six mr were performed over weeks in . the mr is performed within days after admission. on average, . information sources per patient were used for the bpmh: mainly drug prescription (dispensed in pharmacies community); analysis of emergency medical records and patient interview. for the patients included, drugs were listed. discrepancies were observed and were studied (status stopped or changed only): one documented intentional discrepancy, undocumented intentional discrepancies and unintentional discrepancies (ud). these uds affected patients ( - medication errors per patient) and corresponded to a non-prescribed drug in % of the cases. vitamins, antihistamines, anti-reflux and proton-pump inhibitors were involved in % of cases; cardiovascular drugs in % and antiinfectious in %. through this pilot study, the methodology was validated: (a) need to have a minimum of three sources to achieve a relevant bpmh and to confirm each information with two sources; (b) need for a dedicated time with trained staffs; (c) development of tools to improve the traceability of information obtained from each source and traceability of medication reconciliation activity. conclusion: the mr establishment in the internal medicine department was helpful in identifying medication errors that have been corrected. it is proposed to archive the treatment reconciliation form in the patient file to contribute to the traceability of information on treatment. this study strengthens the deployment of this method and mr tools to other services of the hospital. alma mulac * please specify your abstract type: research abstract background and objective: clinical pharmacists have an important role in improving healthcare services. there is lack of knowledge of clinical pharmacists' experiences in interprofessional collaboration. our objective was to explore the challenges and barriers experienced by clinical pharmacists in interdisciplinary teams in norway and incorporation of expanded pharmacist roles in hospital settings. setting and method: this qualitative study was conducted using semi-structured interviews. a total of clinical pharmacists from four (government) hospitals were included in the study. the interviews were audio recorded using a digital recorder. the recordings were transcribed verbatim. main outcome measures: challenges and barriers clinical pharmacists experience in interdisciplinary teams in hospital setting. results: the main findings are that the pharmacists' role is little known to other health care professionals, particularly at hospitals with short tradition for clinical pharmacy services. clinical pharmacists have great motivation from being able to influence drug treatment for patients. from the perspective of the participating pharmacists they succeed in interdisciplinary cooperation when their professional knowledge solves the patients' drug-related problems. communicating recommendations to physicians with professional credibility has great importance for the intervention to be implemented. using the theoretical framework of communicating tensions, we argue that the pharmacists in our study use indirect communication to prevent physicians defensiveness to recommendations. lack of education in interprofessional cooperation and communication is apparent in this study. the participants also stated that there should be some form of quality assurance or education requirements before one can work as a clinical pharmacist. conclusion: training in communication for graduates and interprofessional collaboration during the undergraduate pharmacy education, can possibly help pharmacists with integration in interdisciplinary teams. increased attention to teamwork from the hospital leadership is essential for the implementation of interprofessional collaboration in a larger context. please specify your abstract type: descriptive abstract (for projects) background and objective: antifungal therapy in the icu, particularly therapy targeting resistant aspergillosis, mucormycosis and systemic candida, is often of lifesaving importance. posaconazole and voriconazole are the antifungal agents of choice. our aim was to compile a tool that can be used at the icu to address aspergillosis, mucormycosis and systemic candida in an optimal manner. design: female patient, age + , liver transplant, crp [ mg/ l, creatinine [ lmol/l. abdominal x-ray imaging revealed four large abscesses and laboratory analyses confirmed mucormycosis. posaconazole intravenous ( mg one times daily) and liposomal amphotericin b ( mg/kg/day) were initiated. the inflammatory markers remained unchanged days following initiation of therapy with no change in size or number of abscesses and the patient developed sepsis. amphotericin b dose was increased to mg/ kg/day. after week the inflammatory parameters and size of abscesses began to fall. the dosage form of posaconazole was switched from intravenous to mixture. the dose remained the same and within h the crp rose to mg/l. results: pharmacist intervention revealed a missing loading dose of intravenous posaconazole as well as incorrect dosage of the per oral form due to bioavailability variation. posaconazole mixture dose was increased to mg two times daily. through serum concentration analysis of posaconazole was suggested prior to the dose increase. the serum concentration was . mg/l (range [ . - . ) . through serum concentration days later was . mg/l. both crp and abscess size were on the decline. a dosage and tdm pocket card for posaconazole therapy of mucormycosis, aspergillosis and candida was compiled. conclusion: optimal systemic fungal infection therapy is essential, especially in the critically ill. of special importance is tdm and correct dose adjustment when dosage-form changes occur. please specify your abstract type: research abstract background and objective: potentially inappropriate prescriptions and omission of prescription, respectively ip and op, are common issues in the pharmacotherapy, especially in vulnerable population, such as elderly and children. there are many available tools detecting ip and op for geriatrics, however, similar tools are less common in paediatrics. therefore, a first target tool for paediatric population: popi «paediatrics: omission of prescriptions and inappropriate prescriptions» was created and was validated by delphi method in . we aim to evaluate inter-rater reliability between health care professionals, who apply popi. our study also assessed their satisfaction and the accessibility of this tool. setting and method: twenty cases with or without ip or op were selected. these cases were identified in a previous retrospective ip-op prevalence study on . patients. these patients were admitted to the emergency department of a university mother and child hospital, between october and march . one doctor and one pharmacist, who participated in the creation of popi tool, identified ip and op in cases and composed ''standard answers''. these cases were then reviewed independently by eleven clinicians (including generalists, paediatricians, pharmacists, residents, general practitioners), who did not experience this tool before. inter-evaluator agreement was calculated by using the agreement kappa test. the satisfaction of users was also evaluated. main outcome measures: inter-evaluator agreement, the median time of use and the satisfaction of users. results: a high level of agreement of ip and op detection was recorded (ip: k median = . ; op: k median = . ). the easy use of popi was approved by % evaluators. the median time of use was min s per case (quartiles : . - . ) . as a result, there were % of clinicians satisfied with the provided popi and they would like to apply this tool in their daily practice. conclusion: popi demonstrated a good interrater reliability and is easy to use. this strong validation by many specialists prove popi is a reliable tool. it can be applied daily at work in paediatric section by doctors and pharmacists. other multicentre and prospective study should be conducted to evaluate economical and clinical impacts of popi. please specify your abstract type: descriptive abstract (for projects) background and objective: drug dosing during cvvh is challenging due to changes in pharmacokinetic parameters brought about by the patients' deterioration in health and factors associated with the physical process of filtration. this is of particular significance in the icu. in addition, there is the issue of the patients' diuresis or lack of such. this will affect the total clearance (cl total ) of the drug. the dose of antibiotics must therefore be calculated individually taking into account all of the above as well as changes of filtration parameters. our aim was to illustrate how such dosage calculations can be undertaken. design: a -year-old male patient, weight kg, diagnosed with stenotrophomonas maltophilia infection. the trimethoprim/sulfamethoxazole dose was . mg trimethoprim/kg/day every h as specified for anuric patients on cvvh. patient was initially anuric for days after which diuresis was started. the dose was recalculated. results: creatinine clearance (crcl) related to cvvh during the anuric period was calculated accounting for ultrafiltration rate, sieving coefficient, blood-flow, haematocrit concentration and pre-dilution. the value was ml/min. following diuresis on day , remaining kidney function was assessed by measuring urine and serum creatinine. the value for crcl renal ( ml/min) was added to the extracorporeal clearance, and gave a total clearance of ml/min. this warranted dose adjustment of trimethoprim/sulfamethoxazole since this drug requires normal dosage at crcl [ ml/min. conclusion: during cvvh, the presence or absence of diuresis must be taken into consideration when dosing antibiotics. in anuric patients, the cvvh-machine set up constitutes crcl total , but in patients with diuresis, the remaining crcl renal should be added. please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of the study is; to evaluate patients' home (prescribed and non-prescribed) and hospital medication during hospital admission by computing medication regimen complexity index and investigating possible drug-drug interactions. design: patients (aged and older) who applied internal service during months ( days/a week) were included to the study. patients' medical profile were obtained from patients' file. their home medication and hospital medication were calculated with medication regimen complexity index ( ) and checked drug interactions with micromedex drug interaction program. results: a total of from of patients who applied to the internal service (male . %, female . ; the mean age of patients was . ± . .) were included to this study during months. of them, . % had low education level (\ education years), . % had and more chronic diseases of them, % hospitalized last months before this hospital admission. the most prevalent diagnoses documented at admission were kidney disease ( . %), cardiovascular disease ( . %) and cancer ( . %). the mean of patients' home medication number was . ± . and the mean of their mrci scores was . ± . . % in patients hospitalized in the last months. at least one possible drug-drug interactions were found in . % of patients at home medication and in . % of patients at hospital medication, respectively. the mean number of possible drug-drug interactions at patients' home medications was . ± . , while the mean number of possible drug-drug interactions at patients' hospital medications was . ± . . of them, . % had polypharmacy at home medication. the frequency of possible drug-drug interactions and the score of medication complexity index was found high among patients' hospital medications when compared with their home medications. conclusion: the potential role of pharmacist including medication reconciliation and medication review could improve rationale drug use during hospital admission. coronavirus. experts' local committee has approved to use oral ribavirin for the treatment of these respiratory viral infections. we aimed to assess the effectiveness and safety of oral ribavirin as main treatment in respiratory viral infections. setting and method: from may to october , we performed a retrospective monocentric study including patients who received oral ribavirin for non-hcv infections. main outcome measures: viremia negativation was used to determine the response rate to oral ribavirin. specific toxicities (anaemia, cytopenia, liver dysfunction) and renal function were assessed biologically. results: thirty-five immunocompromised patients (f/m: / , age: ) were included. underlying conditions were lung transplant (n = ), heart transplant (n = ), pulmonary fibrosis (n = ) and acute myeloblastic leukaemia (n = ). the median duration between transplantation and infection was . years ( . - . ). nine patients were exclusively infected by rsv, by hpiv ( hpiv- ; hpiv- ; hpiv- ; hpiv- ; non-identified hpiv), by hmpv and by coronavirus. there were six co-infections: rsv/ hpiv- , rsv/coronavirus, hpiv- /hpiv- and hpiv- or /coronavirus ( patients). all the patients were admitted in pulmonary division, except for the patient with heart transplant who was in cardiac intensive care unit. the administered dose was mg tid or mg tid if there was renal insufficiency ( patients). the median duration of the treatment was days . four patients prematurely discontinued the treatment due to severe toxicity or therapeutic change; three didn't respond to the treatment (no data for the last one). four patients were re-treated despite having a virological response to the first cure. one patient treated for a hpiv- /coronavirus coinfection had an hpiv- relapse days after ribavirin discontinuation. concerning the three other patients, they received a second cure to treat a new infection (coronavirus, hpiv- and hmpv, in opposition to hpiv- twice and hpiv- respectively). virological response rate was % ( / for rsv, / for hpiv, / for coronavirus and / for hmpv). two non-negative viremia patients (rsv and hpiv- /coronavirus) received intravenous ribavirin after oral ribavirin therapy. no patient died from viral infection. twelve patients presented specific toxicity: one hepatic cytolysis and cholestasis, eight haemoglobin decrease, two pancytopenia and one mucositis. conclusion: despite the poor number of patient, our study shows that oral ribavirin seems to be efficient to treat hpiv, hmpv and coronavirus in immunocompromised adults. we observed known side effects that could generally be managed. oral ribavirin may thus represent a therapeutic strategy in several respiratory viral infections. please specify your abstract type: descriptive abstract (for projects) background and objective: reconciliation of medicine lists is important to ensure correct medical treatment of patients both in hospital and other healthcare levels. while reconciliation upon admission is part of the normal routine at surgical ward b, molde hospital, there has been less focus on reconciliation at discharge. as such, this study aimed to ensure reconciliation and correct transfer of medical information at discharge. design: medicine lists of all patients discharged from surgical ward b, molde hospital between week and in (n = ) were investigated. the forms were gathered and counted based on the tasks signed for to ensure completed reconciliation and sufficient information given to the patient. the count was performed every - weeks, and the forms in each count was pooled together as one point of measure. the quality of medicine lists in discharge lists was evaluated based on the norwegian patient safety program criteria. medicine lists in discharge lists from week to (n = ) were pooled together and compared to medicine lists in weeks - (n = ). results: the results of reconciliation was divided into the subsections of surgical ward b, and represent the number of completed tasks as signed for in the reconciliation form. the surgical subsection showed a significant increase in patients with pre-checked medicine lists and reconciled medicine lists over the measured time period. similar results were not found in the orthopaedic subsection. as for the quality of medicine lists in the discharge lists, significant improvement was seen in all set criteria, with the exception of ''source'' in the surgical subsection. in the orthopaedic subsection however, no significant improvement was seen in any of the criteria other than ''indication for use''. conclusion: the implementation of reconciling medicine lists at discharge was successful. however, both subsections need to work further to ensure continuation and improvement of the process. furthermore we found varying results in the writing of medicine lists depending on subsection. still, regardless of the individual results of the two subsections there is big room for improvement to ensure that sufficient medical information is included in the discharge papers. please specify your abstract type: descriptive abstract (for projects) background and objective: from july clinical pharmacists began conducting medication histories and reviews (pharmacist notes) at the emergency surgical ward (esw), north zealand hospital (nzh). inclusion criteria are acute patients using c drugs or c risk drug (antidiabetics, anticoagulants, antipsychotics, benzodiazepines, opioids and digoxin). the aim of the service is to identify drugrelated problems and secure correct medication reconciliation between the medicine the patient is admitted with and the medicine in the electronic medication system (ems) in the hospital. the service ensures that the patients' medication follows across healthcare sectors. the objective is to determine if the discrepancies between the medicine the patient is admitted with and the medicine in ems (documented in the pharmacist notes) are used by the physicians. in addition to determine if the pharmacist interventions increase the physicians' acceptance rate of the discrepancies. design: data were collected at the esw at nzh (capacity of beds). data consist of pharmacist notes conducted from august to may . pharmacist notes were compared to the patient record and ems to identify if the pharmacist notes were considered by the physicians. in order to increase physicians' acceptance rate of the discrepancies suggested in the pharmacist notes, interventions were made according to the model for improvement. throughout the period, the focus was on oral delivery of the pharmacist notes. in december the pharmacist optimized the clinical relevance of the discrepancies, by creating and testing a list of products (including vitamins, herbal drugs, glucosamine etc.) which the pharmacist should not intervene on. in december the pharmacist also started to follow up on the pharmacist notes not considered by a physician the previous day to ensure that the physician considered the discrepancies. results: there were identified discrepancies between the patients' actual medication at admission and ems at the hospital in patient records ( . discrepancies per patient). in total discrepancies were accepted by the physicians ( . discrepancies per patient). the physicians' acceptance rate was based on the acceptance of one or more of the discrepancies in the pharmacist note. baseline data were collected from august to november , where out of pharmacist notes were accepted by the physicians resulting in an acceptance rate of . %. from december to may the interventions made by the pharmacist contributed to an increase in acceptance rate to . % ( out of notes accepted). if the pharmacist notes were not delivered orally to the treating physician the acceptance rate was % ( out of notes accepted). conclusion: the pharmacist interventions contributed to an increase in the physicians' acceptance rate of discrepancies from . to . %. a result indicating that the pharmacist notes contributes to an increase the quality of the medication process across sectors. hp-pc : how the centralization of medicines manufacturing enable to generalize the pharmaceutical validation? samantha oses * , soizic vandierdonck, vincent servant, dominique breilh please specify your abstract type: research abstract background and objective: the centralization of the reconstitution of injectable anti-infective drugs enhance to decrease costs and several risks. this minimization of the risks operates at several levels such as i) reduction of the staff exposure and external contamination of preparations during the reconstitution phase (with controlled atmosphere areas, isolators, etc.), ii) improvement in the quality of the management of infective diseases thanks to a pharmaceutical validation systematically performed after the prescription and before the reconstitution phase. the main objective of the study was to describe and quantify pharmaceutical validation on injectable anti-infective drugs prescriptions restored in a pharmaceutical reconstitution unit. setting and method: an observational descriptive study was carried out on each prescription with at least one injectable anti-infective drug that has to be reconstituted before administration. the process was as follows: -prescription by the physician on an electronic prescription software, -pharmaceutical validation and if necessary pharmaceutical intervention (pi) made by phone call, -reconstitution at the pharmacy, -administration to the patient. the pharmaceutical validation methodology followed the french society of clinical pharmacy (sfpc) guidelines ''prescriptions screening and analyses level'' published in the good practices of clinical pharmacy and one resident and one pharmacist were devoted to the activity every day. main outcome measures: the pharmaceutical validation was quantified by the number of pi by patient, which were categorized according to the sfpc guidelines. results: during months, a total of pi were collected. they concerned patients with an average of . pi per patient. among them, . % ( ) concerned paediatric population. antibiotics were involved in . % ( ) then followed by . % ( ) cases ( . % ( ) biological assessment issues, . % ( ) absence of therapeutic drug monitoring (tdm) and . % ( ) the drug hasn't been adapted to the weight), dosage adjustments in . % ( ), information missing concerning the treatment indication in . % ( ) and miscellaneous pi in . % ( ) such as wrong clinical service on the prescription, etc. approval rate of physicians was . % ( ). conclusion: this study has shown that even if prescriptions were secured by electronic prescription software, the pharmaceutical validation remains essential. in that case, the centralization of the reconstitution of injectable anti-infective drugs enabled to generalize this activity on all prescriptions of the hospital. however, the pharmaceutical validation was focused only on anti-infective drugs, that was not fully efficient and must be extended to the whole prescription. it is a priority to develop a comprehensive and exhaustive validation on every medical prescription; however, this activity is highly time consuming and needs larger and more trained staff. hp-pc : the start/stopp criteria as a helping tool to the pharmacists' medication review in the acute admissions unit of the regional hospital in horsens hans pedersen * please specify your abstract type: research abstract background and objective: polypharmacy occurs often increasing the need for patients' medications to be reviewed. the start/ stopp criteria help detects potentially inappropriate prescriptions in older people. in this study we aimed to measure and categorize the different start/stopp criteria found in medication reviews in the acute admissions unit of horsens and the acceptance rate. setting and method: patients admitted to the acute admissions unit were selected based on their age and the number of prescriptions in a period of months. patients years or more which received six or more drugs were included in the study. only patients who later were transferred to another medicine ward were included in the study. the pharmaceutical medicine review was performed by a clinical pharmacist using minimum two different sources; the electronic medical record and medication-lists. the guideline of pharmaceutical medicine review in the hospital pharmacy central denmark region was used as the standard-guideline. in addition, thestart/stopp criteria version was used. main outcome measures: the number of start/stopp criteria found in medication reviews. the different start/stopp criteria were scored equally with one point each. results: patients, males and females, out of , were included. the mean age was years and the patients received in average prescribed drugs. at admission the average number of stopp criteria were . ± . and . ± . for the start criteria. in average, % of the purposed stopp criteria were accepted by the physicians. the most frequently accepted stopp criteria were in the category of drugs that predictably increases the risk of falls in older people. the benzodiazepines where the most common drugs to be discontinued. in the start category, % of the suggested start criteria were accepted, which included: calcium and vitamin d supplement, beta- agonist and bisphosphonate. conclusion: the present study demonstrates that it was possible to integrate the start/stopp criteria as a helping tool in the medication reviews in the acute admissions unit of horsens. the start/stopp criteria were found within the different categories, however only a minor part of the registered start/stopp criteria were accepted by the physician. please specify your abstract type: research abstract background and objective: the objective of this work is to assess prescribing practices of somatostatin analogues in a surgery department, and to analyse the conformity of switching from immediateacting octreotide to the long-acting release (lar) form, in accordance to laboratories' guidelines. setting and method: retrospective observational study. a focus was realized on patients admitted in a digestive surgery unit between january and december , . the patients' medical records were reviewed for clinical features, diagnosis workup and treatment strategies. main outcome measures: medical records for patients with diagnosis of gastro-entero-pancreatic or endocrine tumors who had received injections of lar octreotide during hospitalization were reviewed and the economic impact of prescriptions errors has been evaluated. results: of the evaluated patients, ( %) were hospitalized in surgery digestive unit; mean age at first administration of octreotide was years and % were male. the male and female ratio was . : . reasons for hospitalization were: digestive system neoplasms ( %), fistula ( %), intestinal obstructions ( %) and other pathologies ( %). of the patients treated with octreotide, ( %) received a lar form. only four patients received doses in accordance with guidelines: one at mg/month lar form and three at mg/month lar form, after having respectively been treated by intravenous octreotide at and mcg/day during - days. medical prescriptions of the remaining patients did not comply: all patients received mg/month after an intravenous treatment of mcg/day, instead of mg/month. from a financial perspective, these misuses have led to an additional cost of . euros for the hospital, excluding tax ( mg: . €/unit and mg: €/unit). conclusion: despite the publication of octreotide release form proper use recommendation in our hospital, % of patients of digestive unit are not right treated. a new guideline will be written added by doses of long-acting release and economic data. this work will be transmitted to specialists by clinical pharmacists. hp-pc : pharmaceutical process for intrathecal analgesia in clinical oncology practice vivien pigeon * , guillaume binson, claire grignon, antoine dupuis please specify your abstract type: descriptive abstract (for projects) background and objective: in some cases, patients with cancer pain remain painful despite the use of high dose of intravenous opioids and intrathecal analgesia becomes the ultima recourse to manage acute pain. until , intrathecal syringes were prepared by nurses in the unit care which involve a risk for patients. therefore, the aim of this work is to describe the set-up of the prescription and preparation process with the potential benefits for the safety. design: multidisciplinary concertation took place between pharmacists, physicians and surgical teams and several points were discussed to secure the process: • identification of patients with high level of infection risk; • identification of critical points of the pharmaceutical process; • validation of quality control and drug stability studies regarding drug compounding involving morphine, ropivacaine, baclofen and clonidine, alone or in admixture. results: multidisciplinary concertation lead us to define the most important points to set up the pharmaceutical process for intrathecal analgesia: • chosen patients are cancer patients; • implementation of a prescription software to secure the prescription step; • production of syringes by the pharmacy department implying several criteria: • preparation in controlled atmosphere area; • training of pharmacy technicians; • implementation of quality control and drug stability studies at °c in syringe over h and at °c in pumps over month; • microbiological control and bacterial endotoxin level. the implementation of a pharmaceutical process for intrathecal analgesia gave us the opportunity to reorganize the care of cancer patients tolerant to high dose of opioids. in this process, the pharmacy department plays a major role leading to decrease the risk of infections and errors of dosing. ingrid plessala *, , xavier deviot , thomas sidibe , zohra mostefaï , michèle minvielle , marta wyrtwal , roselyne gervais pharmacy, geriatrics, saint-denis hospital centre, saint-denis, france please specify your abstract type: descriptive abstract (for projects) background and objective: proton pump inhibitors (ppis) are indicated in gastro-oesophageal reflux and peptic ulcer disease. they are widely prescribed, often in off-label indications. the objective of this work was to reassess ppis prescriptions in collaboration with geriatricians. proton pump inhibitors (ppis) are indicated in gastro-oesophageal reflux and peptic ulcer disease. they are widely prescribed, often in off-label indications. the objective of this work was to reassess ppis prescriptions in collaboration with geriatricians. design: prospective study in three geriatric wards. the study included ppis treated patients from these three geriatric wards. dose, indication of the ppi, age, gender and duration of treatment have been recorded for each patient. the relevance of each ppi treatment has been reassessed by a geriatrician, a pharmacist and a junior pharmacist, regarding the indication and the patient's clinical condition. following this re-evaluation, three situations arose: • to maintain ppi at the same dose ( mg or mg) • to maintain ppi but half dose (from mg to mg) • to stop ppi corrective actions have been recorded in patients' files to allow their traceability. results: patients were included in the study. % of ppis prescriptions were off-label, % had no indication mentioned in patient's file and % were conform to the marketing authorization. % of patients have been on ppis medication longer than months, which is the recommended treatment' duration in france, % longer than a year and % longer than years. in collaboration with the geriatricians, ppi prescriptions were maintained for % of patients. we reduced the dose in % of cases. finally, we decided to stop a third of the ppis prescriptions. conclusion: ppis prescriptions are often longer than recommended. this can lead to side effects for patients. in france, lack of new recommendations since may explain this frequent misuse of ppis. there is also a reserve from doctors to stop these treatments, especially with fragile patients. in our case, the relevance of each ppi treatment was re-evaluated in three geriatric wards and we succeeded in shortening and stopping ppis medications in half of the situations. to assess the impact of this action on our geriatricians, a new review of ppis prescriptions relevance is programmed in . ppis prescriptions are often longer than recommended. this can lead to side effects for patients. in france, lack of new recommendations since may explain this frequent misuse of ppis. there is also a reserve from doctors to stop these treatments, especially with fragile patients. in our case, the relevance of each ppi treatment was re-evaluated in three geriatric wards and we succeeded in shortening and stopping ppis medications in half of the situations. to assess the impact of this action on our geriatricians, a new review of ppis prescriptions relevance is programmed in . hp-pc : oral anticoagulants and heparin for children: standardized protocols for prescription, dispensation and administration alexandra liauzu , marie-françoise hurtaud-roux , ronan bonnefoy , caroline farnoux , philippe sachs , theresa kwon , olivier bourdon , sophie ajzenfisz , sonia prot-labarthe *, pharmacy, hématologie clinique, ap-hp hôpital robert-debré, cardiologie, néonatologie, ap-hp hôpital robert debré, réanimation pédiatrique, ap-hp hôpital robert-debré, néphrologie, pharmacy, ap-hp hôpital robert debré, coordonnateur de la gestion des risques associés aux soins/ responsable du système de management de la qualité de la prise en charge médicamenteuse, ap-hp hôpital robert-debré, paris, france please specify your abstract type: research abstract background and objective: high-alert medications (ham) are medications that are associated with a high risk of serious harm if used improperly. we already identified paediatric ham used in our institution to identify safety measures for their use. anticoagulants and heparin were part of these high-alert medications. we aim to write standard protocol of use for low weight heparin and oral anticoagulant used in our mother-child teaching hospital. our secondary objectives were to decrease medication errors, anti-xa and inr unexplained variability and to help nurses to administer the drugs (standard dilution, oral solution available) setting and method: we carried out a literature search on pubmed Ò , on websites of several learned or professional societies and agencies. the results of the literature search were compiled on written protocol and presented to our institute drug safety-steering committee composed of four doctors, two head nurses, two pharmacists, and one risk manager. main outcome measures: not applicable. results: the protocols concerned enoxaparin, tinzaparin, warfarin but we chose to also include protamine. the most difficult issue was to have standardized dilution and protocol for all ages and weight: from premature to adolescents and all units of care (from cardiology to intensive care unit, nephrology and neonatology). we took into account the administration errors we had in our hospital and the preexisting protocol to avoid any drastic error-prone change. the final version of these protocols will be presented on the final communication with web link to upload them. conclusion: for now we did note evaluate the impact of these protocols but a before/after analysis of error reports and users evaluation will be done. however, these protocols can help all health professionals working in paediatric units for benchmarking. hp-pc : does a hospital formulary system impact timely medication administration and quality of inpatient care? anne-valérie putallaz *, , vera jordan-von gunten , pierre-auguste petignat , pierre turini , johnny beney division of pharmacy, institut central des hôpitaux, division of internal medicine, medical coordinator for quality of care and patient safety, hôpital du valais, sion, switzerland please specify your abstract type: research abstract background and objective: the prevalence of drug omissions is often underestimated but their impact can be clinically relevant. we hypothesized that delays in the administration of non-formulary/nonstored drugs could impair the quality of care. the aims of this study were: °to determine the time between the prescription and the administration of the first prescribed dose and, if applicable, to calculate how many doses were omitted. °to analyse the clinical relevance of the identified delays. setting and method: three months retrospective study of electronic records of patients hospitalized on the internal medicine wards of a network of hospitals supplied by a centralized pharmacy. this pharmacy is located in one of the sites; other sites are - km apart. main outcome measures: . for the main hospital site and the three distant sites: • median time between the prescription and the administration of the first prescribed dose • mean number of omitted doses for formulary and non-formulary/ non-stored drugs. . categorization of patient's harm caused by the delays of timecritical drugs, according to the ncc-merp taxonomy of medication errors. results: ' prescriptions were analysed. calculated delays for non-stored/non-formulary drugs were longer than for formulary drugs. however, the median time to administration is less than h for both formulary and non-stored/non-formulary drugs; and more than % of formulary drugs and around % of non-stored/non-formulary drugs were administered within h following their prescription. there was no significant difference in the mean number of omitted doses or in the delays between the site where the centralized pharmacy is located and the other sites, except for one of them. a delay representing . or more omitted doses was found for ( . %) prescriptions. among them, only were considered potentially clinically relevant. none of them caused severe harm to the patients involved. conclusion: in our setting, non-stored/non-formulary drugs take more time to be delivered than formulary drugs, but more than % of formulary drugs and around % of non-stored/non-formulary drugs are administered within h following their prescription. none of the patients who experienced delays underwent severe harm. our study showed that delays also occur for formulary drugs but no systematic cause of omission was identified; further studies should focus on all dose omissions during hospitalization. penelope randuineau *, , roger jeremy , lauriane cornuault , anne lecoeur , franck lemercier , isabelle javerliat , thomas tritz service de pharmacie à usage intérieur, service de chirurgie vasculaire, hôpital ambroise paré, boulogne-billancourt, france please specify your abstract type: descriptive abstract (for projects) background and objective: a french national survey of inpatient adverse events reveals that nearly half of adverse drug events (ade) are preventable. medication errors behind these ade occur mainly during the transition steps of care pathway. in this context, medication reconciliation process has been implemented in our vascular surgery department. the objective of this study is to identify unintentional discrepancies (uid) and assess their potential clinical impact design: a pharmacy resident or a pharmacy student reconciliated patients: aged older than or with at least five chronic treatments at admission or suffering from chronic diseases. patients were considered reconcilable if at least two reliable sources on usual patient's treatment were available. these many sources of data (patient interview, prescription or interview of general practitioner, reference dispensary, drug box …) were compared to the admission prescription during the first h of hospitalization to detect and correct uid. based on gravity scale promoted by the french high authority of health, two pharmacists (a resident and a senior) and a vascular surgeon reviewed every uid in order to define their potential clinical impact. the uids were considered minor if it leads to no consequence for the patient, clinically significant if it leads to essential monitoring, major if it could cause temporary clinical consequences, and critical if it could result in permanent clinical consequences or the involvement of the prognosis. results: between february th and may st , a total of patients have been reconciled. patients were excluded due to a lack of reliable sources. mean age was . years old (± . ) and sex ratio m/f was . . % of the reconciliated patients' admissions were scheduled. the mean number of medication was . (± . ). patients ( %) had at least one uid and the mean uids per patient was . (± . ). the most common types of uids were omission ( %), incorrect dose ( %) and incorrect administration frequency ( %). more than % of these uid presented a potential clinical impact: an adverse effect (high blood pressure, hyperglycaemia) was observed for nine patients and lead to therapeutic optimization and monitoring; uid were considered to have potential clinically significant impact ( %), a potential major impact ( %) and a potential critical impact. conclusion: these results appear consistent with those reported in literature. vascular surgeons have appreciated the approach and would like systematic medication reconciliation before surgery. as a major part of admissions were scheduled, we would like to establish the reconciliation before the patient's hospitalization every time it's possible. this new organization should facilitate the care pathway before surgery and decrease preventable postoperative adverse events. hp-pc : delirium in elderly patients: successful use of melatonin gaëlle jouin , aurélie reiter-schatz , pierre bentzinger , fatem-zohra laalou , bénédicte gourieux *, pharmacy-sterilization, orthopedic's intensive care unit, university hospital of strasbourg, strasbourg, france please specify your abstract type: descriptive abstract (for projects) background and objective: postoperative delirium happens to about one-third of elderly patients and is a major cause of morbidity and mortality. it is reported that haloperidol, an antipsychotic, has been the agent of choice for managing delirium. however, it induces cerebrovascular adverse effects and greater mortality. the hyperactive type of delirium is known to be associated with a low melatonin level and the loss of a normal melatonin secretion rhythm. the postoperative administration of melatonin to elderly could decrease the symptoms of delirium. the purpose of this study was to evaluate melatonin effectiveness in a cohort of patients suffering from postoperative delirium. design: a retrospective study of melatonin prescriptions has been conducted over a months period. medical background, type of surgery, symptoms of delirium, use of antipsychotics and benzodiazepines have been studied in all patients who received melatonin in an orthopaedic surgery unit. length of hospital stay, time between delirium and melatonin administration and the effect of melatonin had been evaluated. results: a total of patients were included: average age was . years ( - ), sex ratio m/f = . twelve patients ( %) were hospitalized because of an infection (prosthesis or osteoarticular). in % of cases (n = ), the prescription of melatonin was started when the patients were hospitalized in our intensive care unit. nine patients ( %) were under chronic treatments like benzodiazepines or antipsychotics. the average length of hospital stay was days ( - ). melatonin was started on an average of days after surgery , and administered at the dose of mg xr in the evening, during an average of days ( - ). cognitive impairments requiring a prescription of melatonin were: confusion ( %, n = ), agitation ( %, n = ), daytime sleepiness ( %, n = ), temporal-spatial disorientation ( %, n = ), nocturnal awakening ( %, n = ), hallucination ( %, n = ), difficult falling asleep ( %, n = ). the average time to recover from confusion was days, agitation days, daytime sleepiness days, temporal-spatial disorientation days, nocturnal awakening days, hallucination days and falling asleep days. melatonin treatment helped stopping benzodiazepines treatment in six patients ( %). conclusion: after administration of melatonin, delirium symptoms were improved for all patients and benzodiazepines treatment stopped for six patients. earlier prescription of melatonin could regulate sleep-wake cycle and reduce the duration and incidence of delirium. please specify your abstract type: research abstract background and objective: denosumab (xgeva Ò ), a fully human monoclonal antibody targeting rankl, which inhibits bone resorption, is indicated to prevent skeletal complications in patients with solid tumors and bone metastases. about % of patients develop hypocalcaemia, a common adverse event that may induce spasms, muscle cramps, paraesthesia, prolonged qt interval, tetany, convulsions… we report the management of ionic supplementation and physicochemical incompatibilities in a case of hypocalcaemia due to denosumab. setting and method: the clinical case was analysed with the pharmacovigilance regional center. main outcome measures: a year old patient, with nodal and bone metastasis in prostate cancer, was treated with denosumab (stopped with the last injection months before, on the th of march). he went to emergency on the th of may with asthenia, anorexia, nausea, diarrhoea, qt prolongation. biological results showed hypocalcaemia (corrected calcaemic = . mmol/l) and hypophosphatemia (phosphorus \ . mmol/l). concomitant calcium and phosphorus intravenous supplementation started with loading doses ( g of calcium and . g of phosphorus) and then a week of following daily intakes: phosphorus ( g iv and . g oral); calcium ( g iv and . g oral). however, low-serum corrected calcium and phosphorus levels persist at . mmol/l and . mmol/l. results: incompatibility between phosphorus and calcium by formation of soluble or not-soluble complexes is described in literature. in our case, calcium and phosphorus were mixed in a same infusion. after a week of supplementation, calcium infusion is continued with increased dose ( g/day) and phosphorus infusion is stopped. phosphorus oral supplementation remains stable ( . g per day); calcium oral supplementation is increased ( . g per day). h between intakes is applied to avoid digestive complexation. h later, corrected calcium levels are normalized at . mmol/l and phosphorus levels are still low. therefore, as hypocalcaemia due to denosumab induced a secondary hyperparathyroidism and thus hypophosphatemia; phosphorus levels are expected to increase subsequently. conclusion: this case report shows that recurrent hypocalcaemia with denosumab is possible few months after administration. supplementation with large amount of calcium is needed and administration methods may impact the effectiveness of supplementation. indeed, it seems that the incompatibility between phosphorus and calcium did not allow an effective supplementation. gunnhild langdal *, , , ida rudberg , lone holst , anne-lise sagen major , central norway hospital pharmacy trust, Å lesund, centre for pharmacy, university of bergen, bergen, møre og romsdal health trust, Å lesund, norway please specify your abstract type: descriptive abstract (for projects) background and objective: drug interactions (dis) can cause side effects and lack of therapeutic effect. the objective of this study was to describe the prevalence of dis at the medical department of Å lesund hospital, and to investigate how dis were managed by clinical pharmacists and physicians. design: at the medical department, Å lesund hospital, clinical pharmacists serve seven out of ten wards, from which patients were included during a five weeks period. the clinical pharmacists selected patients for screening for potential dis (www.interaksjoner.no) as int j clin pharm ( ) : - usual (= pharmacist group). detected dis were classified according to a predetermined classification system, and it was registered whether the physician implemented suggested changes in prescription. for patients not selected by clinical pharmacists (= non-pharmacist group), a pharmacy student performed the search for dis. results: in total patients were admitted. on average, each patient had . dis, and . % of the admitted patients had at least one di. the prevalence of dis was significantly higher among the patients in the pharmacist group compared to the patients in the non-pharmacist group (median@@@ vs. , respectively, p \ . ). the groups differed significantly regarding number of drugs used, age, duration of hospital stay and number of warfarin users. . % of the dis detected in the pharmacist group were discussed with the physician. the remaining . % were considered not necessary to discuss for various reasons e.g. because they were considered not clinically relevant ( %) or already adjusted for in clinical practice ( %). for dis the clinical pharmacist suggested a change in prescription, and of these suggestions ( %) were implemented by the physician. conclusion: just over half of the patients were selected by the clinical pharmacist for screening of dis, and the pharmacist seemingly made a reasonable priority of patients with many drugs, old age, a long hospital stay and users of warfarin. only of dis was discussed with physicians. this indicated that pharmacists do a considerable work in assessing the relevance of dis before discussing with the physicians. it also seemed that changes in prescription suggested by the clinical pharmacist were reasonable. hp-pc : securing the paediatric use of oral chemotherapy: a proactive risk assessment samia mouffak *, , linda an , anne fratta , anne auvrignon , , nadia marquis , karine morand pharmacy, risk management committee, hematology, armand trousseau hospital -aphp, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: oral chemotherapy is an important part of the therapeutic strategy in childhood cancer or haematological malignancy. it also represents an emerging risk area in oncology practice. several medications errors involving oral chemotherapy were reported in children of our onco-haematology department, fortunately without clinical consequences. nevertheless, the potential severity of such errors led us to implement a failure analysis of the paediatric oncology care pathway in order to identify and prevent potential risks, and secure the paediatric use of oral chemotherapy. design: we conducted a failure modes, effects and criticality analysis (fmeca) which is a proactive risk assessment approach. first, process maps were detailed for each step of the oncology care pathway. it was performed by a multi-disciplinary group composed of physicians, coordinating nurse, hospital pharmacists and pharmacy resident. then, for each step of the medication-use process, the team identified the failure modes, their main causes and effects. finally, participants rated the expected severity, frequency and detectability for each failure mode, assigning a score on a five-point scale. a risk priority number (rpn) was then calculated by multiplying those three indexes. the risks getting a high rpn were categorized as critical risks and have been the object of safety improvements. results: failure modes were identified, including critical risk failure modes. critical failures were related to hospital discharge prescriptions and were about the dispensation of oral chemotherapy by pharmacy assistants. most failures were due to prescriptions heterogeneity, lack of clinical information reported on prescriptions, and lack of training of pharmacy assistants in reading oral chemotherapy prescriptions and in mistake detection. two improvement strategies were implemented. first, physicians' awareness led to the harmonisation of practices and to the standardisation of discharge prescriptions. then, to enhance pharmacy assistants' abilities, an educational program on oral chemotherapy dispensation was planned. conclusion: the implementation of a fmeca has highlighted the most critical risks of oral chemotherapy medication-use process. the awareness of all caregivers and the targeted changes in our practices allowed us to improve the safety of the paediatric oncology care pathway. please specify your abstract type: descriptive abstract (for projects) background and objective: the purpose of this study was to investigate if medication reconciliation and medication review, by using the integrated medicines management (imm) model, were suitable to assure the quality of patients' medical treatment at a gastrointestinal surgical ward. furthermore, to analyse frequency, type, handling and clinical relevance of medication discrepancies (mds) and other medication related problems (mrps). design: patients, above years of age, from two departments at a gastrointestinal surgical ward at a norwegian university hospital were included consecutively. medication reconciliation was performed at admission by a clinical pharmacist. the resulting medication histories were compared with the medications documented in the medical records. mds were detected and categorized. thereafter the clinical pharmacist identified mrps by reviewing the medical records systematically and categorized the revealed mrps. mds and mrps were presented for the physician with proposed solutions. the physician's actions to manage the mrps were registered. later a multidisciplinary team assessed the clinical significance of mds and mrps in a subset of patients. results: a total of patients were included. overall, mds and mrps were identified. at least one md was revealed in % of the included patients, whereas at least one mrp was identified in % of the patients. the most frequent type of mds was omission, whereas mrps most often were related to medications that were considered unnecessary. totally, % of the mds and mrps were discussed with the treating physician. the physicians followed the pharmacist's input in % of the discussed md-cases and % of the mrp-issues. longterm consequences of mds and mrps were considered more serious than short-term consequences for the patients. conclusion: medication reconciliation and medication review revealed, solved and prevented a large number of mds and mrps in this study. the results emphasize that pharmacist involvement, by using the multidisciplinary imm-model, contributed to more correct medical records and furthermore to quality assurance of the patients' medical treatment at a gastrointestinal ward. hp-pc : prevention and management of drug interactions in oncology day-hospital: results from a months study involving drug assessment and pharmaceutical report to oncologist pauline-saraï zeller *, , chloé hugard , céline mongaret , , juliette vella-boucaud , antonin maréchal , olivier bouché , dominique hettler , florian slimano , pharmacy, oncology day hospital, university hospital reims, clinical pharmacy, faculty of pharmacy, reims, france please specify your abstract type: research abstract background and objective: quality during transitions of care is a major concern in drug safety for patients. traditional hospitalization allows to reconciliation medication but there is not possible for dayhospitalization (patient's hospitalization short time and no outpatient medication prescribe by oncologists). however, lack of communication between health professionals may expose patients to drug-drug interactions (ddi). while ddi between oral antineoplastic and other drugs are well known, there is a lack of knowledge about ddi between parenteral antineoplastic (ak) and other drugs. in this pilot study, we aim to investigate prevalence and characteristics of ddi between ak and other drugs in real life and to propose a pharmaceutical report model to enhance patient's drugs safety. setting and method: during months, all new oncologic patients (thoracic and digestive) receiving chemotherapy in day-hospital have been recruited by clinical pharmacist. first it was conducted a patient-clinical pharmacist interview and carried out the best possible medication history (bpmh) by contacting at least three different sources of drug information. then, the bpmh has been confronted with oncologic treatment (including concomitant medications such as like antiemetic) with support at least with two different database of ddi analysis. finally pharmaceutical recommendations in order to manage potential relevant ddi were reviewed with oncologists then reported and inserted in personal health record (phr). main outcome measures: prevalence and description of potential clinically relevant ddi in an ambulatory oncology population. results: from november, to april, n = oncologic patients were included with following characteristics: mean age of . , sex ratio : , majority of oncologic thoracic localization ( %). number of oncologic concomitant medications per patient was . ± . (mean ± standard deviation). patients present an average of . ± . comorbidities (excluding cancer) and . ± . linked medications per patient. pharmaceutical analysis revealed potential clinically relevant ddi ( . ± . per patient): % of them concern antiemetics (ondansetron and aprepitant): pharmaceutical interventions were formulated (including recommendations to adapt chronic treatment) and % of them involved biological monitoring (for renal function, inr, potassemie or magnesemie). conclusion: our pilot study confirms high prevalence of ddi between oncologic and non-oncologic drugs. clinical pharmacy services with bpmh performing and pharmaceutical recommendation appears to be useful to enhance patient' drug safety in oncology dayhospital. we currently are deploying our study in order to convey a pharmaceutical letter to general practitioner and community pharmacist. hp-pc : loading dose of anti-infectives: elaboration of a tool helping pharmaceutical analysis julie soyer *, , cécile sanchez , guillaume beraud , nicolas venisse , pauline lazaro , antoine dupuis pharmacy, infectiology, pharmacokinetics, university of poitiers, poitiers, france please specify your abstract type: descriptive abstract (for projects) background and objective: the recent data on vancomycin and ceftazidime confirm that continuous infusion is the best way of administration of these antibiotics. moreover a loading dose before the administration is required for the antibiotics to prevent from the infratherapeutic period at the start of infusion and limiting the risk of resistance emergence. long half-life antibiotics and antifungals also require a loading dose to be effective. the aim of this study is to analyse the prescriptions of anti-infective requiring a loading dose in order to develop a tool to help pharmaceutical analysis. design: a prospective observational study was carried out during days in units. initially, pharmacists, residents and students were trained (role of the loading dose, drugs concerned). then, all patients with anti-infective requiring loading dose were included. some data were collected: weight patient, creatinine clearance, loading dose or not, dose, administration mode, monitoring of steady state concentrations (vancomycin and ceftazidime) and dose adjustment. the results were analysed and compared to bibliographic research before discussion during a multi-disciplinary meeting (pharmacists, infection control specialist and pharmacokinetic specialist). finally, a list of relevant pharmacist interventions was selected. results: out of the patients, were enrolled for prescription of anti-infective requiring loading dose. twenty-six prescriptions including vancomycin, ceftazidime, the others fluconazole, caspofungine, voriconazole and posaconazole. concerning vancomycin, the loading dose was prescribed in % of case, monitoring of steady state concentrations was performed in % of case and dose adjustment after first dosage was required in % of case. selected pharmacist interventions were: • to favour continuous infusion (excepted paediatric) • to keep loading dose at full dose even in patient with renal failure • to monitor steady state concentrations after the first h in patient with renal failure or obesity • to adapt dosage when the target concentration is not reached concerning ceftazidime, the interventions were: • to recommend continuous infusion: g/ h after loading dose of mg/kg • to monitor steady state concentrations in patient with renal failure a total of interventions (dosage, adaption of posology at the monitoring, patients with renal failure, obese, paediatric patient, administration…) were identified by the group of experts. conclusion: this study allowed creating a recap data sheet for students and hospital pharmacists. the selected interventions will allow the harmonization of practices. these recommendations have been validated by the commission of the anti-infective. finally, this study shows that the pharmacist has a key role in the management of antiinfective requiring loading dose. hp-pc : assessment of potentially inappropriate medications in orthogeriatric patients using the rasp list the detection of inappropriate prescribing. the objective of this study was to investigate if the rasp list (rationalization of home medication by an adjusted stopp list in older patients), an explicit screening method adapted to the belgian context, can be used to reduce the number of potentially inappropriate medications (pims) in orthogeriatric patients. setting and method: single-centre, interventional study conducted at the orthogeriatric department of the uz brussel, a -bed university hospital. the rasp list was first applied by a last year pharmacy student to the admission medication of orthogeriatric patients hospitalised in october . after potential adaptations to the medication by a liaising geriatrician, the rasp list was additionally applied by the same pharmacy student to the discharge medication of these patients. main outcome measures: detection and reduction of the number of pims. results: in total, orthogeriatric patients, from whom an informed consent was obtained, participated in this study. on admission, a total of pims were detected in this population. at discharge, the number of pims decreased to . the median number of pims per patient decreased from (on admission) to (at discharge). this difference was statistically significant (p \ . ; wilcoxon signed rank test). drugs of atc class n (nervous system) were responsible for the highest number of pims. conclusion: pims can be detected and reduced in the hospital using the rasp list. a structured and collaborative medication review between (student) pharmacists and physicians appears a good approach to reduce the number of potentially inadequate drugs. nevertheless, more research is necessary to substantiate this further as well as to assess the clinical impact of the findings. hp-pc : impact of implementing ward based dispensaries across a hospital site on both service delivery and patient care michelle sullivan, paul wright, christopher watson, malcolm smith, sotiris antoniou * please specify your abstract type: descriptive abstract (for projects) background and objective: waiting for medication at discharge is often quoted as a key factor for delaying patients leaving hospital. feedback from service users (patients and healthcare professionals) was for a more patient facing pharmacy service. this led to a phased installation of remote dispensaries on wards within the hospital to supply medicines. this new and innovative service enabled the supply function to be fully co-ordinated on the ward. this model was initially implemented on wards, which coupled with one-stop dispensing meant % of discharges require nothing to be supplied at the point of validation, % of discharge prescriptions meeting key performance indicator of being dispensed and ready within h with average turnaround time of min for a discharge prescription and a reduction in missed doses- . % in september to . % in march . this success prompted further installation of remote dispensaries in all clinical areas on site. design: implementation included; purchasing hardware, pharmacy labellers, locating appropriate computer terminals and stock cupboards. the main pharmacy labelling and stock control system was fully integrated at ward level, enabling the automatic reordering of replacement stock. identification of items and quantities to stock for remote dispensaries was also needed prior to role out. there was a need to scope staffing requirements including the redeploying of roles from a main inpatient pharmacy to patient facing areas. results: over items are supplied at ward level each month via satellite pharmacies for all wards, equating to more than % of the total dispensing workload for the site allowing for pharmacy staff to be redistributed from dispensary to the ward. this offered the benefit of being more patient facing and supporting other initiatives such as patient counselling and medicines reconciliation. the project has impacted the pharmacists as it has enabled them to focus on clinical aspects of service delivery, including attendance of ward rounds as well as supporting a ward team approach with the pharmacy technician. results of missed dose audit from june shows across the site % ( ) wards scored below the national . % target and ( %) wards had no unintentional missed doses. conclusion: ward based dispensing has led to pharmacists and pharmacy technicians being % ward based. as a constant presence on the ward, the team offer consistency within the pharmacy service for patients, nursing and medical staff. impact of pre-discharge planning has been beneficial to nurses, patients and work flow of the pharmacy teams. ward based dispensing has improved supply at discharge as well as promoting a more patient facing pharmacy service that has seen the pharmacy team instilled as integral to service delivery at ward level. kutay demirkan * , nursel surmelioglu, aygin bayraktar-ekincioglu clinical pharmacy, hacettepe university, ankara, turkey please specify your abstract type: research abstract background and objective: hacettepe university hospitals clinical pharmacy unit was established in april . this unit runs its services by clinical pharmacy postgraduate students under the supervision of two qualified clinical pharmacists as part-time and oncall basis, in adults, paediatrics and oncology hospitals. the aim of this study was to identify drug related problems and describe its management strategies in inpatient and outpatient settings by pharmacists in clinical pharmacy postgraduate education program. setting and method: during a total of months study period (period i: february-july , and period ii: november-february ), clinical pharmacy postgraduate students followed patients for - times in a week in different services in hospitals (internal medicine, internal medicine intensive care, infectious diseases, neurology intensive care, paediatric bone marrow transplant/haematology unit, paediatric intensive care, geriatrics and nutrition units) and drug related problems were identified and pharmacists' recommendations were listed. main outcome measures: determination and evaluation of drug related problems by pharmacist in hospital. results: a total of recommendations was provided for patients. those recommendations were classified as alteration or discontinuation of drug treatment ( . %), dose adjustment ( . %), change in drug administration time ( . %), inadequate treatment ( . %), healthcare staff training/consulting ( . %), patient education ( . %) and error/deficit in therapeutic drug monitoring ( . %). a majority of recommendations (n = ) were related with alteration or discontinuation of drug treatment provided mainly in departments of internal medicine (n = , geriatrics (n = ), neurology intensive care (n = ) and infectious diseases service (n = ). the following main reason for pharmacist's recommendation was related with dose adjustment (n = ) which were provided in departments of internal intensive care (n = ), infectious diseases service (n = ), neurology (n = ) and internal medicine (n = ). conclusion: clinical pharmacy practices are being carried out effectively in many services, particularly in internal medicine services, internal medicine intensive care unit and infectious diseases services. a collaborative and bed-side education in postgraduate programs in clinical pharmacy help to increase the knowledge and skills of students in real life circumstances and also maintain safe and effective drug therapy by an involvement of clinical pharmacists in hospital services. hp-pc : development of a tool to help pharmaceutical analysis in patients with hepatic failure barbara troussier *, , eric gautier , astrid bacle , florian charier , christine silvain , pauline lazaro pharmacy, gastroenterology, hepatology and gastroenterology, university hospital of poitiers, france, poitiers, france please specify your abstract type: descriptive abstract (for projects) background and objective: hepatic impairment can cause significant changes in the pharmacokinetics of many medicines. however hospital pharmacists can be helpless in performing pharmaceutical analysis behind the lack of precise guidelines. we need a strategy to first detect accurately patients with hepatic impairment, then lead us in dose adjustments. the objectives of this project were to develop a helping tool for hospital pharmacists in the pharmaceutical analysis of patients with hepatic failure's prescription and to select relevant pharmacist interventions. design: we first planned an investigation of patients with hepatic failure's management, with multidisciplinary experts groups. the study was conducted during one week in post-surgical, gastro-enterology, endocrinology, cardiology, pulmonology, geriatric departments and reanimation care units. a flowchart based on hepatic's biomarkers helped us including patients. criteria used to assess hepatic impairment could be: a stage c child-pugh score, prothrombin score inferior to %, bilirubin superior to micrograms per millilitres of blood without haemolysis, aspartate and alanin aminotransferases superior to three times the high normal value, and presence of a vitamin k antagonist interfering with those results. after a review of each included patient's prescription, we checked the major pharmacokinetic elimination pathway of each prescribed molecule (biliary or renal) and if hepatic biotransformation was expected. we also checked if the molecule could cause hepatic side effects. results: out of patients, patients were included for liver failure ( . %) and for a cholestasis ( . %) mainly in reanimation care units ( . %) and gastro-enterology ( %). among the lines of prescribed medicines, the main pharmacological classes encountered were cardiology, ( %) pain ( %), psychiatry ( %), haemostasis ( %) and antibiotics ( %). at the end of the investigation, the expert group decided on the relevant pharmacist interventions. these were based on dose adjustment of anti-infectious, psychotropic drugs, painkillers, oral anti-diabetics, anti-coagulants and corticosteroids. alternatives are proposed for each class. conclusion: to conduct a better pharmaceutical analysis, steps are necessary. first, any liver failure or cirrhosis must be detected thanks to the patient's biological results and medical record. then the patient's prescription can be analysed in order to highlight drugs that need a dose adjustment in a context of hepatic impairment. finally, the physicist and the pharmacist discuss about dose adjustments or alternatives if presence of contraindication with the drugs prescribed. soon the designed tool will be available to all pharmacists to harmonize clinical pharmacy practices. please specify your abstract type: research abstract background and objective: data regarding adherence rates to oral chemotherapy in lymphoma patients is limited. the aim was to assess pharmacist intervention on adherence to oral chemotherapy in patients suffering from hodgkin's (hl) and non-hodgkin's lymphoma (nhl). setting and method: following ethics approval, hl and nhl patients attending chemotherapy sessions at the medical investigations and treatment (mitu) at mater dei hospital accepted to participate. a questionnaire was compiled to evaluate adherence to oral chemotherapy and to assess pharmacist intervention. the questionnaire was divided into sections (a-c). the same questionnaire was used for both the first interview (t = ) and after weeks (t = ). an additional section (d) was incorporated at t = to evaluate pharmacist intervention. section a consisted of questions regarding patient management of lymphoma. section b incorporated the morisky -item medication adherence scale (mmas- ) to evaluate adherence to oral chemotherapy. a total mmas- score of zero indicates high adherence, a score between and indicates medium adherence and a score between and indicates low adherence. section c consisted of additional questions regarding medication adherence. between t = and t = , pharmacist intervention involved providing each patient with an information leaflet which was developed in this study, an individualised treatment chart and verbal advice. ibm Ò spss version and the wilcoxon signed-rank test were used to assess changes in medication adherence between t = and t = . main outcome measures: evaluation of pharmacist intervention on adherence to oral chemotherapy in patients suffering from hl and nhl. results: out of the patients with hl at t = , 'never' missed a dose, missed a dose 'once in a while' and 'sometimes' missed a dose. for the patients with nhl at t = , 'never' missed a dose, missed a dose 'once in a while' and 'sometimes' missed a dose. the reason for missing a dose was forgetfulness. all nhl and hl patients indicated the haematologist as their source of information about the management of lymphoma. of the nhl patients, scored low adherence and scored medium adherence at t = and after weeks (t = ) all nhl patients who participated scored medium adherence. of the hl patients, scored low adherence and scored medium adherence in the first interview (t = ) and after weeks (t = ) all hl patients who participated scored medium adherence. there was a statistically significant increase (p \ . ) in the number of patients who scored medium medication adherence between t = and t = for both nhl and hl patients. conclusion: this study shows how pharmacist intervention and extended professional services could be implemented in the clinical setting to impact on the management of hl and nhl patients. please specify your abstract type: descriptive abstract (for projects) background and objective: in may , an activity of medication reconciliation was implemented in the gastroenterology service to carry on the optimization of the medication care of patients due to the recent computerization of their prescriptions. design: this project, worked in collaboration with the gastroenterology service has been introduced in two medical committees. this activity gathers pharmacy students, the pharmacist, senior and junior doctors. reconciled patients are selected according to several criteria (advanced age, poly pathological, poly-medicated and those for whom a drug background is difficult to retrieve for the medical team). a minimum of information sources is used for the collection of the drug background. all information are synthetized on a paper, validated by the pharmacist and discussed again with the prescriber. results: on a -month period, patients were reconciled with on average age of . the reconciliation is executed on average . days after the entry in the service. . % of reconciliations are retroactive. the main sources of information used for the collection of the drug background are: in . % of the cases an oral interview with the patient and/or the family; in % of the cases the prescriptions, the hometown pharmacist ( . %) and a medical letter ( . %). . drugs are on average on the hospital prescription, and . % ( / ) of the patients are concerned with at least one non intentional divergence (nid). on average there are . nid/patient and . intentional divergences (id)/patient. the main types of nid are omissions ( . %), drug dose errors ( . %) and errors in administration frequency ( . %). after the detection of nid, the proposed modifications to the prescribers are accepted in more than % of the cases ( / ). the average time of a reconciliation is min. exchanges on the id and nid are made with the junior doctors in . % of the cases. conclusion: some nid are occurring for . % of the reconciled patients. it is therefore necessary to extend this new activity to reconciliation in other services in order to increase the interception of eventual medication mistakes and allow their correction. please specify your abstract type: research abstract background and objective: diuretic therapy is routinely used in the management of congestive heart failure (chf).,compliance with clinical practice guidelines is reported to result in improved outcomes for patients with chf such as reduced exacerbations. the aim was to assess the effect of pharmacist intervention on adherence to diuretic treatment in a hospital and community pharmacy scenario. setting and method: the study was undertaken at karin grech hospital (kgh), a geriatric and rehabilitation hospital, and in one community pharmacy. inclusion criteria for patients recruited from kgh were age over years, suffering from chf and on bumetanide therapy. the validated -item morisky medication adherence scale (mmas- ) was administered to patients on admission (t = ), repeated after two weeks hospital stay (t = ) and again one-month post-discharge (t = ). a total mmas- score of zero indicates high adherence, a score between and indicates medium adherence and a score between and indicates low adherence. in the community setting patients on diuretic therapy were chosen by convenience sampling. the same adherence scale was administered prior to pharmacist intervention (t = ) and one-month after pharmacist intervention (t = ). pharmacist intervention in the community setting involved dissemination of an informative leaflet regarding chf and diuretic therapy developed for the purpose of this study. main outcome measures: impact of pharmacist intervention on adherence to diuretic therapy in chf patients. results: a total of patients were recruited from the hospital setting, of whom were female and were male with a mean age of years (range - years). on admission (t = ), patients scored high adherence, scored medium adherence and scored low adherence to bumetanide therapy. following weeks at the hospital (t = ), the number of patients scoring high adherence increased from to and the number of patients scoring low adherence decreased from to . one-month post-discharge (t = ), patients scoring high adherence decreased from to and patients scoring low adherence increased from to (p \ . ). a total of patients were recruited from the community pharmacy, of whom were female and were male, with a mean age of years (range - years). after pharmacist intervention (t = ), the number of patients scoring high adherence increased from to , while the patients scoring low adherence decreased from to (p [ . ). conclusion: pharmacist intervention in the hospital setting improved adherence to bumetanide therapy. in the community pharmacy setting, there was a slight improvement in the compliance. pharmacist monitoring and patient support is important post-discharge to ensure patient compliance to therapy. conclusion: surveillance of aeds may be followed by combination of data from adverse drug reaction databases and drug utilisation data from prescription databases. focus on reporting adverse reactions is important for pharmacists and clinicians, especially for newly approved drugs. awareness of increased exposure of aeds to new groups of patients followed by data regarding safety aspects is important and contributes to improved pharmacovigilance. please specify your abstract type: research abstract background and objective: the medication review of polymedicated patients is a priority shared among all healthcare professionals. a multidisciplinary approach of these patients is necessary to achieve the best results for their treatment ( ) . the objective was to analyse the rate of acceptance of the recommendations made by the primary care pharmacist (pcp) to the general practitioner (gp) regarding the treatment of polymedicated patients. setting and method: setting: a primary health care centre ( , population). method: a review of the medical records of polymedicated patients (c chronic drugs for c months). the patients' data were collected from january to june from their clinical records. statistical descriptive analysis of data was performed. main outcome measures: drug related problems (drp) for each patient: interactions, contraindications, inadequate dosages, nonindicated drugs, omission of a necessary drug, duplications, medication with low therapeutic effect, and inappropriate medication for patients c years old. treatment alternatives proposed to gp's by pcp were also measured. results: patients were included in the study (average age: . ± . , % women). out of the patients, interventions were laid out to reduce the risks of drp's and to improve the efficiency of treatments. % of patients presented some drp or some intervention to improve the efficiency of their treatment, this mean an average . interventions for patient. the prevalence of intervention proposals were: non-indicated drugs ( %), interventions for improve the efficiency of treatments ( %), interactions ( %), inappropriate medication for patients c years old ( %), contraindicated drugs ( %), duplications ( %), medication with low therapeutic effect ( %), inadequate dosages ( %) and omission of a necessary drug ( %). % of these intervention proposals were accepted by the gp: % of the accepted proposals were carried out and from the remaining , . % led to a prescription from a specialist physician. in % of the cases, the patient did not accept the changes. . % were not carried out due to other issues. the main drug related problem was the prescription of non-indicated drugs and the most involved drug was omeprazole. conclusion: acceptance by gp's to changes proposed by primary care pharmacists was high. a significant number of changes was not accomplished due to the negative response by some patients and led prescriptions from a specialist physician. the gp greatly values the multidisciplinary aid in approaching the complexity of polymedicated patients. background and objective: case-reports provided evidence that influenza infections, particularly severe episodes, may exert neuronal damage in the cns and thereby increase the risk of depression. it was the aim of this study to analyse the association between influenza infections and the risk of developing incident depression. setting and method: we conducted a case-control analysis using the large uk-based primary care database clinical practice research datalink (cprd). this database contains anonymous longitudinal data from primary care. at present, it contains over million person-years of data from some million active patients. the study encompassed , patients below the age of years with an incident major depression diagnosis between and , and we matched each case to one control patient on age, sex, general practice, number of medical encounters, and years of history in the cprd prior to the index date. main outcome measures: major depression diagnosis was identified by read-codes based on icd- codes (f ), with a minimum of three prescriptions for antidepressant drugs recorded after the diagnosis. we calculated relative risk estimates of developing depression in association with previous influenza infections, stratified by the number, timing and severity of such events, and we adjusted for a variety of comorbidities, smoking status, alcohol intake, body mass index, use of oral corticosteroids, and benzodiazepines. results: patients with a previous influenza infection had an increased risk of developing depression (or . , % ci . - . ) compared to patients with no history of influenza infections. a recent influenza infection recorded within - days prior to the index date yielded an adjusted or of . ( % ci . - . ), and an increasing number of previous influenza infections was associated with increasing odds ratios (c recorded influenza infections, adjusted or . , % ci . - . ). we did not see any differences in the relative depression risk associated with influenza with regard to a previous influenza vaccination. conclusion: this study suggests that influenza infections are associated with a moderately increased risk of developing depression. please specify your abstract type: research abstract background and objective: warfarin is known for its interactions with many drugs. elderly patients are particularly sensitive to warfarin interactions. to evaluate the incidence of potential drug interactions when prescribing new drugs to elderly patients on warfarin, a prospective observational study was conducted. setting and method: patients on warfarin older than years were included and monitored for months in community pharmacies in croatia. data regarding new prescribed drugs was obtained from pharmacy records at the moment of dispensing or by patient selfreporting. the potential interacting drugs were identified using the lexicomp Ò lexi-interact online software. only the clinically significant (levels c, d, x of clinical significance as classified by lexicomp Ò lexi-interact online) interactions were included in this analysis. main outcome measures: number of new proscribed drugs, level of interaction with warfarin, mechanism of interactions. results: we included elderly patients with an average age of years. in the follow-up period, new drugs were prescribed to patients ( . %). there were prescriptions of new drugs and ( . %) of those were drugs with a clinically significant interaction with warfarin. there were prescriptions of drugs with level c of interaction ( . %), and ( . %) with level d. there were no drug interactions of level x. in the group with level c the most prescribed drugs were antibiotics with prescriptions: amoxicillin/clavulanate %, clindamycin %, ciprofloxacin %, norfloxacin %, azithromycin %, cefuroxime %, clarithromycin %, doxycycline %. the remaining prescriptions included tramadol with paracetamol %, rosuvastatin %, simvastatin %, fluvastatin %, levothyroxine % and torasemide %. the dominant mechanism of the potential interactions was pharmacokinetic. in the group with level d the most prescribed drugs were nonsteroidal anti-inflammatory drugs with prescriptions-diclofenac %, ibuprofen %, indomethacin %. among other drugs, prescriptions were antibiotic sulfamethoxazole with trimethoprim %, fenofibrate %, miconazole %, and fluconazole %. the dominant mechanism of the potential interactions was pharmacodynamic. conclusion: pharmacists should actively monitor prescribing of new drugs to elderly patients on warfarin in order to reduce the risk of clinically significant drug interactions. please specify your abstract type: research abstract background and objective: explicit criteria of potentially inappropriate medications in the elderly (pims) have been published in the usa, canada, australia and many eu countries. there is a lack of studies describing prevalence of pim use in central and eastern europe. the aim of the eu cost action initiative wg b ( wg b ( - is to evaluate the registration rates and use of pims in central and eastern europe compared to other eu countries participating in this initiative. this abstract describes preliminary findings on different registration rates of pims in different eu countries. setting and method: researchers/members of the eu cost action initiative from the czech republic, serbia, hungary, spain, turkey and portugal were asked to fill in evaluation tables for the list of pims in the period - / . items available in these evaluation tables related to: registration of individual pims on the pharmaceutical market, registered doses, drug forms, availability of pims on prescription or as otc drugs, prescription limits and the most frequently used brand names. data were evaluated using comparative descriptive statistics. main outcome measures: overall prevalence of registered pims in different countries, cross-country differences in availability of individual pims. results: of pims . % were registered in at least participating country. for the czech republic ( . %), turkey ( . %), spain ( . %) and hungary ( . %) overall prevalence rates of registered pims were found to be similar. however, these prevalence rates substantially differed in serbia (low prevalence- . %) and portugal (high prevalence- . %). substantial differences were found also in the lists of individual pims registered in different countries. these lists were similar in spain and portugal compared to the czech republic, hungary, serbia and to turkey. conclusion: although overall prevalence rates of registered pims were similar in the majority of evaluated countries (except serbia and portugal), availability of individual pims was substantially different. our pilot results confirmed that there are substantial geographical/ regional differences in europe in the lists of pims available (in spain and portugal compared to central and eastern europe and compared to turkey). please specify your abstract type: research abstract background and objective: inappropriate prescribing is a common circumstance found in polymedicated patients. screening tools for identifying potentially inappropriate prescription (pip) and pharmacist interventions for evaluating them have been developed to decrease this ( ) . the aim of this study was to evaluate the effectiveness of a pharmacist provided intervention to reduce pips in polymedicated patients. setting and method: the design was a quasi-experimental study focusing on a single group before and after intervention. the study took place from july to december of at three primary care centres ( , population). polymedicated patients were those using c chronic drugs for c months. main outcome measures: reduction in the rate of pip per polymedicated patient (number of pips found divided by the total number of polymedicated patients) before and after intervention, and the influence of the following variables: type of pip (inappropriate medication for patients c years old, medication with low therapeutic effect, duplication of benzodiazepines (bzd) or angiotensinconverting enzyme (ace) inhibitors, combination of anticoagulant and antiplatelet, combination of non-steroidal anti-inflammatory drug (nsaid) with a diuretic and ace inhibitor, nsaid in cardiovascular disease, chronic antipsychotic in dementia, chronic bzd, or chronic nsaid), gender and age of patients with at least one pip, and the main prescribed drugs involved in the pips based on atc classification system of world health organization. results: there were and polymedicated patients before and after intervention, respectively. . % (n = , before) and . % (n = , after) of the total patients had at least one pip. the number of pips was reduced from to , while the rate of pip per polymedicated patient decreased from . to . , achieving the limit established by the regional health authority. . % (before) and . % (after) of patients had more than one pip at the same time, up to pips per patient. before and after intervention, more than half of patients with at least one pip were c years old, and approximately out of were c years old. also before and after intervention, out of patients with chronic nsaid and with bzd duplication were women. out of patients with combination of anticoagulant and antiplatelet were men. the main pips before and after intervention were, respectively: chronic prescription of bzd ( . vs. . % of the total pip), medications with low therapeutic effect ( . vs. . %) and inappropriate medication for patients c years old ( . vs. . %). the main atc group involved in the total of pips was drugs for the nervous system, and the five most prescribed drugs were all bzd (lorazepam being the first). conclusion: pharmacist provided intervention was able to reduce pip in polymedicated patients. gender, age and atc classification of drugs involved were factors in the pips. please specify your abstract type: research abstract background and objective: up to % of women are exposed to selective serotonin reuptake inhibitors (ssris) during pregnancy. information on their effect on birthweight and gestational age remains conflicting. the aim of this sibling-controlled prospective cohort study is to address shared genetic and family-level confounding to investigate the effects of prenatal ssri exposure and maternal depression on birthweight and gestational age. setting and method: we used the norwegian mother and child cohort study (moba) and the medical birth registry of norway (mbrn). our study population consisted of siblings; were prenatally exposed to ssris and were unexposed to any antidepressant medication. random and fixed effects analysis with propensity score adjustment was used to evaluate the effects on birthweight and gestational age. main outcome measures: birth weight. gestational age. results: ssri exposure during two or more trimesters was associated with a decrease in birthweight of g [ % confidence interval (ci) to ] and a decrease in gestational length of . days ( % ci . to . ). neither maternal ssri use in one trimester, lifetime history of major depression nor depressive symptoms during pregnancy were associated with these pregnancy outcomes. conclusion: prenatal exposure to ssris during two or more trimesters may decrease birthweight and gestational length. our results indicate that neither maternal depression nor shared genetics and family environment fully explain this association. please specify your abstract type: research abstract background and objective: the drugs burden index (dbi) is a tool to evaluate the burden of medications with anticholinergic and sedative effects and this exposure has been associated with poorer physical and cognitive function in older people. objectives were; to determine the cumulative burden of anticholinergic and sedative medicines in older adults with intellectual disability (id) using the dbi, to examine the relationship between dbi score with demographics and comorbidity. setting and method: data from wave of the intellectual disability supplement to the irish longitudinal study on ageing (ids-tilda), a nationally representative study of ageing people with id in ireland. dbi scores were calculated for all participants with available medication data (n = ). bivariate associations between dbi and demographic and clinical characteristics were examined with a significance level of . main outcome measures: dbi scores of participants categorised into low ( ), medium ( - ) and high (c ). dbi score categories were related to demographics, cognitive effects and to a modified functional comorbidity index (fci), which is associated with physical function in older adults. results: of participants, . % ( ) had dbi exposure; . % were exposed to any anticholinergic medication, . % to any sedative medication; mean number of dbi medications . (± . ), mean dbi score: . (± . ). ( . %) participants had dbi score , ( . %) - , and ( . %) c . antiepileptics accounted for the greatest contribution to cumulative score ( . %), antipsychotics ( %) and antidepressants ( %). there was no significant association between higher dbi score and sleep difficulties (p = . ). there was a significant age gradient associated with higher dbi score (p = . ) and significant association between higher scores and increased comorbidity scores; mean fci of . in those with dbi c , . in dbi - and . in those with dbi . conclusion: cumulative exposure to sedative and anticholinergic medicines was high in older adults with id. higher dbi scores were associated with higher comorbidity and associated poorer physical function. optimising use of medications with anticholinergic and sedative effects through medicines review by pharmacists as part of multidisciplinary teams using a tool such as the drug burden index may reduce functional decline and improve quality of life among older adults with id. please specify your abstract type: research abstract background and objective: poor adherence to pharmacotherapy may have considerable consequences for the patients' health and for healthcare costs to society. there was observed that diabetes patients have higher risk of later health complications development. it is necessary to be adherent to non-and pharmacological recommendations as well, to improve the clinical outcomes and decrease the cardiovascular risk (cvr). the aim of this study was to evaluate the medication adherence and cvr in group of patients with diabetes, and to find an association between them. setting and method: the methods were based on a questionnaire survey using a modified -item morisky score and score charts ( ). medication adherence and cvr were evaluated in the whole group (n = , males and females, range - years) as well as in subgroups according to age, gender, (no-/ex-) smoking, level of education, residence, number of used medicines, exercises, compliance to the diabetic diet, and total cholesterol levels. the survey was realized in three ambulatory diabetic centres in slovakia. the study has been approved by ethics committee of university hospital bratislava -ruzinov. all participants signed an informed consent. main outcome measures: the results of medication adherence were evaluated as follows: points = full adherence, - points = partial adherence and - = non-adherence. the cvr (estimating -year cardiovascular attack risk) was evaluated according to score charts using data from questionnaire and medical records-gender, age, smoking, total cholesterol levels and blood pressure. the results showed a partial medication adherence in the study group in average ( . ± . ). the average value of cvr in the study group was . %. the highest average medication adherence has been observed in males b years ( . ), with elementary education ( . ), in ex-smokers ( . ), in patients with regular physical activity-at least times a week ( . ), in patients non-adherent to the diabetic diet ( . ), in patients using medications ( . ), and in patients with satisfactory ( . - . mmol/l) total cholesterol levels ( . ). the lowest cvr has been observed in females b years ( . %), in no-smokers ( . %), with elementary education ( . %), in patients with irregular physical activity ( . %), in patients adherent to the diabetic diet ( . ) , in patients using medications ( . %) and in patients with satisfactory ( . - . mmol/l) total cholesterol levels ( . ) . on the other hand, the highest cvr has been observed in males [ years ( . %), smokers ( . %), secondary educated patients ( . %), without any physical activity ( . %), in patients partially adherent to diabetic diet ( . %), using medications ( . %) and, surprisingly, in patients with satisfactory (\ . mmol/l) total cholesterol levels ( . %). conclusion: our survey has showed that medication adherence in our study group has been decreased and cvr has been increased. cvr and adherence to pharmacotherapy in the study group did not correlate with each other. the medication adherence, cvr and their relationships are specific in every patient. please specify your abstract type: research abstract background and objective: studies show that quality of life (qol) of patients with diabetes mellitus can influence medication adherence, satisfactorily improving clinical outcomes and reducing the morbidity and mortality rates and disease progression. this applies even upside down-medication adherence could significant contribute to improving patient qol. the aim of this study was to evaluate the medication adherence in group of patients with diabetes, to evaluate their qol and find a correlation between them. setting and method: the methodology was based on a questionnaire survey using a modified -item morisky score and questionnaire eq- d- l, including visual analogue scale (vas). medication adherence and qol were evaluated in the whole group (n = ) as well as in subgroups according to age, gender, level of education, monthly income, number of used medicines and type antidiabetic treatment. the survey was realized in three ambulatory diabetic centres in slovakia. the study has been approved by ethics committee of university hospital bratislava-ruzinov. main outcome measures: the results of medication adherence were evaluated as follows: points = full adherence, - points = partial adherence and - = non-adherence. the qol in levels of dimensions results were evaluated as follows: the lowest qol in every dimension = point, the highest = points. the highest vas evaluation has been points and every patient should mark number on the scale - to indicate his/her health on current day. results: the results showed a partial medication adherence in the whole group in average ( . ± . ). the average value of the qol in the study group was . and vas . . the highest medication adherence has been observed in males ( . ± . ), patients \ years old ( . ± . ), with primary education ( . ± . ), with monthly income over € ( . ± . ) and in patients using medications ( . ± . ). the highest qol and vas (qol; vas) has been observed in males ( . ; . ), patients \ years old ( . ; . ), university educated ( . ; . ) , with monthly income over € ( . ; . ) . qol has been highest in patients using medications ( . ), vas has been highest in patients using medication ( . ). we have observed the highest level of medication adherence in patients treated with combined therapy-with oral antidiabetic agents and insulin ( . ), the lowest in patients treated with only insulin therapy ( . ). highest qol was recorded in patients treated with oral antidiabetic agents ( . ), and the lowest qol in patients with insulin therapy ( . ). the highest vas has been observed in patients using only oral antidiabetic agents ( . ), the lowest in patients using combined therapy ( . ). conclusion: survey has showed that medication adherence and qol in our study group has been decreased. qol and adherence to pharmacotherapy in the study group did not correlate with each other. the medication adherence, qol and their relationships are specific in every patient. the role of health care professionals should be in education and counselling with patients to improve qol and medication adherence as well. please specify your abstract type: research abstract background and objective: to assess the appropriateness of antibiotic prescriptions used for urinary tract infections (uti) in the elderly. setting and method: we included patients aged years and older, hospitalized in the geriatric department and for whom a urine culture was performed between march and may . a prescription was qualified as inappropriate: when the antibiotic prescribed was not the narrowest compared to the culture result, or when there was a contra-indication, or when the treatment duration was shorter or longer than recommended. prescriptions were consistent with the guidelines when they were identical to those adopted by the french society for infectious diseases in december . main outcome measures: appropriateness of antibiotic prescription (type and duration) results: elderly patients were included (women: . % (n = ), mean age: . years). % of antibiotic choices were appropriate and % of treatment durations were consistent to the guidelines. urinary clinical signs were mentioned in the medical files for . % of the cases (n = ). patients received an empirical antibiotherapy ( . %). . % (n = ) of urine cultures were positive with bacteria, escherichia coli being the most prevalent (n = ). the urine culture results led to a change in antibiotics for . % of the cases. for cystitis, . % of the antibiotics chosen were appropriate (n = ). the main reasons of non-conformity were the lack of deescalation (to amoxicillin or pivmecillinam), and the prescription of ciprofloxacin when the bacteria was in vitro resistant to other fluoroquinolones. the average duration of effective antibiotherapy for cystitis was . days (appropriateness: . % (n = )). for pyelonephritis, . % of the antibiotics chosen were appropriate (n = ). the average duration of effective antibiotic treatment was . days (appropriateness: . % (n = )). . % of the patients had a transurethral catheterization (n = ). another infection was diagnosed for . % of the patients (n = ). conclusion: according to these results, it appears important to reemphasize to the prescribers the guidelines around the uti diagnosis and treatment in order to improve the prescriptions appropriateness in elderly patients. it is particularly necessary to promote the de-escalation of antibiotherapy (with pivmecillinam for example which has recently become available in our hospital) and to insist about the recommended durations of treatment. please specify your abstract type: research abstract background and objective: to measure the use of potentially inappropriate medications (pim) in the general elderly population several criteria lists exist, e.g., beers criteria. last year, a set of explicit criteria for assessing pharmacologically inappropriate medication use in nursing homes was developed; the norwegian general practice-nursing home criteria (norgep-nh). the aim of this study was to investigate the prevalence of pims in nursing home patients using this new assessment tool. furthermore, we studied possible associations between the use of pims and factors like gender, age, geographical area and the number of drugs used. setting and method: cross-sectional study comprising nursing home patients from two geographical different regions in norway; tromsø city (n = ) and lofoten islands (n = ). data was collected from november to january . pims were identified by norgep-nh. we used logistic and poisson regression to examine possible associations between the use of pims and factors like gender, age, geographical area and the number of drugs used. main outcome measures: number of pims per patient, and odds ratios (or) and marginal effects for associations. results: nursing home patient used a mean (sd) of . ( . ) drugs; . ( . ) regularly and . ( . ) as needed. at least % of patients used one pim. concomitant use of three or more psychotropic drugs was the criterion most commonly identified ( %), followed by the use of antidepressant ( %) and hypnotics ( %). an increasing number of regularly used drugs increased the odds of having pims (or: . ), as well as it lead to . more pims per extra drug used. on average, patients c years had . fewer pims than patients \ years. no statistical significant associations were seen between having pims and gender, nor geographical area and the use of as-needed medication. yet, statistical significant differences were identified in some criteria. conclusion: this is the first study that explicit uses norgep-nh. our results confirm that nursing home patients often use potentially inappropriate medications. this is an area where further work is necessary, not to measure the prevalence of pim, but to develop interventions in order to prevent pims from being used. pe : use of pharmacy dispensing data to measure adherence and identify nonadherence with oral hypoglycaemic agents please specify your abstract type: research abstract background and objective: a framework for calculation of adherence for oral hypoglycaemic agents (ohas) based on data from health-insurance claims is available. pharmacy dispensing data aid identification of nonadherent patients in pharmacy practices. however, use of these data for calculation of oha adherence requires additional methodological categories. we examined the impact of different methodological choices on estimation of oha adherence using pharmacy dispensing data. setting and method: a framework for adherence calculation for pharmacy dispensing data was developed from health-insurance claims. a basic scenario was developed from methodological categories. consequences of choices for different parameters within these categories on the scores of the three adherence measures were calculated from dispensing data. main outcome measures: for oha use between july and july , three adherence measures were calculated: ( ) average medication availability (ama); ( ) mean rate of adherent patients with an ama c % (mrap ); ( ) please specify your abstract type: research abstract background and objective: ulcerative colitis (uc) is a chronic inflammatory disease usually affecting young adults and impacting on patient's quality of life. although many biological agents (bas) have been approved for the treatment of moderate-to-severe uc in patients who have responded inadequately to conventional therapy, the selection of bas is controversial due to the lack of head-to-head trials. indirect economic comparisons of these costly drugs are available from national healthcare perspectives that are not the italian ones. therefore, the objective is to evaluate cost-utility of bas for the treatment of refractory moderate-to-severe uc both in italy and in the lombardy region. setting and method: a markov model (considering transition states: remission, clinical response, relapse) was constructed using the software r . . markovchain-package to evaluate incremental cost-utility ratios (icur) of adalimumab, infliximab, infliximab biosimilar, golimumab and vedolizumab treatments of patients over a ten-year time horizon from the perspective of the italian (n) and lombardy region (r) healthcare system. clinical parameters were derived from clinical trials. costs (which have been actualised- . %) were obtained from the national database and regional public tender. utility was expressed as qaly (quality adjusted life years). main outcome measures: icur. results: costs per treatment were different from a n and r perspective (adalimumab - %; infliximab - . %; infliximab biosimilar - . %; golimumab - . %; vedolizumab - %). direct healthcare costs (treatment cost, visits, lab tests, hospital admissions) were calculated over years of treatment per patient: adalimumab (n: € , . , r: € , . , - . %), infliximab (n: € , . , r: € , . , - %), infliximab biosimilar (n: € , . , r: € , . , - . %), golimumab (n: € , . , r: € , . , - . %), vedolizumab (n: € , . , r: € , . , - . %) with associated qaly respectively of . , . , . , . , . . from a n perspective, infliximab biosimilar was dominating compared to all other treatments. the icur of vedolizumab/infliximab biosimilar was € . for years (willingness to pay (wtp) € . /qaly). from a r perspective, adalimumab was dominating compared to all other treatments. the icur of vedolizumab/adalimumab was € , . for years (wtp € , . /qaly). conclusion: national and regional cua produced different results. as regional price discounts can occur, local analyses are needed to estimate the economic impact of therapies to ensure optimal choice. please specify your abstract type: research abstract background and objective: automated dispensing systems (ads) have been implemented to reduce overall medication errors related to picking, preparation and administration of drugs. costs of drug storage between ads and classic dispensing system (cds) had not been yet performed in france. our objective was to assess economic impact of ads compared to cds. setting and method: retrospective quasi experimental study was conducted in university hospitals in , one with ads ( beds, ads) and one with cds ( beds, cds ( ) for ads and ( ) for cds (p \ . ). mean number of costly drug per system was for ads and for cds. the global stock value in the wards was , € in ads and , € in cds representing respectively . and . % of total pharmacy stock value. conclusion: our data demonstrate that despite the same storage capacity, ads allow the storage of more expensive drugs such as innovative drugs fully reimbursed up to national reimbursement prices, due to the lower risk of pilferage. this preliminary study was focused mainly on stock value. subsequently, another study is conducted to evaluate cost of these two drug storage systems, satisfaction of pharmaceutical technicians and nurses and time allowed for systems reloading. please specify your abstract type: descriptive abstract (for projects) background and objective: in france, pharmacists are not entitled to substitute an original biological drug with its biosimilar, due to specific issues of efficiency, safety, and patient monitoring. our hospital referenced a biosimilar of infliximab on january . according to the french medication safety national agency's recommendations, it has been decided that naïve patients would be treated with biosimilars, and changes between specialties would be proscribed. the objective is to compare prescribing practices between infliximab and its biosimilar, year after its introduction. design: a database tracking patients treated with infliximab was set up. data comparing prescribing practices of biosimilar and reference treatment were analysed between june and may . regional and national infliximab consumption between january and february were used to compare the practices of our hospital with other hospitals. the past and future savings were estimated from repayments data of the regional health agency. results: infliximab was administered to patients, of which ( %) were naive. patients were treated with biosimilar (i.e. . % of all patients), of which were naive. in the end, nearly % of naive patients actually received the biosimilar and . % of patients treated with infliximab switched specialties during treatment. in % of cases, biosimilar prescriptions were consistent with the recommendations (vs. % for infliximab). in % of cases the off-label prescriptions of the biosimilar were explained in the patient record (vs. % for infliximab). in february , the share of biosimilars was % in france, % at regional level and % locally. in year, infliximab and its biosimilar's consumption in our hospital have increased by % in quantity and only % in expenditure (+€ m expenditure). negotiating a lower purchase price and costs has enabled the hospital to save € , (vs. € , during the previous year). because of the decline of refund rates, the gains would have been zero without using the biosimilar but € , if it had been prescribed to every naive patient. conclusion: current data from the literature on security and effectiveness of infliximab biosimilars are very reassuring and the french medication safety national agency doesn't exclude the possibility of changing specialties during treatment. in our hospital, there is room to improve the efficiency of treatment with infliximab. feedback on prescribing practices will be given to prescribers and a campaign to widespread prescriptions of biosimilars will be made. the arrival of biosimilars on the market is a real economic opportunity for hospitals, which are increasingly financially constrained in particular by the arrival of therapeutic innovations which are more and more expensive. setting and method: the study used health claims data on prescription ppis from st january to st july obtained from the health insurance institute of slovenia. to assess medicine use and costs before and after trp implementation data were aggregated into four periods: jan-dec , pre-baseline period; jan-dec , baseline period; jan-sept , transition period between announcement and introduction of trp; oct to jul , period after trp enforcement. main outcome measures: medicine costs; defined daily doses (ddds) dispensed per inhabitants per day; market share; herfindahl-hirschaman index (hhi); number of active substance switches; number of exceptions when medicine is fully reimbursed since physicians may choose option ''not to switch medicine'' when adverse consequences are predicted. results: average monthly cost of ppis declined from € , , in pre-baseline period to € , in period after trp introduction although the consumption increased from . to . ddds/ inhabitants/day. cost of ppis decreased the most in baseline period ( %), however trp induced . % cost reduction compared to the transition period. the reference pantoprazole was market leader already in the transition period, but its use increased significantly after trp introduction and represented % of total ppis consumption. manufacturers' market shares were constant before trp, whereas trp caused decrease of the largest market share for %. still, this resulted in the minor market concentration change; hhi was on average . before and . after trp introduction. further, at least one active substance switch was detected in approx. and % of patients before and after trp introduction, respectively. similarly, the proportion of exceptions when medicine was fully reimbursed increased from . % in transition period to . % in period after trp introduction. conclusion: enforcement of trp for ppi contributed to approx. € m annual cost savings. from the payer's perspective the new policy was proven to be effective in reducing pharmaceutical expenditure; however trp also affected physician prescribing pattern and use of ppis. pec : blood coagulation factor: improvements of the supply chain samantha oses * , serri traore, sonia caroline sorli, lea damery, philippe cestac, sylvie pomies, julien tourel please specify your abstract type: descriptive abstract (for projects) background and objective: most of the antihemophilic factor (ahf) must be held by a teaching hospital to face serious bleeding events. to ensure better availability, offsite-stocks at critical points are required (emergency unit, intensive care unit, etc.). however, this management system increases the risk of economic loss and alteration of the quality due to expired products. in this context, we carried out an optimization of the supply and management system of the ahf. to identify critical points of the supply and management system and to implement improvement solutions. design: a multidisciplinary working group belonging to a regional management centre of haemophilia was set up. two lines of improvement were discussed: i) optimization of stocks ii) optimization of the supply system. results: the optimization of stocks has led to the modification of the threshold of the lowest stock (ls) for ahf out of . in % of cases, this stock modification has exceeded %. the overall cost of ls has been reduced by . % ( , €) for the general stock at the central hospital pharmacy (hp) and by . % for offsite-stocks ( , €). the ahf mainly involved in this reduction was fvii mg ( , €), then followed by the strengths of mg and mg ( , € for each). in order to improve the ahf management, several propositions have been implemented: ( ) developing an online, easily accessible and monthly updated spreadsheet that displayed several accurate data such as the shortest expiry date and the storage location. this operative tool is shared between all pharmacists involved in ahf management in order to facilitate a stock rotation and decrease economic losses, ( ) regular reminders to physicians and health care staff concerning the guidelines for inventory management and the importance of checking the drug expiry date, ( ) presentation of the financial results and raising awareness on ahf costs to the medical consultant[ppip ] and ( ) optimizing stock distribution based on consumption on the different hospital sites for better patient care management (pcm). conclusion: this optimization of stocks and improvement of the supply chain have led to a direct cost saving of , €. however, a more accurate assessment has to be performed to quantify the direct and indirect impact on pcm and cost saving. this work has been done in a context of a sharing operative network at a regional level. the aim of such project is to share, to optimize and to improve practices, knowledge, human and medical health resources at a widespread level to enhance the security and quality of health services and to promote cost and time saving. please specify your abstract type: descriptive abstract (for projects) background and objective: the overall pharmaceuticals consumption in hospitals is rising, which has led to an increasing expenditure, challenging health care professionals and threatening patients safety. clinical trials in hospitals have increased over the past few years and currently play an important role, giving access to new investigational medicinal products and also avoiding costs with standard treatments. the objective of this study is to evaluate the savings of centro hospitalar do porto, a central university hospital with beds and currently clinical trials, with patients included in clinical trials between january and may . design: retrospective observational study over months. all the clinical trials ongoing between january and may were analysed and the data was collected based on: pathology and doses established; number of treatments per patient and the medium prices of standard treatments that patients would be receiving if they were not in the clinical trial. results: there were clinical trials ongoing between january and may , but only were selected to be included in this study. the total number of patients included was . the clinical trials selected for this study were conducted in medical specialties: in dermatology, in immunology clinical unit, in hemato oncology, in gastroenterology, in ophtalmology and in neurology. during these months, with all ongoing clinical trials, centro hospitalar do porto was able to save, in medical products, more than million euros. conclusion: during the period of time established, of the clinical trials ongoing, were not selected due to: not including patients or not having an alternative treatment. hospitals and patients can benefit from clinical trials not only financially but also by preserving resources and medication. on centro hospitalar do porto, the pharmacists specialized in clinical trials, as members of the study team, are more and more required to perform specific tasks, their contribution has been increasing over the years and also have become more aware of all the advantages from participating in clinical trials. these savings can be used to provide a better assistance and contribute, in general, to a higher quality health care. please specify your abstract type: descriptive abstract (for projects) background and objective: several studies show a misuse of opioid maintenance treatment (omt) in detention. in fact, buprenorphine (bup) when it's misused, could present the same effects as heroine. in order to reduce misuses, the pharmacist decided to switch all the patients under bup to buprenorphine/naloxone (bup/nlx). bup/ nlx prevents patients from misusing by a withdrawal syndrome when it's issued by another route of administration than sublingual route. in france, bup/nlx is more expensive than bup which may explain why this therapeutic strategy is not often observed. the purpose of this study is to evaluate the extra cost after switching patients from bup to bup/nlx in order to decide if this choice could be maintained. design: to identify our population, we used the administration reports drugs written by nurses. please specify your abstract type: research abstract background and objective: haemophilia b is an x linked genetic disorder characterized by spontaneous or prolonged haemorrhages due to factor ix (fix) deficiency . within the next few years, new treatments are willing to hit the market. among them are recombinant extended half-life products that will reduce by half the number of injections and will potentially improve the patient quality of life. the aim of the study is to describe the development of haemophilia treatments market between and and to forecast the potential impact of these new therapies on the haemophilia market. setting and method: national and french hospitals of paris (aphp) consumption data of fix between and have been studied. new therapies in development or soon to be marketed have been identified. potential benefits and interest in the therapeutic care of these new products were discussed with haemophilia's medical experts. main outcome measures: quantity (ui) and value (euros) of fix aphp and national consumption. results: in , recombinant (rfix) and plasma-derived factors (pfix) were on the french market. the ap-hp's purchases of these factors represent almost million ui and million euros, which comprise % of national fix expenditures. in france and aphp, ambulatory care is a major part of the use of these treatments with nearly % of the fix purchases in . french rfix consumptions are higher than pfix consumptions ( % against %). in the ap-hp hospitals, rfix even account for % of consumptions against % for pfix. both national and ap-hp rfix purchases have steadily increased between and . the added competition arising from new treatments may lead to more competitive market procedures in hospitals and may reduce costs of haemophilia treatments. according to haemophilia doctor, long-acting (la) fix would offer obvious benefits like fewer infusions and presumably fewer bleeds. these treatments will mainly be used in a prophylactic wayin ambulatory care-than in a curative way (such as surgical use). conclusion: the therapeutic extent of these new treatments is still hard to define. the choice of treatment must remain consensual between physicians and patients. please specify your abstract type: descriptive abstract (for projects) background and objective: good practice about medicines imposes to health institutions a close monitoring of prescriptions, especially off-label prescriptions. patient care should take into account clinical profile, respect of guidelines and health expense control. we report here a case highlighting the significant role of the clinical pharmacist in care units to ensure medication good use in a castleman syndrome, a rare disease due to human herpesvirus (hhv- ) and associated with human immunodeficiency virus (hiv) infection. design: case report. results: our patient, a years old man (creatinine clearance rate (crcl): ml/min), was diagnosed with hiv infection in february (cd at ui/l), leading to introduce a therapy by emtricitabine-tenofovir, darunavir, and ritonavir. the evolution was hampered by repeated episodes of acute renal failure (arf; crcl: ml/min) and pancytopenia (hemoglobinemia at . g/dl, leucopoenia at . g/l, and thrombopenia at g/l). because of hhv blood pcr at copies/ml, transient crises with pancytopenia, arf, and hiv infection, a diagnostic of kaposi sarcoma herpesvirus (kics), an atypical castleman syndrome, was retained. given the lake of data in literature for this rare disease, a multidisciplinary team (medical specialists and clinical pharmacists) was gathered to choose an appropriate therapeutic strategy. treatment regimen consisted of: day , intravenous etoposide at mg; day , rituximab at mg/ m ; following one week later by rituximab day and oral etoposide at mg the day after. good communication between medical specialists and pharmacists enables the patient to get an optimal and personal treatment. relaying the information by clinical pharmacists in care units to pharmacists in charge of good practice facilitate the reimbursement. conclusion: clinical pharmacists in care unit help to optimize therapeutic strategies according to their experiences and scientific works. cooperation with physicians is improved, as well as prescriptions follow-up of off-label drugs, and health patients fully respected. quality and relevance of prescriptions are strengthened, with a better control of economic expenses. please specify your abstract type: research abstract background and objective: the maltese government launched the hpv vaccination scheme in and the national healthcare system (nhs) has since provided the cervarix Ò vaccine free of charge to girls aged . the aim of this study was to assess the cost of the administration of hpv vaccines in the healthcare system of malta. this study was based on the scheme provided by the nhs. the number of girls born per year was used to estimate the annual cost for vaccinating year old girls, based on the wholesale price and tender price respectively. the estimated yearly cost using the wholesale price was approximately € , while the average estimated cost based on the tender price was approximately € , . this signifies that cost savings based on the tender price compared to wholesale costs were of approximately € , . the cost for the cohort who completed the three dose schedule using the tender price on average was of € , per year. this result proved to be more than the anticipated cost. a reason for this could be that the number of girls aged increased possibly due to an influx of immigrants. including boys in the vaccination scheme would increase costs by an average of € , per year. conclusion: this study shows that procuring branded vaccines using the tendering process reduces expenditure for the government and the tax payer. wholesale prices were found to be more expensive than tender prices. this proves that the tendering system in malta is a potent system with many advantages for the tax paying public. the impact of the tendering process must therefore, be safeguarded. please specify your abstract type: research abstract background and objective: with the old age, presence of comorbidities, and overcrowding in mass gatherings such as the annual hajj pilgrimage in saudi arabia, there is a high risk of spreading infectious diseases among pilgrims and then within their country of origin. knowledge and application of hygiene principles in such an environment is therefore important to reduce the transmission of infectious diseases. up to date, there have been no studies to evaluate pilgrims' knowledge, attitude and practices toward mers-cov during the annual hajj pilgrimage in order to see whether there is a need for these aspects to be improved. setting and method: a cross-sectional survey study was conducted with a convenience sample of participants. participants were pilgrims, aged over , and able to speak arabic or english. a selfadministered structured questionnaire was distributed during hajj season in mecca. descriptive and multiple linear regression analysis were used in data analysis. main outcome measures: assessing pilgrims' knowledge, attitude and practices regarding mers-cov. results: two hundred and fifty-seven participants completed the study, % of whom were female, and the median (iqr) age was ( . - . ) years. pilgrims had moderately correct knowledge and accurate attitudes towards mers-cov with median scores of (iqr - ) and (iqr: - ) respectively. they were less educated about management ( %), hallmark symptoms ( %), high-risk individuals ( %) and source of coronavirus ( %). almost % of participants showed a negative attitude towards the use of protective measures such as avoiding food prepared under unsanitary conditions and contact with live animals. some participants ( %) were unable to comply with hygiene practices, particularly washing hands with soap and water or disinfectant after sneezing/coughing and wearing a face mask in crowded areas. educational level and employment status were significantly associated with knowledge whereas gender and age were significantly associated with attitude and practices respectively (p \ . ). the correlation between knowledge, attitude and practices was significant (correlation coefficient: . ; p \ . ). better knowledge was found to be a predictor for positive practice. conclusion: these findings aided in the assessment of the adequacy of current pilgrims' educational measures. they will also provide insight when designing future interventions to promote specific messages to improve knowledge, change attitude and improve practice regarding mers-cov. please specify your abstract type: research abstract background and objective: the prevalence of type diabetes significantly increased in the paediatric population, which is affected by obesity worldwide. today, type diabetes accounts for % of all cases of new-onset diabetes in adolescents. preventive health care particularly taking place at community pharmacies may involve risk assessment for the children and the adolescents, early referral for seeking relevant medical care and patient education on healthy lifestyle choices. the aim of the study is to conduct a type diabetes risk assessment program for the kids b years of age of whose parents visited the community pharmacies involved in the study and also to identify the behavioural parameters that might be associated with this risk. setting and method: the study was conducted in community pharmacies. all patients with kids aged b years who visited the study pharmacies during one-week period were informed about the study and invited to participate in the study. patients who gave their informed consent were included in the study. all data were provided by the parents. demographic data, height and weight of the kid, as well as data regarding the behavioural features (eating habits, exercising, time spent in front of a screen, etc.) of both the children and the parents were collected using standardized forms. type diabetes risk test consisted of questions and identified subjects at risk. the parent of the kid who was identified to have risk for type diabetes was referred to a physician for further examination. also, information regarding type diabetes and the importance of preventive measures such as converting to a healthy life-style was provided. main outcome measures: main outcome measures were the percentage of kids identified to be at risk of developing type diabetes and the behavioural parameters associated with type diabetes risk. results: the study involved subjects. of the subjects % were identified to be at risk of type diabetes. more girls than the boys had the risk ( vs. . %). those with type diabetes risk were older, taller, heavier and had higher body mass index. they were spending more time in front of a screen (tv, pc, tablet, smart phone); . % were spending more than h a day. although the kids' eating habits were similar for those with and without risk, the parents' of the kids with risk ate out more frequently, consumed rice, pasta and pastry more frequently. both the kids with risk and their parents exercised more regularly and frequently. conclusion: this study shows that pharmacist have a vital role in identifying children and adolescents at risk for type diabetes; thus at early management of this condition. identifying and addressing the behavioural parameters associated with the risk will be helpful in lifestyle modification interventions. please specify your abstract type: descriptive abstract (for projects) background and objective: analyse and promote the reporting of adverse drug events (ade), to improve the quality and safety of care to be able to control the risks. design: a software is available on the intranet website of the institution, to enable health professionals to report ade. the drug and medical devices commission (comedims) of the hospital, centralizes these statements and always makes a multidisciplinary and overall analysis of the event, using a collection sheet which is based on the pdca model (plan, do, check, act). it proposes the nursing and medical teams axes of improvement. results: in , only ade were reported and analysed by the comedims, including from the paediatric centre ( %), particularly sensitized to this issue. health professionals are divided as follows: healthcare executives ( %), nurses ( %), pharmacists ( %), residential students ( %), doctors ( %) and others ( %). the main impacted steps of the drug circuit are: administration ( %), prescription ( %) and the use or implementation of a sterile medical device ( %). identified causes include related following factors: operational tasks and procedures ( %), health professionals ( %), work environment ( %), organization and management ( %), drugs or associated medical devices ( %). the number of ade reports, taking into account the size of the institution, remains very low. in january , the comedims decided to broadcast a communication campaign to promote ade reporting, on the hospital website via the intranet. three months after the release, this document was viewed times, and the number of reports increased by % compared to the same period in . conclusion: in front of the low number of returns of adverse drug events, and relying on the charter of non-punishment, the come-dims wants to increase health professionals' awareness. in our hospital, where e-learning about drug-related iatrogenesis is already available, the communication campaign with poster and analysis of adverse events seems to be a useful complementary tool to enhance awareness of medication safety concerns. please specify your abstract type: research abstract background and objective: the migration of modern social networks to the internet has facilitated the transition of traditional pharmacy networks online. the ubiquitous nature of social media (some) combined with merging of personal and professional personas have led to organisations publishing guidance on online behaviour and responsible use of social media. the research to date on the use of social media as a support for professional practice in general is limited. as the pharmacy profession evolves to embrace the technologies which underpin core services and mainstream online daily social activities, it is important that research tracks and evaluates its use and impact within the profession. the objective of this research was to explore and describe how and why pharmacists interact with hosted networks on social media. setting and method: two one-hour online hosted micro-blogging twitter chats were held in december via the #weph network. topic guides were developed around 'exploring the use of twitter and wepharmacists' in line with the wenetwork guidelines (#wecommunities), informed by existing literature, discussion with the #weph moderator after review by an expert panel. all research was carried out in accordance with university governance processes and association of internet researchers guidelines. themes were inducted from analysing the textual content of the chats using the topic guide as a framework. the research was approved by the school of pharmacy and life sciences ethics committee. main outcome measures: tweets per chat results: each of the chats had over million impressions with participants representing international pharmacy practice. themes of e-professionalism and online privacy emerged as concerns; however, the benefits included using social media for education, networking, support mechanisms and career development. tweets highlighted personal experiences of 'trolling' (angry, offensive behaviour) and the effect on user interaction with social media. twitter was also recognised as a career development tool and, in particular, collaborative outcomes around mentorship networking early career pharmacists with more experienced colleagues. conclusion: results support the responsible use of social media as a force for inclusion, breaking down geographical barriers in support of pharmacy practice. further research is underway including a systematic review of guidance on the use of social media by registered healthcare professionals. please specify your abstract type: research abstract background and objective: it is estimated that half of the , persons with diabetes in norway have not been diagnosed. with early treatment, life expectancy can be increased and the incidence of longterm complications and health costs reduced. community pharmacies may be able to help uncover undiagnosed diabetes, but being diagnosed with diabetes can lead to strong emotional reactions, and how the diagnosis is given may influence the experience. the aim of this study was to explore how norwegian people living with type diabetes (t d) experienced being diagnosed, and what led up to the diagnosis. in addition, their attitudes towards a planned community pharmacy service to identify undiagnosed t d was investigated. setting and method: three focus group interviews with people with t d were conducted using a semi-structured interview guide. eleven participants were recruited through a course about type diabetes. the interviews were audio-taped and transcribed in modified verbatim form and analysed in accordance with malteruds principles of systematic text condensation. the study was approved by the norwegian data protection authority, and did not require approval from the regional committee for medical and health research ethics. main outcome measures: how people with t d describe their experiences of being diagnosed with t d, how the disease was revealed and reactions towards using community pharmacies to perform risk assessment for t d. results: none of the participants were diagnosed due to their own suspicion of having diabetes. some saw their doctor because of unspecific symptoms such as fatigue and thirst, and were thereafter diagnosed with t d. others were diagnosed through a routine checkup. negative reactions like shock, discontent and denial were commonly used to describe the experience of being diagnosed with t d, but some participants also expressed a more relaxed attitude, especially if they were familiar with the disease through family members. participants expressed a strong wish for more and better information following the diagnosis. ''it's a jungle out there'' was used to describe how difficult they felt it was to find trustworthy and understandable information. they described change of lifestyle, side effects from drug use, and stigma as challenges following the diagnosis. while in general the participants were positive to using community pharmacies to uncover undiagnosed diabetes as this could help reduce the number of people who were undiagnosed, some were sceptical. they questioned whether the pharmacy staff had the necessary competence of the for this type of service, and saw it as the doctor's responsibility. conclusion: more information and support when people are diagnosed with diabetes may lead to that the experience being diagnosed will be more adaptable and that the challenges living with diabetes are reduced. community pharmacies are important healthcare providers, and risk assessment of t d at the pharmacy can be valuable. however, the pharmacies may also be helpful to reduce the information gap. please specify your abstract type: research abstract background and objective: chemotherapy-induced nausea and vomiting (cinv) is a disruptive and unpleasant side effect in chemotherapy patients and is associated with decline in patients' quality of life and decrement in the adherence to effective chemotherapy regimens. setting and method: chemotherapy naive patients were included in this study. consistency with guidelines were assessed according to mascc/esmo . flie questionnaire was administered to patients before chemotherapy, and days after receiving chemotherapy to assess the difference in the quality of life due to chemotherapy administration. main outcome measures: patients were categorized into two groups as consistent with guidelines group (acute (gcga) and delayed (gcgd)) and inconsistent with guidelines group (acute (giga) and delayed (gigd)). flie score differences between the two groups were assessed. results: the median flie score for patients prior to chemotherapy was and a dramatic decline was noticed post chemotherapy (flie score ; p \ . ). the post-chemotherapy score were for nausea and for vomiting ( . , respectively). although the flie score differed significantly between gcgd and gigd (p \ . ), these differences were not significant in gcga and giga. conclusion: the significant drop in flie scores in the study ( pre-to post-chemotherapy) reflected substantial declination in patients' quality of life. the lower postchemotherapy flie score of nausea emphasized the negative impact of nausea, and to a lesser extent vomiting on the patients ability to complete normal daily activities such as enjoying meals and maintaining social activities. although there were no significant differences in flie scores between giga and gcga groups for acute cinv prevention, significant differences were noted between gigd and gcgd (p \ . ). the flie score was lower for gigd patients. this result implied guideline inconsistency associated with high incidence of nausea which negatively affect patient quality of life. as for the degree of compliance with gp, the results are expressed as percentage of compliance compared to the ideal of %. prescription criterion was fulfilled to %: all requirements of pntb were performed using standardized procedure. in what concerns validation, % of pntb prescriptions were validated by a pharmacist. the invalidated prescriptions were made outside opening hours of the pharmacy service, which is open monday to friday from : to : and on weekends and holidays from : to : . % of the dispensations were individualized and not pntb stocks were found in hospital wards. as for preparation, % were supplemented with micronutrients. pntb of kabiven peripheral administration ml are not supplemented in our centre. of the remaining prescriptions central administration, % were supplemented. in all cases, the addition of micronutrients was performed in laminar flow hood in pharmacy service and the corresponding galenic validation was performed. finally, in the process of administration, % of pntb identified with a complete label: name of the patient, medical record number, type of pntb, qualitative and quantitative composition, date of administration and infusion rate. conclusion: use practices of pntb of our centre are far from those recommended by the sefh standards. this initial evaluation will serve for improvement measures that increase the quality of prescribing and safe use of pntb, in order to minimize errors that can occur with the use of this therapeutic modality. please specify your abstract type: research abstract background and objective: methadone maintenance treatment was developed in malta in and is provided to patients by sedqa, the national agency against drug and alcohol abuse. methadone is the most frequently prescribed opioid in opioid substitution treatment and is dispensed through a centralised service through the substance misuse outpatients unit. in , patients were in opioid substitution treatment, of who were on methadone. in , the government introduced a take-home methadone program. the prescribing, purchasing and dispensing of methadone are regulated by subsidiary legislation . . the objectives were to determine whether community pharmacists in malta would be willing to dispense and supervise the consumption of methadone and to investigate the involvement of community pharmacies in the development of a regionalised methadone dispensing service. setting and method: the study was set in community pharmacies. a cross-sectional study, through the use of a questionnaire, was performed to quantitatively analyse whether pharmacists in malta would be willing to dispense methadone. the questionnaire consisted of questions divided into sections, with each section assessing a particular aspect of community pharmacists' attitudes towards methadone dispensing. community pharmacies were then chosen via a systematic sampling procedure. a hard copy of the questionnaire, addressed to the managing pharmacist, along with a cover letter, instructions on how the questionnaire was to be returned, and a prepaid self-addressed envelope was distributed via postage to community pharmacies. an online format of the questionnaire was also circulated to community pharmacists through the pharmacy council. data was analysed using spss version . main outcome measures: community pharmacist's attitudes towards methadone dispensing. results: a total of responses were obtained and a response rate of . % was achieved. eighteen percent of the pharmacists (n = ) who responded to the questionnaire worked in a community pharmacy located in the north of malta, % in the centre, % in the south, % in the southeast and % in gozo. thirty-two percent of community pharmacists were willing to dispense methadone to drug misusers. the number of community pharmacists who are willing to dispense methadone increased to % if they were provided with appropriate education and support. twenty-nine percent of community pharmacists were prepared to handle the duty of supervising the consumption of methadone while % had never learnt about methadone and its clinical application within opioid substitution treatment. conclusion: community pharmacists should be provided with education and training regarding methadone substitution treatment before embarking on a new regionalised methadone dispensing service within community pharmacies. this would allow more community pharmacists to become involved in a new dispensing methadone service. pt : evaluation of regorafenib in patients with colorectal cancer please specify your abstract type: research abstract background and objective: the colorectal cancer is the second more frequent cancer in europe and the third in the world. regorafenib is only approved in adult patients with metastatic colorectal cancer who are previously been treated with available therapies or are not considered suitable candidates to these treatments. regorafenib is an oral anti-tumor drug that blocks the kinases involved in the tumor angiogenesis (vegfr , - , - , tie ), the oncogenesis (kit, ret, raf- , braf, brafv e) and the tumor microenvironment (pdgfr, fgfr).in this study, we are reviewed the reports of the patients with colorectal cancer who are been treated with regorafenib in our hospital and analysed the information in order to evaluate the efficacy and safety of regorafenib. setting and method: descriptive and observational study about the use of regorafenib from april to the present day. the variables studied, obtained from the software applications archinet and diraya, were: sex, age, pathology, location of metastasis, posology and adverse effects of regorafenib, tumor markers (cea y ca . ) before and after the treatment with this drug and the mutational state of kras. main outcome measures: the tumor markers cea and ca . only decreased in the . % of the patients after the regorafenib treatment. results: regorafenib was taken by patients ( %men).the average age of these patients was . ± . years old. the patients took regorafenib to treat: metastatic and non-intervened gastrointestinal stromal tumors (gist) e-iv that progressed with the previous treatment of imatinib and sunitinib ( . % patients), intervened colon adenocarcinoma e-iv ( . % patients), sigma adenocarcinoma e-iv ( . % patients) and unresectable and non-intervened rectal adenocarcinoma e-iv ( . % patients).all patients presented metastasis in different locations on the body: liver ( . % patients), diaphragm ( . % patients), intestine ( . % patients) and lung ( . % patients).the % of the patients started the treatment with mg of regorafenib, administrated once a day for weeks followed by one week without this drug; while the . % of the patients started the treatment with mg. however, the . % had to decrease the initial dose and the . % of the total patients had to get off the treatment because of the development of side effects. the most frequent adverse effects were: hypertension associated with headache, hyperbilirubinemia, elevation of ast and alt, intense asthenia. the . % of the patients presents native kras. the native kras was presented in the % of the patients treated with regorafenib who had an appropriate development of the illness (decrease of cea and ca . ) conclusion: the decrease of cea in the . % of the patients and the high development of side effects reveal that regorafenib has low effectiveness and security in the control of the progression of colorectal cancer. in addition, it is supposed that this drug has better results in native kras patients. however, more studies are necessaries in order to demonstrate the effectiveness of regorafenib in this pathology. pt : evaluation of nintedanib in patients with non-small-cell lung carcinoma (nsclc) please specify your abstract type: research abstract background and objective: the nsclc means a high rate of mortality in developed countries. patients diagnosed with nsclc who debut with advanced or metastatic disease have a median survival of months. one of the innovative drugs approved to improve survival in nsclc is nintedanib: an inhibitor of multiple tyrosine kinases, which can be found in some receptors on the surface of cells involves in the growth and spread of cancer cells (''pdgfr'', ''fgfr'' and ''vegfr''). nintedanib is not yet marketed in spain. hospital pharmacists are responsible for applying this treatment as ''expanded drug'', only after the elaboration of an exhaustive report. in this study, we have reviewed all the reports and classified the information in order to present our clinical practice. the objective of this study is to evaluate the effectiveness and safety of nintedanib in patients with nsclc treated in a tertiary hospital. setting and method: descriptive observational study of the use of nintedanib from november to september . sex, age, body mass index (bmi), pathology, smoking habits, line of treatment, posology and adverse reactions of the treatment with nintedanib and tumor markers (cea an ca . ) before and later the treatment with nintedanib were collected from medical history through archinet informatic application. main outcome measures: the tumor marker cea decreased in % of the patients and ca . no decreased in any patient after nintedanib treatment. results: nintedanib was used in patients ( % men and % smoker).the average age of these patients was years old. the average bmi was kg/m ( - ).all patients received nintedanib together with docetaxel for metastatic nsclc with adenocarcinoma histology and with non-mutated egfr and alk in third line treatments. posology: all patients started the treatment with nintedanib mg/ h from day to day every weeks; but patients had to reduce the initial dose to mg/ h ( patient) and mg/ h ( patient) because of some adverse reactions. the side effects were: asthenia, diarrhoea, alteration of transaminases, muscle pain and cramps, weight loss and mucositis. conclusion: the decrease of cea in % of the patients reveals that nintedanib is effective in controlling nsclc progression which involves an increase of the survival and the quality of life of these patients. however, more studies are required to demonstrate the efficacy of nintedanib in this illness. please specify your abstract type: research abstract background and objective: patients with sore throat symptoms often seek fast, meaningful relief when presenting to their local pharmacy. flurbiprofen is a non-steroidal anti-inflammatory drug, which has been developed as a spray and lozenge to provide targeted relief for the main underlying process responsible for the symptoms of sore throats, inflammation. to study the relief provided by flurbiprofen . mg delivered as a spray or lozenge, we conducted a multicentre, randomised, double-blind, double-dummy, parallel group, activecontrolled, single-dose, non-inferiority study. setting and method: adult patients with acute sore throat were randomly assigned to take one dose of either flurbiprofen . mg spray plus a placebo lozenge, or flurbiprofen . mg lozenge plus placebo spray at sites across russia. main outcome measures: patients rated sore throat relief using the sore throat relief rating scale (strrs; a -point scale, = no relief, = slight relief, = mild relief, = moderate relief, = considerable relief, = almost complete relief, = complete relief) at timed intervals throughout h starting from min post completion of first dosing ( min after administration of the spray, and min after the lozenge had fully dissolved). adverse events (aes) were recorded over h post-dose. results: patients were assessed (n = for spray, n = for lozenge). [ % of patients in either treatment group experienced some relief (a score of [ on the strrs) at min post-dose, which increased to % of patients by h. - % of patients reported 'at least moderate relief', which is a well-recognised measure of a clinically meaningful effect at min post-dose, which increased to - % of patients by h. over the h post-dose, a total of drugrelated aes were reported by patients across both treatments and no severe adverse events were reported. conclusion: flurbiprofen . mg delivered as a lozenge or spray provides fast, clinically meaningful relief from sore throat. pt : analising antiangiogenics prescription in an ophtalmology service after a protocol implementation silvia cornejo-uixeda * , ivan de la vega-zamorano, celia aparicio-rubio, olga carrascosa-piquer, manuel prieto-castello, agustin sanchez-alcaraz pharmacy, hospital universitario de la ribera, alzira, spain please specify your abstract type: descriptive abstract (for projects) background and objective: after some years using antiangiogenics in our hospital, we observed a large variety of use. considering the high cost of these treatments, we proposed ophthalmology service to develop a protocol of use, attending efficiency criteria. in this paper, we analyse the protocol implementation repercussion. design: a protocol of use was designed with the main of unify criteria and to use the most efficient treatment depending on the specific situation on each patient. once it was implemented, we compared two periods, the period after the implementation (january-may ) and the period before of it (january-may ). the protocol designed is the following: the cost for each injection and patient was the following: aflibercept €, bevacizumab €, ranibizumab €. results: in the period, patients were treated with antiangiogenics. ( %) with aflibercept, ( %) with bevacizumab and ( %) with ranibizumab. in the period, patients were treated, ( %) with aflibercept, ( %) with bevacizumab and ( %) with ranibizumab. the consumption of aflibercept decreased a %, bevacizumab consumption increased % an ranibizumab increased a %.we also observed, some patients had more than one diagnostic at the same time. once the protocol was implemented, the percentage of use was the following: % . please specify your abstract type: research abstract background and objective: drug prescribing is the most common medical intervention in the elderly. however, elderly patients are more sensitive to the drug's effects due to pharmacokinetic and pharmacodynamic changes associated with aging. chronic diseases and co-morbidities often require the use of a large number of medications. therefore, when prescribing drugs for the elderly, the choice of suitable drugs, dosage and duration of treatment should be carefully considered as well as clinically significant drug interactions. inappropriate prescribing is often associated with an increased risk of adverse drug reactions, increased morbidity and mortality, and health care costs. the aim of this study was to determine the incidence of potentially inappropriate medications (pim) prescriptions in the elderly (c years) using the original protocol developed by mimica matanovic and vlahovic-palcevski. setting and method: we enrolled patients hospitalized in clinic of internal medicine. data about patients' medications was collected during patient interview taken by the pharmacists on hospital admission. pharmacotherapy was analysed using the original protocol developed by mimica matanovic and vlahovic-palcevski in order to detect pims. main outcome measures: number and type of potentially inappropriate medications, potential clinically significant interactions. results: the average age of patients was years (range - ), and the average number of drugs per respondent was . (range - ). a total of patients ( . %) were taking at least one pim. the most common pim were long-acting benzodiazepines, central antihypertensive moxonidine and non-steroidal anti-inflammatory drugs (nsaids) in patients with hypertension. in the study population, patients ( . %) have taken at least one combination of drugs that could result in a clinically significant interaction. the most common combinations included application of nsaids and antihypertensive drugs or diuretics, concomitant use of multiple medications with effects on the central nervous system and drug combinations that can cause hyperkalaemia. conclusion: this study revealed the high prevalence of inappropriate prescribing. clinical application of this protocol could be an effective method for improving and optimizing drug prescription with the aim to reduce the number of side effects and the morbidity and mortality associated with the drug use in the elderly. please specify your abstract type: research abstract background and objective: to reduce adverse effects of conventional amphotericin b formulation (deoxycholate or d-amb) it can be infused in intralipid Ò (a fat parenteral nutrition), or lipid-based formulations can be used (i.e. amphotericin b lipid complex (ablc), amphotericin b colloidal dispersion (adcd) and liposomal amphotericin b (l-amb)). studies evaluating safety profiles present conflicting results. the aim of our study was to gather evidence on nephrotoxicity rates of d-amb versus lipid-based formulations in immunosuppressed patients susceptible to invasive fungal infection. setting and method: a systematic review, including randomized controlled trials (rcts) that compared the use of d-amb and amphotericin b lipid-based was performed. a search was conducted in pubmed, scopus, web of science and scielo. results were synthetized and meta-analysis was performed using software review manager . . main outcome measures: nephrotoxicity rates. results: eighteen rcts were identified (n = participants). the result from the meta-analysis favours the treatment with the lipidbased amphotericin b formulations (or: . ( . , . ) and presents a low heterogeneity (i = %). about % of patients from lipid-based treatment group presented an increase in serum creatinine of one to two times, which corresponds to stage one or two of acute renal failure (arf). and % presented an increase of tree times in serum creatinine achieving a stage three in arf (severe) which will require dialysis. while in group treated with conventional formulation int j clin pharm ( ) all of these patients, except one whose treatment adherence was inadequate, were cirrhotic ( / ), liver transplanted ( / ) and/ or presented hepatocellular carcinoma ( / ). / patients were coinfected with hiv. / patients ( %) were genotype . the total genotype patients treated with daas (svr /relapsed) were , which means that . % ( / ) of all genotype patients has had a relapse. / patients ( %) were treated with ledispavir/sofosbuvir ( . % of a total of patients (svr /relapsed) treated with this option). % of patients who suffered a relapse were treated with daas sofosbuvir, simeprevir, daclatasvir, previously to the introduction of the newest antivirals (dasabuvir + ombitasvir/ paritaprevir/ritonavir, ledispavir/sofosbuvir), which represents . % of the total of patients treated with the older option. conclusion: relapses rate was . %, slightly lower than reported in other studies. according to the references, these results show that genotype is the one presenting more relapses. all the patients presented a deteriorated performance status, except for one whose treatment adherence was inadequate. patients treated before april , when the newest daas where introduced, showed more relapses. more studies have to be developed in the near future since other daas will appear, the treatment options will be amplified and the number of relapses is expected to decrease. please specify your abstract type: research abstract background and objective: the inappropriate use of antibiotics remains a major issue since it causes bacterial resistance, longer hospital stay and increased mortality. antibiotic prescriptions must be monitored: the clinical pharmacist has a key role in ensuring patient safety and quality of pharmaceutical care. therefore, an antimicrobial stewardship program has been implemented as part of a national project of the italian society of hospital pharmacy (sifo). the objective is to describe the results obtained at the hospital. setting and method: a multidisciplinary antimicrobial management team has been implemented including clinical pharmacists, microbiologists and infectious disease specialists. the pharmacist examines drug charts on a daily basis in the department of medicine and supports clinicians to improve the appropriate use of antibiotics. data from time-points were extracted from medical records and collected in an excel database: t (november -january ) and t (february -april ). main outcome measures: type of infection, antibiotic consumption data, type of isolated pathogens, patient allergies, clostridium difficile infection assessment and adverse drug reactions (adr). results: records were analysed (t -t ), of which contained at least one antibiotic prescription. the most frequent infections were urinary tract ( %), respiratory ( %) and gastro-intestinal ( %). antibiotic therapy was started in . % of cases due to aspecific increase of c-reactive protein (crp). ddds were calculated for each treatment and were grouped by type of infection and setting (empiric vs targeted): ceftriaxone, meropenem and metronidazole were the most widely used antibiotics for empiric therapy. at t , an increase in the use of piperacillin-tazobactam instead of meropenem was observed. the ddd of ceftriaxone for targeted therapies decreased significantly, while an increase was observed for carbapenems, levofloxacin, glycopeptides and, in case of mdr bacteria, tigecycline. three allergies to antibiotics were reported in medical history. there were clostridium difficile infections ( relapses), confirmed by antibiogram. a total of adrs were identified: of these were related to antibiotics. conclusion: antimicrobial stewardship is a fundamental step to optimise antibiotic management, ensure patient safety and improve quality of care. the results obtained so far demonstrate the added value of a multidisciplinary team in controlling bacteria resistance and in the improving the use of antibiotics. please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of this study was to analyse effectiveness and safety of pirfenidone, an anti-inflammatory and antifibrotic agent used for treatment of idiopathic pulmonary fibrosis. design: a retrospective, descriptive, observational study including all patients treated with pirfenidone at the hospital between march and june ( month) was carried out. to identify patients and collect data the outpatient medication dispensation software farhos Ò and the electronic medical record software hcis Ò were used. statistical analysis was carried out using microsoft excel Ò . demographic (age and sex), clinical (forced vital capacity (fvc), diffusing co capacity (dlco) and six-minute walk test (wt m)) and therapeutic (dosage and adverse reactions) variables were collected. results: throughout the study period, a total of patients ( males) started treatment with pirfenidone, with a median age of . years ( - ). during this period patients were excluded for lack of monitoring. the median fvc, dlco, wt m values prior to pirfenidone therapy, were % ( [ %), . % ( [ %) and m ( - m) respectively. all patients met the inclusion criteria of capacity trial according to fvc and wt m; however of them didn't meet the dlco criteria (at least %).'' all patients were monitored every months. the median in fvc percentage change at the end of the study was - % (- % to + %). patients ( %) showed an improvement on fvc during treatment with a median change of %. in the other eight patients fvc value decreased with a median of - %. only one patient would be candidate to discontinue treatment due to a lack of efficacy, according to discontinuation criteria established at the hospital (absolute decrease of c % in fvc during first year of treatment). dlco percentage was measured in patients, with a median change of % (- % to + %). dlco decreased in patients. wt m was monitored in patients, with a median change of - . m (- m to + m). adverse effects related to pirfenidone were gastrointestinal disorders ( / ), increase of hepatic ggt ( / ), and dermatologic toxicity ( / ). six patients ( %) required a dose reduction because of gastrointestinal adverse effects. five patients ( %) discontinued treatment with pirfenidone due to hepatotoxicity ( ), gastrointestinal ( ) and dermatologic effects ( ). one patient died. conclusion: half of the patients improved fvc during the period of the study. the other half, showed a decrease in fvc value which was similar to the median obtained in capacity trial. gastrointestinal disorders were the most frequent adverse effects and cause of discontinuing treatment. treatment monitoring is important to achieve therapeutic benefit and control the adverse effects. the national centre for epilepsy, oslo university hospital, oslo, norway please specify your abstract type: research abstract background and objective: systematic medication reviews in interdisciplinary teams can help to identify potential and actual drugrelated problems (drp). the centre for development of institutional and home care services in oslo, norway, conducted medication reviews for polypharmacy patients with mental disabilities in - , based on a lack of knowledge about drug-related problems in this patient group. the objective was to examine prescribing patterns, frequencies and types of drp in patients with mental disabilities. setting and method: the forms for medication reviews were developed by the national patient safety campaign in norway. the nurse/social educator recruited eligible patients, observed them, and ordered test if needed. the clinical pharmacist (jwa) reviewed the medications to identify drps. the interdisciplinary case conference took place at the different general practitioners' offices being responsible for the individual patients. the general practitioner, the nurse/social educator and the pharmacist were present, and in some cases, also patients took part. main outcome measures: an independent researcher (aqm) collected and analysed the data based on the drp-forms containing information on the prescribed medicines, strength, dose, indication, a description of drp and suggested interventions to resolve them. results: overall, patients with mental disabilities, aged - years, consented to have a medication review. they used on int j clin pharm ( ) : - average medicines (range - ). the team identified drp in of the patients (average . , range - ). overall, % of all drp were resolved. for one-third of the medicines, an action was taken to improve the prescribing. the most commonly medicines were analgesics ( %), antiepileptics ( %) and anxiolytics ( %). the most frequent drps were unnecessary drug choice ( %), side effects ( %) and too low dose ( %). drps were most common in antipsychotics ( %), antidepressants ( %) and anxiolytics ( %). conclusion: patients with intellectual disabilities take more medicines and have many drps compared to other patient groups. they are also more prone to taking combinations of cns-active medicines and therefore more at risk of side effects and drug interactions. pt : protocol feasibility and patient findings when using a dry extract of zingiber officinale roscoe (ginger extract gr ) during pregnancy please specify your abstract type: research abstract background and objective: there is limited information about the use of dry extracts of ginger root. the objectives of this study are ( ) to evaluate the feasibility of a pilot study with a food supplement among pregnant women ( ) to learn what the patient findings are when using the dry extract of ginger during pregnancy. this abstract deals with the intermediate evaluation of a study conceived to investigate the safety of the ginger extract gr during pregnancy. setting and method: a prospective, interventional and real life pilot study with pregnant women between and weeks of gestation and having symptoms of nausea and vomiting or digestive complaints. the included patients can use the ginger extract gr for digestive comfort during pregnancy when needed. during the use, the score of digestive discomfort is noted and the researcher reports adverse events. main outcome measures: ( ) number of included patients as an indicator of feasibility: including a number of patients was taken as a target ( ) analysis (qualitative and quantitative) of the patient diaries, more particularly patient behaviour, wellbeing and impressions. results: within twelve weeks, patients were included with an average age of . years and a median age of ( - ) years. patients used gr : patients were dissatisfied, patients had a neutral opinion and patients were satisfied to very satisfied. one miscarriage occurred at a gestational age of almost weeks (only tablets of gr were used, with no relevant medical history in preceding pregnancies). two patients were hospitalized, of which with hyperemesis gravidarum. one patient complained about heartburn and one patient experienced a bad taste and heartburn. three patients have indicated that they experienced more nausea after taking the tablets. patients experienced no adverse events. the remaining patients were not yet evaluated. of the included patients, six patients decided not to use the product: because their gastrointestinal complaints were not serious enough, because problems of swallowing (using ginger gums instead). one patient was afraid for the negative consequences for her unborn child. the last of the nonusers indicated that she had no confidence in the product. conclusion: conducting a pilot study with the ginger extract gr in case of pregnancy is feasible. the majority of the evaluated patients were satisfied. signing the consent form does not guarantee the intake of the product. pregnant women remain very cautious in the use of unknown products during their pregnancy, even though it concerns a food supplement and not a drug. the severity of symptoms does not give a good indication whether or not and how often the product will be used. please specify your abstract type: descriptive abstract (for projects) background and objective: to analyse effectiveness and safety of ibrutinib, an oral inhibitor of bruton tyrosine kinase, in patients with mantle cell lymphoma (mcl) who have received at least one prior therapy. design: a descriptive observational study was carried out. all patients with relapsed or refractory mcl who started treatment with mg of daily ibrutinib between september and june were included. patients were identified and followed through electronic medical record. demographic and baseline clinical characteristics of patients were collected: age, sex, ecog (eastern cooperative oncology group scale), number and type of prior regimens, simplified mipi status (mantle-cell lymphoma international prognostic index), and disease stage (relapsed or refractory). progression free survival (pfs) and response to treatment were recorded to evaluate effectiveness. adverse effects related to ibrutinib and possible interactions with concomitant medication were documented to measure safety. statistical analysis of the data was carried out using microsoft excel Ò and spss Ò . results: throughout the period of study a total of patients ( males and female) with a mean age of . ± . years started treatment with ibrutinib. the median of previous treatments were ( ) ( ) ( ) ( ) ( ) including first-line treatment with high dose chemotherapy ( %), steam-cell transplantation ( %), rituximab ( %), bortezomib ( %) and lenalidomide ( %). the median ecog value prior to ibrutinib therapy was (range - ). the mipi status was intermediate risk in patients and high risk in , the disease stage was relapsed in % of the patients. partial response was reported in patients. the mean pfs estimated at the end of the study period was months ( % . - . ). adverse effects related to ibrutinib were: fatigue ( %), diarrhoea ( %) leucocytosis ( %) and infections ( %), including upper respiratory and urinary tract infections, sinusitis and pneumonia. one possible interaction between ibrutinib and everolimus was found in a liver transplant patient. close monitoring of everolimus plasmatic levels was recommended. conclusion: the mean pfs estimated in our study was similar to the median obtained in the pivotal phase ii trial. infections were the most frequent adverse effects. concomitant medication to ibrutinib should be checked, as ibrutibib is metabolised by cyp a and interactions may be frequently present. pharmacy, hiv unit, germans trias i pujol hospital, badalona, spain please specify your abstract type: descriptive abstract (for projects) background and objective: dolutegravir (dtg) is one of the preferred options for initial antiretroviral therapy (art) due to its high efficacy, good tolerability and low potential for drug-drug interactions. nevertheless, an unexpectedly high rate of dtg discontinuation (up to %) due to adverse events in the clinical practice has been recently reported. therefore, we aimed at assessing the dtg discontinuation rate and reasons for discontinuation in our hospital. design: single-centre, retrospective study from september to june of patients cohort with art both naive and pretreated. patients who had started dtg-based art containing regimen were identified and the reasons for the discontinuations were analysed. data were collected using the primary care service program and the electronic prescription program. results: out of patients attended by pharmacy department in our hospital, patients ( males, mean age years (range - )) had started a dtg-based art. out of them, patients were art naive and art-experienced. at the moment of starting dtg, mean cd cells were cell/mm (range - ) and hiv- rna load in plasma was detectable in patients. treatment discontinuation was reported in / patients ( . %) with a median treatment time of days (range - ). / patients ( . %) were naïve and / patients ( . %) pre-treated. most of the patients ( ) were in single tablet regimens (str) containing dtg in combination with abacavir and lamivudine, whereas the rest were in combination with other antiretroviral drugs. the main reason for treatment discontinuation was toxicity in / patients ( . %). the rest of the patients discontinued due to other motives (clinical trial inclusion ( / ), treated in another hospital ( / ), exitus ( / ) and others ( / ). reasons for the discontinuation were classified in different side effects: / ( . %) related to central nervous system (cns) (insomnia, psychiatric disorders such as anxiety, nightmares and depression), / ( . %) gastrointestinal effects, / ( . %) headaches, / ( . %) musculoskeletal effects, / ( . %) fatigue, / ( . %) allergy and / ( . %) for other reasons. some patients reported various toxicities at once. conclusion: more than % of patients treated with dtg discontinued by toxicity reasons. it is important to note that half of these patients had cns adverse effects. please specify your abstract type: research abstract background and objective: hcv therapy has been revolutionised recently by the approval of antiviral agents direct-acting (daa) facilitating the treatment of patients coinfected with hiv/hcv. however, potential drug interactions and overlapping toxicities of both treatments represent the major challenges in adapting therapy. to analyse the prescription profile of direct acting antivirals (aad) in patients coinfected with hiv/hcv. setting and method: retrospective observational study from january to january in a specialty hospital. the data were collected from the hospital program of clinical stories, archinet Ò , and the outpatient program farmatools Ò . the results were analysed using the statistical program r-commander. main outcome measures: inclusion criteria: adult patients coinfected with hiv/hcv with undetectable viral load. the following variables were collected: age, gender, hcv genotype, degree of fibrosis, patient type (naïve or pre-treated), baseline cd count, cd levels end of treatment, sustained viral response (svr) and hcv treatment. results: patients, of whom were men, mean age years were included. patients received daclatasvir and sofosbuvir for hcv, patients had genotype a and b respectively, patients genotype and patient genotype . patients had fibrosis f f , . of the patients they had not received previous treatment (naïve) and had failed to treatment. hiv treatment was modified in patients, patients achieved svr. the cv was undetectable to hiv treatment change for all patients. cd levels increased in all patients at the end of treatment for hcv with a median of cells/ul and at the beginning and end respectively. patients received ombitasvir/paritaprevir/ritonavir and dasabuvir, who had a genotype a. these two patients had received previous treatment and had a f and f fibrosis. none of them was modified hiv treatment and only one got svr. cv remained undetectable and cd slightly increased after the treatment. patients received ledipasvir and sofosbuvir, patients had genotype a, patients genotype b and patient genotype . patients had f fibrosis and had f . patients had received previous treatment (naïve). the hiv treatment was modified only in one of the patients, patients achieved svr. cv increase in patients after the treatment while cd followed the trend of increasing. conclusion: the aad that caused fewer changes in the hiv treatment were ombitasvir/paritaprevir/ritonavir and dasabuvir followed by ledipasvir/sofosbuvir. sofosbuvir and daclatasvir present a greater number of interactions with hiv drugs so they behaved to a major change. more patients are needed to assess more accurately the aad leading to a minor modification. please specify your abstract type: research abstract background and objective: the simplification strategies reduce the amount of tablets and the toxicity in order to facilitate adherence in patients with virological suppression. the strategy more studied is monotherapy with a ritonavir-boosted protease inhibitors (pi/r). to analyse the effectiveness of monotherapy with pi/r in pre-treated patients infected with hiv. setting and method: retrospective observational study. selected hiv patients treated with pi/r monotherapy at any time of pharmacotherapeutic history to / / , with at least one clinical and analytical control months before the beginning. data were collected from the medical record archinet Ò and outpatient farmatools Ò program. variables included were age, sex, duration of monotherapy, virological failure, treatment failure, cd % during monotherapy. main outcome measures: inclusion criteria: virological suppression for year prior to the start of monotherapy, no previous ip virological failure, high cd count ([ cell/ml) and a high level of drug adherence. the effectiveness is defined as the percentage of patients without virological failure ( consecutive plasma viral load (vl) [ copies/ml) and without treatment failure (any event causing retirement monotherapy). results: patients with monotherapy, which represent % of patients with antiretroviral therapy (art) at our institution were identified. were excluded ( co-infected with hepatitis virus, with insufficient data and no had more than months included), including patients in the analysis, with a mean age of years and % were men. the median of time monotherapy treatment was . years ( . days), ( . %) patients received darunavir/r and ( . %) lopinavir/r. the effectiveness of monotherapy treatment during the follow up period was % with undetectable pvl at follow-up. the median of cd % over the treatment time was cell/ml ( %). conclusion: the effectiveness of treatment with ip/r monotherapy in our hospital obtained good results. according with our results treatment adherence plays a very important role. this is a current and valid strategy that brings benefits to the patient and to the healthcare system. please specify your abstract type: research abstract background and objective: the access to investigational drugs for patients who are not included in a clinical trial and without authorized therapeutic alternatives is known as compassionate use. the incorporation of the evidence-based medicine in the area of oncohaematology has implied that an important part of clinic therapy validated by evidence that could not be controlled from an administrative point of view. this is due to the continuous and progressive development of investigation and information on cancer treatment and the delay of the administration regulation. the use of drugs in this way is regulated by royal decree / ( / ). the objective of the study is to describe the use of cancer drugs through compassionate use in the last years in a specialty hospital. setting and method: descriptive retrospective study on a specialty hospital. all the applications for a compassionate use drugs were analysed from january until october . the data were obtained from medical records programme diraya Ò and from an excel database of medicines in compassionate use of the pharmacy service. main outcome measures: the following variables were registered: • number of patient clinic history • authorized medicine • authorization date • applicant service results: we recorded requests of cancer drugs in compassionate use during the years of study. oncology was the service that recorded more authorizations with %, followed with gynaecology with . % and finally endocrinology and haematology with . %. drugs of the requests were approved ( %) and unauthorized ( %) in the years of study. the year in which more applications were received was ( . %) and the least requests were received in ( . %), being the year where all requests were authorized. in fewer applications were authorized, %. in the years , and were authorized . , and . %, respectively. a total of different active drugs were received during the study, the most requested bevacizumab ( %) for grade iii oligoastrocytoma, ovarian cancer (monotherapy), metastatic gall bladder cancer and metastatic platinum-resistant ovarian cancer, everolimus ( %) for indications of neuroendocrine carcinoid tumour and metastatic breast cancer, nab-paclitaxel ( %) for invasive lobular carcinoma indications of high-grade and metastatic pancreatic cancer, ipilimumab ( %) for the indication of metastatic melanoma, and regorafenib for indications of colorectal cancer and metastatic gist i pre-treat with imatinib ( %). the solicitude of drugs through compassionate use needs effective commissions of pharmacy and therapeutics, along with the medical management to establish an agile and faster requesting circuit and the consequent use monitoring. please specify your abstract type: research abstract background and objective: to describe the standard procedure for the elaboration and control of a magistral formula (mf) to assess their effectiveness in two patients with cutaneous metastases of malignant melanoma refractory to other treatment. setting and method: medication for compassionate use was requested for two patients of and years with histopathologic diagnosis of cutaneous metastases of malignant melanoma in the left thigh and left heel in which the lack of response to first-line treatments made to be valued to start with adesleukina intralesional therapy. the first week was infiltrated mu ( ml) in lesions less than cm, mu ( ml) in the larger lesions and repeating each week until complete remission of the lesions. in the nd patient we proceed in the same way but the second week was infiltrated mu ( ml). the following week, infiltrated mml, in metastases and we turn to weekly infiltrations. the response was assessed by clinical disappearance of the lesions treated. complete response (cr) is defined as a clinical disappearance of lesions and partial response (pr) greater than % reduction of the lesion diameter. main outcome measures: we performed a literature search (pubmed, trissel, spc) for all studies published to determine the standard procedure for preparing and monitoring the mf (processing, preservation, stability, dose and indication). results: the standard procedure of preparation and quality control was carried out following the rules established in rd / . it was made in a vertical laminar flow cabinet. the aldesleukin vial was reconstituted with . ml api ( mu/ml) and then diluted with . ml of a solution of . % albumin, % glucose as stabilizer, to avoid aggregate formation, preparing ml syringes ( mu/ml). it was obtained a homogeneous and clear solution without precipitate or opalescence appearance. stable days in a refrigerator ( - °c), protected from light. initially patients had approximately a total of injuries. after months of treatment it was obtained a cr of most lesions in the first patient and rp of the second patient injuries. treatment was well tolerated. the side effects presented were only a flu-like syndrome in the second patient. conclusion: intralesional administration aldeslukina has been effective in treating malignant melanoma skin metastases in our patients, allowing the extension of its use in patients with the same involvement refractory to other primary treatments. the results are similar to those of the publications consulted. please specify your abstract type: research abstract background and objective: chronic infection with hepatitis virus c (hcv) affects about million people worldwide and is a leading cause of liver cirrhosis and hepatocellular carcinoma. the new direct acting antivirals against hcv have revolutionized the treatment of this disease. due to the high cost of these drugs it is necessary to assess their use in clinical practice. to evaluate the effectiveness of daclatasvir in combination with sofosbuvir in patients with hcv monoinfected in a specialty hospital. setting and method: retrospective observational study of patients who began treatment with the combination of daclatasvir and sofosbuvir from january to january in a specialty hospital. the data were collected from the hospital program of clinical stories archinet Ò and the outpatient program farmatools Ò . the results were analysed using the statistical program r-commander. main outcome measures: the sustained virologic response (svr) was considered the primary endpoint of the study. as secondary variables were analysed: sex, duration of treatment, naïve patients or pre-treated, degree of fibrosis, hcv genotype, concomitant use with ribavirin, viral load (vl) before treatment and medical service. results: there were included patients of whom were men. baseline characteristics were: patients with genotype , genotype b, with genotype a and genotype . the degree of fibrosis in the study was patients with f , f and to f . among the patients infected with hcv genotype , had not received prior treatment (naïve) and had failed therapy. the duration of the treatment was weeks to patients and weeks for patients. only patients receiving ribavirin of these had genotype and genotype b. from ribavirin patients it was greater the number of patients in whom the treatment duration was weeks ( patients versus with p-value = . ). the digestive service attended to patients while patients were followed by infectious. the median cv was , , iu/ml. svr was achieved in . % of patients with hcv genotype in . % with genotype b and % with genotype and a. after weeks of treatment % of patients achieved svr and % after weeks. only one patient died during treatment. the results are similar to those obtained in clinical trials. svr has not been influenced by hcv subtype, duration of treatment, degree of fibrosis, pre-treatment or by concomitant use of ribavirin. further studies are needed to evaluate the efficacy of this treatment. please specify your abstract type: research abstract background and objective: the safety and efficacy of medications can vary significantly between patients as a result of genetic variability. as genomic screening technologies become more widely available, pharmacists are ideally suited to utilize this tool to optimize medication management. the objective of this study is to evaluate the feasibility of implementing personalized medication services into community pharmacy practice and to assess the number of drug therapy problems identified as a result of pharmacogenomic screening. setting and method: the study was designed as open-label, nonrandomized, and observational. two community pharmacies in toronto, ontario offered pharmacogenomic screening as part of their professional services program. prior to initiation, participating pharmacists received structured, comprehensive training in pharmacogenetics. pharmacists then facilitated voluntary subject enrolment among patients who they believed would benefit from screening and met inclusion criteria. eligible patients received a simple buccal swab followed by dna analysis using pillcheck Ò . pillcheck Ò is a genotyping assay that translates genomic data and generates a personalized, evidence-based, report that provides insight into patients' inherited drug metabolic profile. upon receiving the report, pharmacists invited patients back to the clinic for interpretation of the results. clinically significant drug therapy problems were identified and recommendations for medication optimization were forwarded to the primary care physician. main outcome measures: number of clinically significant drug therapy problems identified by pharmacists as a result of pharmacogenomic testing. results: patients were enrolled in the study. average age was . years and patients were taking a mean of . chronic medications. pharmacists cited the most common reasons for testing as ineffective therapy ( . %), to address an adverse reaction ( . %), and to guide initiation of therapy ( . %). an average of . drug therapy problems were identified per patient. pharmacist recommendations included change in therapy ( . %), dose adjustment ( . %), discontinuation of a drug ( . %), and increased monitoring ( . %). generally, physician feedback was positive but did reveal an opportunity for a broader understanding of the technology. conclusion: these results highlight the readiness of community pharmacists to adopt pharmacogenetic screening into practice and their ability to leverage this novel technology to positively impact medication management. community pharmacists are ideally suited to both offer personalized medication services and interpret genomic results. please specify your abstract type: descriptive abstract (for projects) background and objective: visual impairment is a common geriatric syndrome and glaucoma/miotic eye drops treatment is a frequent therapeutic option. pharmacist's role in medication reconciliation is an effective process for reducing medication errors and supporting safe medication use. we observed that mentioned medication reconciliation was occasionally not performed during hospital stay and could be cause of delirium because of visual impairment. the aim of this study was to evaluate the influence of omission errors of eye drops treatment on incidence of acute confusional state. design: we conducted an observational, descriptive and retrospective study in an orthogeriatric unit with an average of patients with hip fractures per year ( % surgically treated). data collection was performed from june to march . reconciling medications at admission was performed by implementing the tools and resources of the canadian patient safety institute (cpsi). we extracted from our electronic database (filemaker pro Ò ): • demographic patient data (age and gender). • name and posology of the glaucoma/miotic eye drops treatment. • medication reconciliation performed and identification of professional in charge (pharmacist, geriatrician or orthopaedic surgeon) registration during hospital stay. • protocolar management of delirium with tiapride occasional intramuscular administration performed if necessary was also registered to establish the incidence of acute confusional state. results: thirty-two patients ( women and men) were included, median age year-old . in patients, eye drops reconciliation treatment was performed by the pharmacist in of the patients, the geriatrician in cases and the orthopaedic surgeon in . in patients, the mentioned medication reconciliation was not performed (pharmacist absentism). considering the patients on eye drops treatment during hospital stay, ( . %) of them suffer from acute confusional state. on the other hand, among the patients without medication reconciliation, delirium was registered in cases ( . %). concerning ocular topic treatment, . ± . active principles per patient were observed, being the most frequent timolol ( . %), brinzolamide ( . %) and latanoprost ( . %). conclusion: we consider of paramount importance the pharmacist evaluation availability at an orthogeriatric unit, minimizing the impact of acute confusional state during hospital stay by medication reconciliation. please specify your abstract type: descriptive abstract (for projects) background and objective: to report the therapeutic management of haemorrhagic rectocolitis onset in a lung-transplanted patient with mycophenolate-induced diarrhoea. design: case report. results: a -year-old-man lung transplant patient for alpha -antitrypsin deficiency in receiving mycophenolate mofetil, tacrolimus and corticosteroid developed chronic diarrhoea worsened by sigmoid and cecal necrosis in , and treated successfully by sigmoidectomy. severe diarrhoea attributed to mycophenolate mofetil reappeared in april , which motivated a switch to mycophenolate sodium. the absence of clinical improvement in june led to stop mycophenolate sodium and introduce azathioprine at mg/day (absence of mutation for the thiopurine methyl transferase gene). one month later, the patient presented melena, diarrhoea, bloating, nausea, and knee pain, attributed to azathioprine. this latter was stopped and mycophenolate mofetil was rechallenged associated with symptomatic treatment (i.e., diosmectite and loperamide). in january , a colonoscopy, performed in a context of profuse chronic diarrhoea with mucus during months, highlighted haemorrhagic rectocolitis. therefore, the patient initiated sulfazalasine therapy with no clinical improvement, and then high doses of oral corticosteroids. because high-dose of oral corticosteroids was not recommended as a long-term treatment, mercaptopurine was proposed as a new therapeutic option. mercaptopurine has no indication as an immunosuppressive treatment in solid organ post-transplant supportive care. however, as the active metabolite of azathioprin, an immunosuppressive drug widely used in transplantation, mercaptopurine has immunosuppressive functions towards t-lymphocytes. after multiprofessional collaboration between gastroenterology, pneumology and pharmacy specialists, it was decided to stop mycophenolate mofetil and introduce mecaptopurine at . mg/kg/day, as immunosuppressant for haemorrhagic rectocolitis as well as lung transplantation. this unusual lung transplant immunosuppressive therapy, associated with tacrolimus, improved digestive disorders and patient's quality of life. currently, mercaptopurine is biologically and clinically well tolerated. the dosage of blood residual concentrations of purinethol metabolites ( -thioguanine and -methylmercaptopurine) is going to be performed. conclusion: immunosuppressive therapy in solid organ transplantation is a real challenge for patients who have comorbidity onset. despite a lack of data in the literature, a multidisciplinary collaboration based on comprehensive pharmacology skills is essential to choose the best therapeutic option in this type of patients. please specify your abstract type: descriptive abstract (for projects) background and objective: the use of complementary medicines (cm) in oncology is the subject of broad but still controversial interest. a large part of patients with cancer uses cm, including complementary drugs, during their treatment period. indeed, according to different studies, this proportion ranges from to %. importantly, the risk of interaction between cm and anti-cancer drugs is not negligible; hence we need to identify these cm to ensure the security of our patients and the success of their treatment. design: to achieve this purpose, a monocentric retrospective analysis was conducted with collection of data by pharmacy students during medication reconciliation of hospitalized patients from january to june . collected data are patients' characteristics, prevalence of cm use and potential cm-anticancer drug interactions. results: patients were included in the study ( men- women); median age was [ - years]. a total of . % (n = ) were using a least one cm, most frequently homeopathy ( %, n = ) or phytotherapy ( %, n = ); some patients were using a combination of two cm ( %, n = ). cm are mainly used by women in comparison to men ( . % versus . % and p = . , chi square test). for phytotherapy, at least different herbs were described by patients and among them the most frequently used were mistletoe (viscum album), propolis and fireweed (epilobium angustifolium). data analysis showed that % (n = ) of patients were at risk of potential cm-anticancer drug interaction. moreover this risk was increased to % if we considered only patients taking phytotherapy. interactions included pharmacokinetic ( %, n = ), such as altered hepatic metabolism, and pharmacodynamics ones ( %, n = ). conclusion: in conclusion, our work clearly demonstrates that the use of cm by patients is associated with high risk of relevant drug interaction with their anti-cancer treatment. even if further investigations are necessary to clarify the clinical impact of these interactions, the use of cm must be considered during prescribing process. please specify your abstract type: research abstract background and objective: since their reimbursement, the direct oral anticoagulants (doacs) are increasingly used for stroke prevention in atrial fibrillation (af). the objective of this study was to identify the proportion of real life patients with af eligible for doac therapy, based on the inclusion and exclusion criteria used in the clinical studies and based on the officially approved indications as mentioned in the summary of product characteristics (smpc). setting and method: data for this retrospective cross-sectional study was extracted from the uz brussel stroke registry, containing anonymized data of patients with a suspected stroke. characteristics of patients with documented af were compared with the patient characteristics in clinical trials and the approved indications in the smpc. main outcome measures: proportion of real life patients with af eligible for doac therapy. results: data of patients with af was analysed. based on the selection criteria of the clinical trials, significantly less patients were eligible for treatment with rivaroxaban compared to dabigatran etexilate ( . % versus . %; p = . ), but not compared to apixaban ( . %; p = . ). based on the indications and contraindications in the smpc, significantly fewer patients were eligible for apixaban compared to dabigatran etexilate and rivaroxaban ( . % for apixaban, . % for dabigatran etexilate and . % for rivaroxaban; p \ . and p \ . , respectively). significantly more patients were eligible for doac therapy based on the indications and contraindications in the smpc compared to the inclusion and exclusion criteria of the clinical trials ( . % versus . %; p \ . for dabigatran; . % versus . %; p \ . for rivaroxaban and . % versus . %; p \ . for apixaban). conclusion: when taking into account the selection criteria from the pivotal clinical trials with doacs for stroke prevention in af, less than half of real life patients are eligible for therapy with one of the doacs. however, the indications mentioned in the smpcs of these drugs are less strict. please specify your abstract type: research abstract background and objective: idiopathic pulmonary fibrosis (ipf) is a disease in which tissue deep in the lungs becomes thick and stiff, or scarred, over time. the formation of scar tissue is called fibrosis. pirfenidone is an anti-fibrotic and anti-inflammatory agent, thus offers a new hope for treating progressive fibrotic diseases. int j clin pharm ( ) : - our objective is to set a description of idiopathic pulmonary fibrosis patients treated with pirfenidone, as well as the adverse reactions observed. setting and method: descriptive study in which all patients have received pirfenidone. the data were obtained through the dispensing program of outpatient (farmatools) and review of medical records of the hospital database (archinet) and clinical station (diraya). main outcome measures: we have extracted from each patient baseline data, comorbidities, dose received, reported adverse reactions and data about haematology and biochemistry. results: we have a total amount of patients treated with pirfenidone, all diagnosed with idiopathic pulmonary fibrosis, including women and men. the age of patients is between and years, with an average of . years. all patients are ex-smokers and one of them is also ex-alcoholic. concerning concomitant pathologies, patients have diabetes mellitus, have arterial hypertension, and one of them has ischemic heart disease. another has upper gastrointestinal bleeding prior, among others chronic pathologies. pirfenidone dose received was the usual dose in of the patients: days - mg every h, days - mg every h and a maintenance dose of mg every h. in one patient due to its low imc the dose received was smaller ( - days mg every h, days - mg every h and maintenance dose of mg every h). in relation with the adverse effects, digestive discomfort were observed in of the patients, causing the interruption of the treatment in one of them (with prior gastrointestinal bleeding). in the other patient it was relieved by lowering the dose received. also, one patient has experienced photosensitivity. alterations in transaminase levels were observed in patients but that didn't force to discontinue the treatment. no alterations were observed in the blood count. conclusion: treatment with pirfenidone is being generally well tolerated by patients. it has improved their life-quality and reached the objective data of a slowdown in disease progression. currently, the number of patients is no enough to give conclusive information in relation to the drug effectiveness. please specify your abstract type: research abstract background and objective: to describe the total amount of patients treated with a magistral formula of sodium cromoglycate mg without excipients: indications, concomitant therapy and the response to therapy. setting and method: we run a descriptive study in which we included the totality of patients in treatment with a magistral formula of sodium cromoglycate mg without excipients in a tertiary hospital. the data were obtained through paracelso (development of magistral formulas program), as well as with farmatools (dispensation program of outpatient) and the review of medical records from the hospital database (archinet), and diraya clinical station. main outcome measures: from each patient we extracted data relative to sex, age, diagnosis, time in treatment with the formula, dose received, response to therapy, concomitant antihistamines treatments and adverse effects. results: a total of patients in treatment with a magistral formula of sodium cromoglycate mg without excipients were reviewed: women and men with a mean age of . years old (range - years). regarding the indication of the prescription, patients have been diagnosed of indolent systemic mastocytosis and the remaining were diagnosed of mast cell activation syndrome. in all cases, the diagnosis was established by examination of the bone marrow in the mastocytosis studies institute of castilla la mancha (spain). on average, patients took the treatment . months, with a range between months and months. the dose received was mg every h in patients, having to be increased to mg times daily in a case with poor response to the therapy. in the remaining patients, the treatment response has been optimal. in relation to the concomitant anti-allergic treatment received, patients took fexofenadine daily during the study. no cases of adverse effects related to the therapy received have been reported. conclusion: both indolent systemic mastocytosis and mast cell activation syndrome are considered rare diseases, and we should indicate that in spain there are no commercial medicines available of sodium cromoglycate without excipients for its treatment. the treatment with this magistral formula of sodium cromoglycate mg without excipients has been effective and well tolerated in all patients, improving the symptoms associated with their condition as well as their quality of life, and also, assuming a solution to the lack of marketing of the drug currently in spain. please specify your abstract type: research abstract background and objective: to analyse the prescription profile, safety and effectiveness of new therapies available for the treatment of hcv genotype b in a tertiary hospital. setting and method: we run a retrospective observational study in which we included a total amount of patients infected with hcv genotype b treated with the new therapies against hcv from february to december in a tertiary hospital. the data were obtained through the outpatient dispensing program farmatools and the review of the medical records from the hospital database, archinet and prescription hepatitis c portal of the andalusian health service. main outcome measures: from each patient the following information was collected: sex, age, viral genotype (gen.), naive/nonnaive, hiv coinfection, presence of cirrhosis, degree of hepatic fibrosis measured by fibroscan, treatment prescribed and duration, adverse effects, sustained viral response (svr) and the service that made the prescription. results: a totality of patients with hcv gen. b were reviewed which . % of them were men with a mean age of . years (range - years). of the patients were naive and only of them were hiv co-infected, there were a . % of cirrhotic patients. regarding the degree of hepatic fibrosis, patients had grade f , f grade patients, patients grade f and f grade patients. the most commonly therapy prescribed was lepidasvir + sofosbuvir in patients ( without ribavirin and with ribavirin ) using a treatment schedule of weeks in of them. the treatment was discontinued in one case because of the adverse effects, achieving svr in the remaining patients. the combo treatment with paritaprevir/ombitasvir/r + dasabuvir was prescribed in times ( without ribavirin and with ribavirin) choosing only in one of them for a treatment period of weeks. there were no treatment discontinuations and svr was achieved in all patients treated in this way. patients received simeprevir + sofosbuvir for weeks ( without ribavirin and with ribavirin), one patient of the left the treatment due to adverse effects. svr was found in the remaining patients who completed treatment. sofosbuvir + daclatasvir was prescribed to patients, associating ribavirin in only one case. a treatment duration of weeks was used in patients and weeks in the remaining two. one patient failed rvs without any incidences of adverse effects in any case. interferon + ribavirin sofosbuvir + was prescribed to patients in -week regimen which was well tolerated achieving svr. digestivo service treated the % of the total amount of patients. conclusion: new therapies for hcv have been used in all the treated patients and the older drugs have been relegated. about the effectiveness, svr was achieved in . % of patients. regarding the safety, only patients have discontinued the treatment due to adverse effects representing less than % dropout rate of the therapy. please specify your abstract type: descriptive abstract (for projects) background and objective: thanks to pharmacogenetics we can identify and predict different responses to the same drug among different individuals. during these last years we have noted a big increase of dosing guidelines and advices about the use of several drugs due to the influence of different polymorphisms. the aim of this study is to describe and evaluate the use of pharmacogenetics in our hospital from april , when we started our first research about pharmacogenetics, to the actual time, using these information in our daily clinical practice; and indeed quantify the number of different tests and the number of different clinical advices done because of pharmacogenetic information, by different healthcare specialty areas and drugs. design: we reviewed all the pharmacogenetic test requests in our hospital from april to april , noting which health specialty and for which drug was asked the test. polymorphisms were genotyped using taqman Ò genotyping assays technology by independent laboratories to confirm the results. results: from april we were asked for pharmacogenetic tests from different healthcare specialty areas: rheumatology ( . %), infectious diseases ( . %), oncology ( . %), cardiology ( . %), vascular surgery ( . %), neurology ( . %), ophthalmology ( . %); this information was asked about different drugs: clopidogrel ( . %), trastuzumab ( . %), ranibizumab ( . %), azathioprine ( . %) and tocilizumab ( . %). from all the genotypes, ( . %) were done after using the drug (study phase) and ( . %) were done previous to the use of the drug in daily clinical practice to make a ''clinical recommendation''; from these recommendations affected to the prescription of clopidogrel. conclusion: during the last years we could implement the use of pharmacogenetics in the daily clinical practice in our hospital in different healthcare areas affecting drugs and we started research studies previous to its use on the clinical practice for other three different drugs. please specify your abstract type: descriptive abstract (for projects) background and objective: the drug burden index (dbi) is a tool used to quantify the anticholinergic and sedative burden of medication on an individual. it has been independently associated with poor physical and cognitive performance in community-dwelling older people. objectives were: to create an inventory of medications used in ireland with clinically significant anticholinergic and/or sedative activity and to decide upon the minimum daily dose (mdd) for each medication. design: medications with potential anticholinergic and/or sedative burden were identified by literature review and examination of the summary of product characteristics (smpc) for all medications registered in ireland. each medicine was classified as anticholinergic or sedative. drugs with both anticholinergic and sedative properties were classified as primarily anticholinergic. the mdd, a key component of the dbi score calculation, was selected by reference to the irish smpc. other options which were also considered for this value include the defined daily dose (ddd) of a medication, as available from the world health organisation (who), and the mdd as outlined in the british national formulary (bnf). mdds were decided upon regardless of indication as the lowest effective therapeutic dose as specified in the smpc for the medication. the final list of medicines and mdds to be included in the inventory was then defined by consensus of three pharmacists. results: in total, medicines with potential anticholinergic and/or sedative activity were considered for inclusion. a final list of medications was identified by consensus ( anticholinergic, sedative). of these, ( %) were agents which act primarily on the nervous system. the three main therapeutic groups contributing to the inventory of dbi medications were antipsychotics ( medications), antidepressants ( medications) and antiepileptics ( medications). conclusion: creation of an inventory of medications with anticholinergic and/or sedative properties, in combination with the individual mdds, was achieved. this is a useful resource for use in analysis of drug burden in an older population. it could help in both identifying patients who would benefit from medication review as well as analysing population medication data. please specify your abstract type: research abstract background and objective: vancomycin is an antibiotic widely used to treat infections such as bacteraemia, infective endocarditis, osteomyelitis, meningitis and pneumonia. nowadays, optimal trough concentration is stablished between and mg/l to avoid development of resistance or - mg/l to improve penetration in complicated infections. some articles have been published explaining the methodology to calculate an expected trough level in steady state. our aim was to compare the trough serum value estimated by the mathematical method with a two-compartimental bayesian forecasting model. setting and method: observational retrospective study carried out in a tertiary hospital from january to december . non obese adult patients with creatinine clearance (crcl) \ ml/min and who have achieved steady state level were included. vancomycin serum values were measured using a chemiluminescence's immunoassay (cmia) and bayesian analysis was performed with abbottbase pksystem Ò (pks Ò ). the statistical analysis was made with medcalc software Ò . bland-altman plot and passing-bablok regression were used to compare both methods. main outcome measures: sex, age, weight, dose, creatinine, and size were collected from clinical history. serum trough values (cminr) were collected from cmia. trough values were estimated using two methods: mathematical method (cminf) and bayesian calculations (cminb). results: patients were included, with a mean age of (± . ) years. % were male and % female. they received a median dose per h of ( - ) mg. the mean of cminr was . mg/l ( % ci . - . ), cminb . mg/l ( % ci . - . ), cminf . ( % ci . - . ) . correlation coefficients (r) comparing both methods were significantly different: r between cminf and cminr was . ( % ci . - . ), while r between cminb and cminr was higher: . ( % . - . ). bland-alman plot analysis showed both methods cannot be used interchangeably. the regression equations estimated by passing-bablok regression were y = - . + . x and y = - . + . x. conclusion: bayesian method has demonstrated better correlation with real measures than mathematical method. most part of our patients could be underestimated or overestimated using mathematical methods which could cause toxicity or lack of efficacy, so this method is unsuitable for clinical use. bayesian estimation remains the best option for optimal dosing of vancomycin. please specify your abstract type: research abstract background and objective: combination therapy with digoxin and acenocoumarol is common in patients with atrial fibrillation (af). getting optimal concentrations of digoxin leads an appropriate response; taking into account its narrow therapeutic range and all the factors which can affect to its pharmacokinetics. interaction between them has been studied, even though its mechanism is not clear yet. patients who are taking both drugs need higher doses of digoxin; because they get lower concentrations by using the same dosage. the objective of this study was to analyse digoxin concentrations in patients treated with this combination compared to expected concentrations according to population parameters. setting and method: retrospective observational study from december to march performed by pharmacokinetic unit. patients included had chronic treatment with acenocoumarol and digoxin, which determination were realized in the steady state before the next dosage. patients with toxics concentrations of digoxin, or who were suspected nonadherence, were excluded. the plasma digoxin concentrations were determined through the autoanalyzer architect c- Ò (petinia). dosage adjustment was realized by the program abbot pharmacokinetics system (pks). a comparative between the real measured concentrations in patients and estimated concentrations were realized based on population parameters. finally, in order to get optimal concentrations, some dosage changes were proposed based on pharmacokinetic monitoring. data collected: population characteristics (gender, age, weight, and height), analytical data (potassium, urea, creatinine and clearance). main outcome measures: digoxin serum concentrations (optimal range . - ng/ml). results: data from patients, . % women with a mean (sd) age of . ( . ) years were included in the study. at baseline, potassium, urea, creatinine and clearance mean (sd) was . ( . ) mmol/l; . ( . ) mg/dl; . ( . ) mg/dl; . ( . ) ml/min. . % of the patients had lower concentrations than expected according to population parameters. finally, digoxin dosage was increased in . % of patients, it was maintained in . %, and it was decreased in . %. conclusion: digoxin concentrations in patients with af in combination therapy of digoxin and acenocoumarol are lower than would be expected in most cases. it is important monitoring digoxinaemia to achieve optimal concentrations and a good clinical response. further studies are needed to determine the relevance of this interaction in clinical practice. please specify your abstract type: research abstract background and objective: tocilizumab (tcz) is a humanized monoclonal antibody inhibitor of il- receptor, indicated in combination with methotrexate in the treatment of rheumatoid arthritis (ra) in patients with inadequate response or intolerance to prior therapy. interleukin is involved in the pathogenesis of rheumatoid arthritis via its broad effects on immune and inflammatory responses. previous studies have shown that c-allele at the - g[c (rs ) polymorphism is related with a bad response to tocilizumab (according to eular criteria). the aim of our study was to explore the potential role of il- genetic polymorphisms as a predictor of tocilizumab efficacy in rheumatoid arthritis (ra) patients and check this association depending on the genotype. setting and method: the il- (g[c) (rs ) genetic variant was genotyped using predesigned taqman Ò genotyping assays technology and analysed on a viia Ò real-time pcr system. main outcome measures: clinical response was evaluated at , , and months according to the eular criteria. patients were classified as ''responders'' (good and moderate response according to eular criteria) and ''non-responders''. the statistical analysis was performed using spss v. . results: we recruited patients with ra treated with tocilizumab, these were aged . ± . (mean ± sd), ( %) were women. the mean das at baseline was . ± . . of these patients, the il- g[c genetic polymorphism was significantly associated with ''responders'' at months after the baseline (cc vs non-cc p = . , or . , % ci . - . ) but not at (p = . ), (p = . ) and (p = . ) months. conclusion: the il- g[c may be useful as a genetic marker of tocilizumab efficacy at months. other polymorphisms, clinical parameters and other pharmacological treatment during the follow-up may be checked about their influence on the response to tocilizumab. tdmp : daptomycin pk/pd profile in neutropenic cancer patients with beta-lactam-resistant gram-positive infection nancy perrottet *, , frederic tissot , laurent decosterd , thierry buclin , guy prod'hom , christina orasch , oscar marchetti , farshid sadeghipour , , thierry calandra , véronique erard pharmacy service, infectious diseases service, laboratory and division of clinical pharmacology, service of biomedicine, institute of microbiology, lausanne university hospital, lausanne, school of pharmaceutical sciences, university of geneva, university of lausanne, geneva, switzerland please specify your abstract type: research abstract background and objective: the pharmacokinetics (pk) and pharmacodynamics (pd) of many antibiotics are modified in neutropenic patients and few data are available on daptomycin in this population. this prospective study aimed to assess the pk/pd profile of daptomycin in the treatment of neutropenic patients with beta-lactamresistant gram-positive cocci infections. setting and method: this substudy was performed in the context of a prospective pilot study on daptomycin versus vancomycin in adult hemato-oncological patients with febrile neutropenia and proven or suspected infection with methicillin-resistant staphylococci or betalactam-resistant enterococci. patients received daptomycin mg/ kg/day ( mg/kg/day for enterococci) for c days as a -min infusion. main outcome measures: pk analysis using a published non-linear mixed effect model with nonmem Ò , followed by comparison of parameters with values published for healthy subjects. pd analysis based on auc/mic (area under the concentration-time curve/minimal inhibitory concentration). according to eucast, an auc/mic ratio [ is required for bacteriostatic effect against staphylococci and [ for a two-log reduction in bacterial count. for e. faecium, an auc/mic ratio of . has been suggested for bacteriostasis and . for a -log bacterial count reduction. results: model-derived mean auc observed in patients was . ± . mg h/l, maximum concentration (cmax) ± mg/ l, minimal concentration (cmin) . ± . mg/l. clearance was . ± . l/h and volume of distribution at steady sate . ± . l, both values found higher than those reported in healthy subjects. all patients ( / ) with a staphylococcal infection achieved auc/mic values predictive of bacteriostatic effect on staphylococci, and out of values associated with two-log bacterial killing. of note, infection relapse occurred in the only patient with suboptimal daptomycin exposure (auc/mic of ). the pd targets were also reached in the two patients with e. faecium infection. an asymptomatic elevation of creatine phosphokinase was reported in two patients ( u/l and u/l) with cmin of . and . mg/l, respectively. conclusion: daptomycin pk profile in neutropenic cancer patients indicated higher total clearance and volume of distribution, along with lower total exposure, compared to healthy subjects. despite this, standard dosages allowed attainment of pd targets in / patients with a staphylococcal infection (two-log drop) and / with e. faecium infection ( -log drop) . please specify your abstract type: research abstract background and objective: individual clinical response to infliximab can be influenced by their pharmacokinetics and immunogenicity, so therapeutic monitoring of drug levels (tdm) can guide these biologic treatments. the objective was to analyse the suitability of serum infliximab trough levels (sitls) in patients with inflammatory bowel diseases (ibd) receiving dose schemes based only on clinical response. setting and method: prospective and descriptive study of patients with ibd treated with infliximab and under tdm. medical records were reviewed. dose schemes were established according to clinical guidelines ( mg/kg every weeks) and optimized based on an index of clinical response (mayo, pcr…). sitls (therapeutic range - mcg/ml) and anti-drug antibodies (ada) were measured in all of patients by elisa (promonitor Ò ). ada presence was considered as a therapeutic failure indicator. informed voluntary consent was obtained from all patients. main outcome measures: sitls and ada. results: a total of patients, with a median age of years (range ), were included in the analysis. infliximab standard dose according to clinical guidelines were administered to patients: . % showed sitls under the therapeutic range ( . % with ada). in eight patients with maintained good clinical response, dose decrease or interval elongation had been implemented: % of these patients showed sitls below the therapeutic range ( % with ada). it had been necessary to increase the dose or shorten the interval in patients due to inadequate clinical response: . % of these patients with sitls below the therapeutic range ( % with ada). conclusion: optimization based on clinical response of infliximab treatments in patients with ibd is not always an effective strategy, since it leads to a high percentage of patients with sitls below the therapeutic range and adas. tdm together with clinical response should guide the optimization of infliximab treatments. please specify your abstract type: research abstract background and objective: in addition to its anticonvulsive properties, valproate is also used as a mood stabiliser in bipolar disorder and as augmentation treatment of other psychiatric disorders. the unpredictable relationship between dose-plasma valproate concentrations and correlation between concentrations-efficacy suggest therapeutic drug monitoring (tdm) of plasma valproate concentrations might be useful. the aim of our study was to evaluate the rationale of a new protocol for measuring valproate concentrations and the incorporation of a clinical pharmacist in the process of valproate tdm service, compared to pre-existing standard measuring. setting and method: in the retrospective study we analysed the process of measuring plasma valproate concentrations at the department of psychiatry and at the unit for forensic psychiatry of a large teaching hospital in slovenia before the enrolment of a clinical pharmacist. for the prospective study we created a protocol for tdm of valproate in adults based on literature research. the protocol included reference range, sampling time, indications for sampling and schedule of other laboratory tests that have to be monitored during valproate therapy. main outcome measures: percentage of plasma valproate concentrations in reference range (c trough = - mg/l) before/after the enrolment of a clinical pharmacist, percentage of measured valproate c trough . results: in the retrospective study randomly chosen patients with measured plasma valproate concentrations were included ( % male, age ± years, length of hospital stay ± days). plasma valproate concentrations were measured . ± . times per patient, % were in the reference range (other % subtherapeutic), % were drawn at c though , . % were drawn for assessing compliance (nontrough). in the prospective study patients were included ( % male, age ± years, length of hospital stay ± days). plasma valproate concentrations were measured . ± . times per patient, % were in the reference range (other subtherapeutic), % were drawn at c trough , . % were drawn for assessing compliance. conclusion: the inclusion of a clinical pharmacist in valproate tdm service increased the number of valproate plasma concentrations in the reference range by almost % and increased the number of concentrations drawn at c trough , when indicated. including a clinical pharmacist in valproate tdm is beneficial and the new protocol is useful for optimising valproate therapy. concurrent and predictive validity of a self-reported measure of medication adherence the effect of pharmacist-led interventions in optimising prescribing in older adults in primary care: a systematic review aflibercept: . neovascular membranes with visual acuity higher than . . one eye affection severe cardiovascular pathology (severe episodes in the last months) non-responders to other anti-vegf bevacizumab: . diabetic macular edema macular edema secondary to vascular pathology setting and method: a longitudinal study was carried out in primary care centres. participants: patients aged c , under treatment with or more drugs and belonging to primary care areas in different towns. patients should have at least one of the following potential safety problems: (a) concomitant use of a non-steroidal anti-inflammatory drug (nsaid) with an antihypertensive drug, anticoagulant or antithrombotic drug; (b) use of two or more benzodiazepines. two clinical management units (cmu) were randomized per area to be included in the study. thirty patients per cmu were randomized to be enrolled and monitored during months number of adverse effects ( . ; p \ . ) and number of clinical problems ( . ; p \ . ).with each year increase in age ) and a significant rise in physician ( . ; p \ . ) and nurse ( . ; p \ . ) home visits. women compared to men resulted in a significant decrease ) but a significant increase in visits to nurses ( . ; p \ . ), hospital admissions ( . ; p \ . ) and hospital visits ( . ; p \ . ). age, sex and npsp had no significant effect on falls, fractures or cardiovascular events. conclusion: the npsp in elderly patients contributes to an increase in the use of health services and comorbidity. effective interventions should be addressed to general practitioners to reduce inappropriate prescriptions bpa was found in the dialysate ( ng/l) and ls ( ng/l) wherein the concentration of bpa decreases over time to reach ng/l at the end of a session. finally, bpa was present in all tested dialysis at concentrations of up to . ng/dialyzer in the compartment mimicking the blood and to . ng/dialyzer in the dialysate despite prior rinsing with l of . % nacl. conclusion: our study is the first one to show the risk of exposure to bpa and bpa-clx hdf-ol. while assessment of the impact of this exposure in a patient under treatment remains to be done, it is now possible to better master contamination by bpa and its four chlorinated derivatives through better practices (choice of medical devices) and improvement of the overall water treatment process san cecilio university hospital, genomic unit san cecilio university hospital, genomic unit, genyo, centre for genomics and oncological research the aim of this study is to compare the apparition of stroke, acs, cardio-vascular death and the need of surgery in patients after percutaneous transluminal angioplasty (pta) or stroke depending on the presence of cyp c * * polymorphisms. setting and method: retrospective cohort study. we recruited patients treated with clopidogrel after a pta of the lower limb or stroke (without surgery) from to in our hospital. data collected: age, sex, cyp c * (rs ) and cyp c * (rs ) genotypes and the primary end-point: stroke, acs, cv death and surgery of the affected vessel during months after discharge. polymorphisms were genotyped using taqman Ò genotyping assays technology. main outcome measures: we recruited patients with stroke ( . % men; mean age . ) and patients after pta ( . % men; mean age . ) treated with clopidogrel after discharge %) suffered the primary end-point during months after discharge; of these patients had the cyp c * allele. among patients with pta of the lower limb: % of them had the cyp c * allele and no one a cyp c * allele; ( . %) of these patients suffered the primary end-point during months after the discharge and of these had the * allele of the cyp c isoenzyme * allele and treated with clopidogrel have a higher risk of the primary end-point than those patients not carrying it spain please specify your abstract type: research abstract background and objective: the engagement of fcgrs by tnf antagonists could affect to macrophage-mediated clearance of immune-complexes. the aim of our study was to evaluate the potential role of fcgr a (a[c) (rs ) single nucleotide polymorphism (snp) as a predictor of tocilizumab efficacy in rheumatoid arthritis (ra) patients. setting and method: the fcgr a (a[c) (rs ) snp was genotyped using predesigned taqman Ò genotyping assays technology and analysed on a viia Ò real-time pcr system. the statistical analysis was performed using spss v the mean age of the patients was . ± . years and % were women. the mean das at baseline was . ± . . we found no statistically significant association between our end-point and the genetic polymorphisms studied tdmp : therapeutic drug monitoring of infliximab biosimilar and anti-infliximab antibodies in inflammatory diseases patients with dermatological conditions and inflammatory bowel disease being treated with ifx-b ( mg/kg/ weeks after the induction dose) were included. the concentrations of ifx-b and ati-b were quantified by two sandwich-type elisa immunoassays (triturus Ò analyser). main outcome measures: plasma levels of ifx-b and ati-b, clinical response and infusion reactions. the clinical response was assessed according to pathology of each patient (based on specific clinical variables for the pathology into the electronic history) pharmacokinetic results (% assessments): (a) . % no ifx-b detection (c \ . mcg/ml) and positive ati-b (c [ ua/ml) ( assessments/ patient). atis = , y ua/ml. no clinical response (nr) in . % assessments. (b) . % ifx-b and ati-b (c b ua/ml) no detection ( assessments/ patient). nr %. (c) . % ifx-b detection (c [ . mcg/ml) and negative ati-b ( assessments/ patients) weight: ( - ) kg. twenty assessments, . ( - ) assessments per patient, ( - ) ifx-b doses, % concomitant treatment ( / -azathioprine, / -corticosteroid) the incidence of ati-b was low. a correlation was observed between the presence of ati-b and loss of clinical response, as infliximab original. tdmp : serum concentration of non-vitamin k antagonist oral anticoagulants (noacs) in older hip fracture patients ina linnerud , mette i martinsen estimation of t of noacs by t / = ln /kel [kel; elimination constant] using two s-concentration measurements. results: we included patients (median age years, . % women). noac use was detected be serum analysis in patients ( . %; % coherent with mr), while patients ( . %) used warfarin. of the noac users ( . %) had s-concentrations of noacs above the reference range at admission, and five patients ( . %) had s-concentrations within the reference range before surgery. patients using noac had significantly longer median waitingtime for surgery than warfarin-users ( vs h, p = . ). blood transfusions were given to . % of noac-users vs . % of warfarin-users (p = . ). mean estimated t of noacs were , . and . h for dabigatran (n = ), apixaban (n = ) and rivaroxaban (n = ), respectively. conclusion: mr is effective in detecting noac use in older hip fracture patients, but importantly s-concentrations are higher than expected in this population. this might reflect the significantly longer waiting-time for surgery this column is supplied with packing material made of totally porous spherical silica coated with a silicone polymer monolayer containing octadecyl (c ) groups. the mobile phase was composed of . % na po h o (ph . ), acetonitrile, and methanol ( : : , v/v/v), which was degassed in an ultrasonic bath prior to use. the flow rate was . ml/min at ambient temperature and sample detection was carried out at nm. plasma samples were obtained from patients with cml receiving nilotinib treatment. sampling was performed at the steady state. blood samples were collected by venipuncture h after oral administration of nilotinib. plasma was separated by centrifugation at g for min and stored at - °c until analysis. plasma samples ( ll) were then extracted as described above. the same samples were also sent to a commercial laboratory (bml, inc.) for assaying nilotinib concentration by liquid chromatography-tandem mass spectrometry (lc-ms/ms). in addition, we applied this method to tdm of cml patients receiving nilotinib at our hospital. main outcome measures: the calibration curve exhibited linearity over the nilotinib concentration range of - ng/ml at nm, with relative standard deviations (n = ) of . , . , and . % for , , and ng/ml, respectively. the detection limit for nilotinib was ng/ml due to three blank determinations (q = ). in addition, we compared the results with those measured by lc-ms/ ms at bml, inc. (a commercial laboratory). as a result, a strong correlation was observed between the nilotinib concentrations measured by our hplc method and those obtained by lc/ms-ms (r = . , p \ . ). in addition, tdm of nilotinib was performed to six cml patients. there was the case which participated in dosage adjustment of nilotinib in hepatic dysfunction and poor glycaemic control. results: we have developed a simple ultraviolet detection method for the determination of nilotinib, which has high sensitivity and large dynamic range please specify your abstract type: research abstract key: cord- -fio cjj authors: nan title: peripheral nerve society meeting july – , sitges, barcelona, spain date: - - journal: j peripher nerv syst doi: . /jns. sha: doc_id: cord_uid: fio cjj nan the peripheral nerve society was founded in from two groups of academic investigators, peripheral nerve study group and peripheral neuropathy association of america, interested in the basic biology and function of the peripheral nervous system and its application to the clinic. their invite only biennial meetings involved - attendees in cloistered settings organized by shoestring and local initiative. from this, we have grown remarkably. we now have an annual meeting of over people including meetings within the meeting for the special interest groups in inflammatory, diabetic and hereditary neuropathy. with this substantial growth and the success of jpns, the journal of the peripheral nervous system the society continues to flourish. has proven to be a year full of exciting changes for the society. pns has transitioned from a biannual, to an annual meeting. next year, the meeting will be taking place at the renaissance baltimore harborplace hotel from - july in baltimore, maryland. the development of a new website has been completed, please visit www.pnsociety.com to see the new face of the society. finally, pns has adopted new executive staff. with their guidance and the leadership of an active and diverse board of prominent professionals in the field the peripheral nerve society continues to grow and anticipates more exciting changes in the year to come. the peripheral nerve society provides annual meetings, teaching courses, guidelines, and other resources to aid in the education of members. becoming a member of pns means collaborating with prominent global professionals in the field to develop and provide the best treatments for people with peripheral nerve diseases and setting standards of care within the field. please participate in our future by joining the pns, volunteering for a project aligned with your interests and sending your ideas for the future to the executive office, or board member. peles e . department of molecular cell biology, rehovot, israel. two schwann cell-dependent mechanisms control the presence of na + channels at the nodes of ranvier: i. clustering of the nodal complex by glia-derived proteins and ii. restriction of nodal proteins within the nodal gap by the paranodal junctions. these mechanisms depend on specific cell adhesion molecules that mediate the contact between myelinating glia and their underlying axons at the forming nodes and the paranodal junction. during myelination, na + channels initially clustered at heminodes that border each myelin segment. this process requires gliomedin, nrcam and neurofascin (nf ), three cell adhesion molecules (cams) that mediate the interaction between schwann cell microvilli and the axon. na + channels clustering activity of gliomedin is tightly regulated by two distinct and functionally opposing proteolytic events. while the clustering activity of gliomedin is enhanced by its shedding from the surface of schwann cells by a furin protease, its activity is negatively regulated by bone morphogenetic protein /tolloid-like (bmp /tld), and tolloid-like (tll ) metalloproteinase. cleavage by these enzymes restricts the activity of gliomedin to the nodal area and prevents the formation of ectopic clusters along axons that are devoid of myelin segments, as well as below the myelin internodes. hence, proteolytic processing of gliomedin facilitates, yet limits, the clustering of na + channels to specific sites along the axon in a timely manner. furthermore, axon-glial contact mediated by gliomedin and nf at the nodes, not only plays a role in na + channel clustering during development, but also contributes to the long-term maintenance of na + channels at nodes of ranvier. in addition to clustering by gliomedin, the distribution of na + channels is restricted between two growing myelin segments by the flanking paranodal junction. at this site, axon-glia contact is mediated by a distinct set of cell adhesion molecules (i.e., caspr, nf and contactin) that also delineate the underlying axonal and glial cytoskeleton. this paranodal junction-dependent restriction of na + channels to the nodes is mediated by the spectrin-based paranodal axonal cytoskeleton. illa i . neuromuscular unit, neurology department, hospital santa creu i sant pau, universitat autònoma de barcelona, barcelona, spain. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune disorder of the peripheral nerves with clinical and immunological heterogeneity. currently, the diagnosis of cidp is based on clinical and electrophysiological criteria and does not take into consideration the presence of immune biomarkers. several autoantibodies against proteins of the node of ranvier in patients with cidp have now been described. these antibodies define specific cidp subtypes sometimes referred to as nodopathies and can have diagnostic and prognostic implications. anti-contactin (cntn ) antibodies. we have described the presence of antibodies to cntn in a small subset of patients with cidp. these patients shared a phenotype and have poor response to ivig. the anti-cntn antibodies are predominantly igg . pathological studies from skin and sural nerve biopsies of patients show morphological changes in the paranodes. experimental data supporting the pathogenicity of anti-cntn igg antibodies include: a) demonstration in vitro that the antibodies disrupt the binding of the cntn -caspr complex to neurofascin- (nf ); b) intraneural injections of antibodies progressively and specifically disrupt the paranodal axo-glial junction; and c) chronic infusion of antibodies induced clinical and electrophysiological worsening in animals with experimental autoimmune neuritis (ean). anti-nf antibodies. antibodies to neurofascins were first reported in patients with guillain-barré (gbs) and cidp and subsequently, antibodies specific to the nf isoform were found in a small group (< %) of patients with cidp. studies by us and confirmed by others have demonstrated that patients with cidp and anti-nf antibodies have a distinct phenotype that often includes a low-frequency tremor and poor responses to ivig. the autoantibodies are predominantly of the igg subtype. the passive transfer of monoclonal anti-neurofascin antibodies (which recognize all neurofascin isoforms) to mice with ean strongly exacerbated the severity of the pathology, but no studies have yet demonstrated that patient-derived anti-nf igg antibodies are pathogenic. a pathogenic role of the antibodies is however supported by sural nerve biopsies from patients with cidp and anti-nf antibodies that showed paranodal demyelination in the absence of inflammation, the loss of septate-like junctions and, the interposition of cellular processes between the paranodal loops and the axolemma. these alterations are reminiscent of those found in nfasc-null mice suggesting that anti-nf antibodies may specifically disrupt the nf -cntn -caspr complex at the paranodes. antibodies to other nodal proteins. recently neurofascin- and neurofascin- were reported as the main targets of autoantibodies in five patients with igg reactivity against the nodes of ranvier; the antibodies were predominantly igg . these patients presented with clinical features distinct from those in patients with anti-nf igg antibodies. four of these patients had subacute onset of sensory ataxia without tremor. the presence of anti-caspr antibodies has been reported in two patients with inflammatory neuropathies, one classified as cidp, the other as gbs. both patients had intense neuropathic pain. the skin biopsy from both patients showed paranodal disruption. some patients whose sera show nodal or paranodal reactivity in teased nerve fiber preparations have antibodies against other nodal proteins, such as gliomedin or neuronal cell adhesion molecule (nrcam) . the skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. indeed some mechanoreceptors can detect even nanometer-scale movements. movement is transformed into electrical signals via the gating of mechanically-activated ion channels at sensory endings in the skin. the sensitivity of piezo mechanically-gated ion channels are controlled by stomatin-like protein- (stoml ), which is required for normal mechanoreceptor function. under pathophysiological conditions following nerve injury or diabetic neuropathy the slightest touch can produce pain. it is at present unclear whether peripheral changes in sensory mechanotransduction may underlie hypersensitivity associated with neuropathic pain. here we have examined the role of the stoml modulation of piezo channels in mechanoreceptors and nociceptors to under pathophysiological conditions. we recently developed small molecules that act as inhibitors of stoml function. peripheral application of stoml inhibitors can alleviate hypersensitivity in models of neuropathic pain. our data strongly suggest that tactile evoked pain in models of peripheral neuropathy may be at least partly driven by sensitization of sensory mechanotransduction driven by stoml . coleman m . john van geest centre for brain repair, cambridge, uk. axons are lost early in many neurodegenerative disorders of peripheral and central nervous system. the degeneration of transected axons (wallerian degeneration) can be slowed tenfold by overexpression of a variety of nad-synthesizing enzymes, such as isoforms of nmnat or the related mutant fusion protein, wld s . wallerian degeneration is also delayed by deletion of tlr adapter protein sarm , a protein recently reported to promote nad degradation. it is important to understand fully the mechanism of wallerian degeneration because related mechanisms contribute to axon loss in a number of disease models, including models of peripheral neuropathies, parkinson's disease, multiple sclerosis and glaucoma. new data also suggest a role in hereditary spastic paraplegia. while depletion of nad is an attractive hypothesis for the mechanism of wallerian degeneration, especially as nad can be increased by dietary methods, it cannot explain a number of key observations. fk , an inhibitor of nampt, blocks the nad salvage pathway and strongly depletes nad, including within axons. however, instead of killing axons as the nad hypothesis would predict, it does precisely the opposite: it phenocopies the protective effect of wld s . moreover, ectopic expression of the bacterial enzyme nmn deamidase, a protein absent in mammals, protects injured axons both in transgenic mice and in primary neuronal cultures, but it has no effect on nad levels either under basal conditions or in degenerating nerves. these observations fit better with a proposed toxic role for the nad synthesis intermediate nmn, a model that can also explain the protective effect of wld s . a full understanding of the pathway should identify a number of points where intervention could be a treatment for multiple axonopathies. as with any medical discipline, expansoin of knowledge about the fundamental science behing a disorder of the human nervous system comes part and parcel with a change in our understanding of the epidemiology of any given disorder or groups of disorders. recent advances in our fundamental understanding of inflammatory neuropathies of the peripheral nervous system have been accompanied by drastic changes in our understanding of the neuroepidemiology of these disorders -the specific populationss affected by peripheral neuropathies, as well as the varying importance / contributions of select peripheral neuropathies to the overall burden of peripheral nervous system (pns) disease, and how this shift in epidemiological understanding influences the clinical approach to diagnosis and management of patients with pns disease. the past few decades have witnessed a paradigm shift in many aspects of pns disease diagnosis and treatment; from the association of human immunodeficiency virus (hiv)-associated neuropathies; to the increassing recognition of hereditary / familial peripheral neuropathies; to the increased recognition of specific neuropathies such as multifocal motor neuropathy with conduction block. in addition, timely events such as the recent, and increasingly irrefutalbe evidence for a link between zika virus and a guillain-barré syndrome, and the rather unexpected resurgence of peripheral neuropathies due to previously 'exotic' etiologies such as lepromatous neuropathy require prompt clinical attention. this plenary session aims to describe the evolving neuroepidemiology of peripheral nervous system disorders, and how these changes may influnece the clinical approach to the diagnosis, prognostication, and treatment of otherwise 'unusual' periphal nerve diseases. oxaliplatin chemotherapy for colorectal cancer is seriously limited by neurotoxic side effects which are not fully understood. oxaliplatin-induced peripheral neurotoxicity (oipn) comprises an acute syndrome and a chronic sensory neuropathy. the acute symptoms, notably cold hyperalgesia, have been attributed to transient ion channel dysfunction, and the worse they are the more severe the chronic neuropathy that ensues. we designed a combined in vitro and in vivo project, using neurophysiology to better understand the pathogenesis of oipn. in the in vitro study, differentiated f cells (rat drg neurons x mouse neuroblastoma n tg- cell line) were incubated for and hours in . m oxaliplatin, and their electrophysiological properties studied by patch-clamp. the treated f cells showed relatively depolarized resting membrane potentials, significantly decreased firing frequencies, and increased sodium current densities. moreover, a decrease in erg (ether-à-go-go-related gene) potassium current was also evident. in the in vivo study, we applied nerve excitability testing (net) to a wistar rat model of oipn. to investigate the acute syndrome, we compared behavioural and neurophysiological data of animal cohorts (controls and oipn rats, n= each) before and after oxaliplatin administration ( mg/kg, iv). twenty-four hours after the injection we observed differences between the groups in behaviour (cold plate test, p= . ) and in superxcitability of motor axons (p= . ). to investigate the chronic neuropathy, we compare a control group (n= ) with a treated group (n= , oxaliplatin mg/kg twice weekly x weeks, iv). both groups are studied with behavioural, neurophysiological (sensory and motor nerve conduction studies, net), and pathological (caudal and sciatic nerve, skin biopsy, drgs) methods. data are collected at baseline, end of treatment and weeks after treatment; to obtain a full net profile of all significant changes. in this highly translational approach to oipn, the in vivo net changes in the acute and chronic rat models can be matched on the one hand to findings from in vitro experiments, and on the other to clinical data, since net is also easily applied in humans. of these results. in denmark there is a unique situation to conduct epidemiological studies facilitated by the danish civil registration and the danish national hospital registry (dnhr). this enables us to identify all gbs patients in denmark in a given period. from the same period as the igos cohort was included we have identified all gbs patients admitted to or seen in outpatient's clinics of hospitals in denmark (september st to december st ). records from the population based danish cohort will be reviewed for demographic and clinical data and compared to the patients included in igos from denmark, as well as with the igos europe/america cohort. during this period patients from denmark have been included in igos. the danish group is comparable to the europe/america group not counting the danish patients (n= ) of the igos cohort in regard to sex and age at entry, gbs disability score at nadir, and percentage of patients needing mechanical ventilation. in the danish igos group % are males, the median age is ( - ) years, the mean(sd) gbs disability score at nadir . ( . ), and % of the danish group needed mechanical ventilation. in the europe/america group % are males, the median age is ( - ), mean(sd) gbs disability score at nadir . ( . ) and % of the group needed mechanical ventilation. at the meeting we will compare and present data from the danish population based cohort as well as epidemiological data. chronic inflammatory demyelinating polyneuropathy (cidp) is a common autoimmune disease of the peripheral nervous system (pns) that causes sensorimotor impairment. mice with a dominant autoimmune regulator gene (aire) g w mutation on the non-obese diabetic (nod) background (nod.aire gw/+ mice) develop spontaneous autoimmune peripheral polyneuropathy (sapp) resembling cidp. in sapp, demyelination is caused primarily by th t cells; however, the contributions of nerve-resident cells such as schwann cells are poorly understood. we identified a population of non-hematopoietic, integrin alpha + (itga +) cells in the pns that increases in frequency and number during sapp. these itga + cells coexpress numerous schwann cell markers including sox , p , s b, myelin protein zero, and peripheral myelin protein , suggesting that itga + cells are schwann cell-like. additionally, during sapp, these itga + cells upregulate the extracellular matrix protein periostin (postn), which has recently been shown to promote macrophage recruitment and activation in inflammatory disease and cancer. our data indicate that macrophages are pathogenic during sapp. therefore, we hypothesized that itg a+ cells promote macrophage recruitment during sapp via postn production. to test this hypothesis, we performed in vitro chemotaxis assays. conditioned media from nod.aire gw/+ nerve promoted significantly more macrophage chemotaxis than conditioned media from postn −/− nerve. furthermore, postn recombinant protein was sufficient to induce macrophage chemotaxis in vitro. our findings show that itg a+ schwann cell-like cells mediate macrophage chemotaxis by upregulating postn during sapp and suggests postn as a novel target for the treatment of cidp. "wear-off" frequency will be analyzed by assessing the proportion of subjects with any given degree of gs and rods intracycle fluctuation and the proportion of cycles in which gs and r-ods fluctuation occurs. to determine the extent of "wear-off" the degree of difference between maximum and minimum gs, r-ods, tugs, onls, and vas scores will be analyzed. currently subjects from different sites have been enrolled ( sites eligible for enrollment). this interim study report will provide preliminary representative data, demonstrating ivig "wear-off" effects on gs and other outcome measures. by better understanding the frequency and extent of ivig treatment-related fluctuations we expect that these results will help facilitate development of cidp treatment optimization strategies. we also expect that this information will be important in forming hypotheses to be tested in future studies (for example, comparing different dosage intervals, optimal ivig taper guidelines, or assessing the long-term outcome of short-term cycle to cycle clinical fluctuations). neuropathic pain is a frequent feature of peripheral neuropathy causing a significant impact on patients' quality of life and health care costs. resolving the genetic architecture of painful neuropathy will lead to better disease management strategies, risk stratification, and counselling. therefore, we aim to develop a reliable technique to rapidly and accurately re-sequence multiple genes in a large cohort of painful neuropathy patients at low cost. whole exome sequencing of thousands of samples remains expensive for clinical use. several targeted enrichment approaches are currently available to selectively enrich for genomic regions of interest. in this study, we compared the sensitivity, specificity, targeting efficiency, reproducibility of performance and cost effectiveness of truseq ® custom amplicon-next generation sequencing (tsca-ngs) and molecular inversion probes-next generation sequencing (mips-ngs) methods. for both methods, we constructed a targeted enrichment kit to capture the coding and exon-flanking intron sequences of nine sodium channel genes (scn a, scn a-scn a, and scn b- b) expressed in nociceptive neurons. probes were designed for the two methods using their respective informatics pipelines. in total, patients with diabetic and idiopathic neuropathy were tested by both methods. among the patients, patients were tested previously by sanger sequencing for scn a-scn a. approximately kb was captured and sequenced. % of the targeted regions showed an average coverage of ≥ x in tsca-ngs, and % in mips-ngs. we managed to identify potential pathogenic mutations and polymorphism variants by mips-ngs and tsca-ngs. moreover, we observed a perfect agreement ( %) between sanger sequencing data and those obtained using mips-ngs and tsca-ngs. both ngs approaches showed user-friendly software to design probes and exhibited a similar on-target efficiency. although the overall coverage per region varied across different dna samples, it was sufficient to detect any variant in these regions. mips-ngs has more versatile assay design, demonstrated a high degree of flexibility with probes re-placement and > x cheaper than tsca-ngs. mips-ngs is a reliable, flexible, and inexpensive method to detect genetic variations in thousands of patients. in our centers, this technology is currently implemented as a routine diagnostic tool for screening of sodium channel genes in painful neuropathy patients. alonso-jiménez a , , belvis-nieto r , diaz-manera j , , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, universitat autònoma de barcelona, spain; centro para la investigación biomédica en red para enfermedades raras, ciberer, madrid, spain; neurology department, hospital universitario dexeus, barcelona, spain. most acute demyelinating polyneuropathies have an immune-mediated pathogenesis and are included within the guillain-barré syndrome spectrum. occasionally, other mechanisms such as metabolic, infectious or toxic may lead to gbs-like presentations. thermatrim ® and pura alegria ® are different brands of the same illegal slimming product that is sold through online vendors and in which the exact composition is unknown. here we present a patient with an acute demyelinating polyneuropathy secondary to the intake of the slimming product "pura alegría". a year-old woman with no remarkable medical history reported days history of distal numbness in her feet that progressed in one week to her knees and her left hand. she had had an upper respiratory tract infection ten days prior to these symptoms. the neurological examination showed absent distal vibratory and arthrokinetic sensations and arreflexia in lower limbs, decreased vibratory sensation in her hands and a ataxic gate. the lumbar puncture showed . g/l of proteins with no cells. the emg fulfilled diagnostic criteria for acquired demyelination. intravenous immunoglobulin therapy was started but the symptoms kept worsening and corticosteroids were started. the patient mentioned then the slimming product. a brain mri showed diffuse leukoencephalopathy that was asymptomatic. steroids and the pura alegria slimming products were withdrawn and the patient recovered completely after one year of follow-up. "pura alegría", "thermatrim" and "thermatrim plus" are slimming products that were forbidden in spain after several cases of acute leukoencephalopathy and acute polyneuropathy. exact composition is unknown although the spanish drug agency detected the pesticide malonoben, a tyrosin kinase inhibitor, among the components. since nine cases of pura alegria/thermatrim neurological toxicity, including leukoencephalopathy and polyneuropathy have been described. all cases had good outcomes after treatment withdrawal, although recovery is slow and may be incomplete. our case highlights the need to carefully consider drug toxicity, including dietary supplements, in the differential diagnosis of gbs specially when evolution does not follow typical patterns. alvarez s , klein d , martini r , moldovan m , , krarup c , . center for neuroscience, university of copenhagen, denmark; department of neurology, developmental neurobiology, university hospital würzburg, germany; department of clinical neurophysiology, rigshospitalet, copenhagen, denmark. charcot-marie-tooth neuropathy type a (cmt a) resulting from peripheral myelin protein kda (pmp ) overexpression is the most common hereditary motor and sensory neuropathy in humans. the transgenic pmp (pmp tg) mouse line c carrying copies of the human pmp gene, has a slowly progressing neuropathy phenotypically like cmt a with thin and abnormally thick myelin profiles and supernumerary schwann cells. in addition, pmp tg nerves showed activated macrophages leading to axon-myelin compartment disruption and maldistribution of k+ channels (kohl b et al, am j pathol. ; : ) . the aim of the present study was to investigate the motor axon excitability in pmp tg versus wt littermates. multiple measures of motor axon excitability under anesthesia were carried out by stimulation of the tibial nerve at ankle and "threshold-tracking" the plantar compound muscle action potential (cmap). at age months, when the post-developmental maturation was nearly complete in the wt, the pmp tg cmap showed an increase in latency by %. the cmap amplitude was decreased by %, although the mean motor unit size (mscan method) appeared unchanged indicating a lack of collateral sprouting. furthermore, pmp tg showed abnormalities in both passive cable properties and voltage dependent parameters. at age month, the cmap latency of pmp tg was increased by % as compared to wt. in contrast to this marked conduction slowing along the tibial nerve from to months of age, the progression of excitability changes localized at ankle appeared modest. nevertheless, when pooling data from to months, the increase in pmp tg latency was correlated (spearman p< . ) with an increase in accommodation half-time during depolarizing electrotonus (+ % of threshold) from to ms and a reduction of the late subexcitable period of the recovery cycle from to % of threshold, both changes consistent with a redistribution of k+ currents consistent with the maldistribution of k+ channels. our data suggest that in the pmp tg cmt a model, a functional, thus potentially reversible abnormality in k+ channel distribution, accumulates along the nerve and aggravates the conduction impairment due to impaired myelin formation and maintenance. alvarez s , krarup c , , moldovan m , . center for neuroscience, university of copenhagen, denmark; department of clinical neurophysiology, rigshospitalet, copenhagen, denmark. mice heterozygously deficient of myelin protein p gene (p +/−) show a mild progressive dysmyelinating neuropathy, with conduction slowing and impaired excitability, phenotypically similar with charcot-marie-tooth disease type b (cmt b). we found that in p +/− the accumulating myelin abnormalities were paralleled by progressive changes in voltage-dependent motor axon function resulting in neurotoxic membrane depolarization (rosberg mr, et. al. neurobiol dis. : ) . the aim of this study was to investigate the relationship between demyelination and motor axon function in p +/−. demyelination of the right sciatic nerve by topic lysophosphatidylcholine (lpc) application was carried out in p +/− and wild-type (wt) mice, in year (mature) and years (aged) groups. multiple measures of motor axon excitability under anesthesia were carried out by stimulation of the tibial nerve at ankle (distal to lpc demyelination) and "threshold-tracking" the plantar cmap responses. live imaging studies by cellvizio (mauna kea technologies, paris, france) confocal laser endomicroscopy were carried out in transgenic mice expressing the fluorescent reporter yfp in peripheral nerve axons under the thy promoter. in mature wt the sciatic morphological and electrophysiological demyelinating features following lpc could be readily observed at hours but disappeared by weeks. no morphological changes could be observed at the tibial level. consistently, no conduction or excitability changes could be observed at the right tibial neve level as compared to the left tibial nerve in wt. in contrast, in p +/− the motor axon function was impaired at the tibial nerve level at weeks after sciatic lpc demyelination. in mature p +/−, although the cmap amplitude appeared preserved, the distal motor latency was prolonged whereas the excitability measures showed reduced deviations during threshold electrotonus and increased refractoriness at the expense of superexcitability of the recovery cycle, both consistent with membrane depolarization. furthermore, in aged p +/− the delayed tibial conduction was associated with a drop in cmap amplitude and a prolongation of the strength-duration time constant. taken together these data suggest that focal demyelination aggravates membrane dysfunction along the entire motor axon in p +/− providing a novel experimental model to explore the link between demyelination and axonal membrane dysfunction in cmt b. amass l , li h , gundapaneni b , schwartz j , keohane d . pfizer inc., new york, ny, usa; inventiv health inc., burlington, ma, usa. a number of factors can influence disease progression in transthyretin familial amyloid polyneuropathy (ttr-fap), a rare, fatal, hereditary amyloidosis. this analysis evaluated the specific role of baseline neurologic severity on neurologic disease progression in ttr-fap. a predictive model was created based on longitudinal data from val met patients who participated in the tafamidis (a selective ttr stabilizer) clinical development program. data from the intent-to-treat population of the double-blind, placebo-controlled registration study (tafamidis group, n= ; placebo group n= ) and its two consecutive open-label extension studies in which all patients received tafamidis were used. the second extension study is ongoing, but a formal, prospectively-planned interim analysis was conducted with the cut-off date of december , . this analysis focused on the first months of treatment for the overall study cohort analyzed. the neuropathy impairment score-lower limbs (nis-ll) was used to assess neurologic functioning at baseline and at subsequent study visits. a linear mixed-effects model for repeated-measures (mmrm) analysis, with baseline nis-ll, treatment, and their interactions with time as fixed effects, was used, and the slope and intercept for each patient were included as random effects. patients were primarily caucasian with early-stage neurologic disease (baseline nis-ll mean [standard deviation]: tafamidis, . [ . ]; placebo, . [ . ] ). across both groups, disease progression increased with increasing levels of baseline severity (nis-ll) (p< . ). however, the predicted magnitude of change from baseline to month for tafamidis was consistently less than that for placebo across a range of observed baseline nis-ll values, suggesting a disease-modifying effect of tafamidis. similar findings were observed for the nis-ll muscle weakness subscale. this mmrm analysis in patients with val met ttr-fap demonstrates that disease progression strongly depends on baseline neurologic impairment and highlights the disease-modifying effect of tafamidis across a range of baseline levels of neurologic severity. clinicaltrials.gov identifiers: nct , nct , nct . amino h , misawa s , sekiguchi y , shibuya k , watanabe k , suichi t , kuwabara s . department of neurology, chiba university, chiba, japan. guillain-barré syndrome (gbs) is a potential life threatening neurological disorder and respiratory insufficiency is one of the critical complications. eramus gbs respiratory insufficiency score (egris) is a method for predicting the chance of respiratory insufficiency in gbs. however, clinical characteristics and courses can vary for subtypes of gbs, whose occurrences differ for each region: acute inflammatory demyelinating polyneuropathy (aidp) is very common in european countries, whereas acute motor axonal neuropathy (aman) is frequently seen in asian countries. the aim of this study is to investigate the usefulness of egris in japan, where aman is more common than in the netherlands. clinical and electrophysiological profiles of consecutive gbs cases, who visited our hospital within days after symptoms onset between and , were reviewed. of the gbs patients, % were classified as aidp and % as aman according to the electrodiagnosis criteria by ho and colleagues. higher egris scores correspond to higher risk of respiratory insufficiency in total of the gbs patients, as well as in aidp patients. however, in patients with aman, egris scores did not always match the chances of respiratory insufficiency: up to % of the patients with low risk of egris showed respiratory failure, whereas only % of the patients with high risk of egris needs intubation/mechanical ventilation. in aman, associations with mechanical ventilation were seen for rapid progression (shorter duration between onset and hospital admission), more decreased vital capacity, and more frequent autonomic involvement. egris is useful also for japanese gbs patients. however, for aman patients, it should be used with discretion. another score to predict respiratory insufficiency might be required in asian countries. anandan c , litchy wj , laughlin rs , leep hunderfund an , naddaf e . mayo clinic, rochester, usa. in patients with suspected ulnar neuropathy, nerve conduction studies (ncs) are commonly requested to help with diagnosis and localization. however, routine ncs are often normal or not localizing. ulnar ncs recording from the first dorsal interosseous muscle (ncs-fdi) is thought to increase the diagnostic yield of electrodiagnostic testing, although not commonly considered. we developed a quality improvement strategy to routinely perform ulnar ncs recording from the abductor digiti minimi muscle (ncs-adm) as well as ulnar ncs-fdi in all patients referred for suspected ulnar neuropathy. we utilized the dmaic (define, measure, analyze, improve, control) model of process improvement to define our problem and create a map of the current process for ulnar neuropathy diagnosis in our electromyography laboratory. we determined baseline performance via review of distal sensorimotor polyneuropathy (dpn) is the most common complication of diabetes and risk factors beyond hyperglycemia have proven important particularly in type diabetes (t dm). only few prospective studies from early-stage t dm exist. we aimed to study the development of dpn during the first years after a screening-based diagnosis of t dm. from the addition-denmark study participants were eligible for this study. dpn was assessed by the michigan neuropathy screening instrument questionnaire (mnsi) at four time-points during follow-up. dpn was defined by a mnsi score ≥ . participants ( %) were positive in mnsi at baseline and thus excluded from this study. by kaplan-meier plot we evaluated the cumulative incidence of dpn and in cox proportional hazard models we calculated hazard ratios (hr) for the intervention groups in the addition trial and for various covariates proposed to influence the development of dpn. models were adjusted in steps for intervention group, age, sex, baseline mnsi, lipid-lowering and anti-hypertensive treatment. this study cohort consists of participants ( % men) with a median age of . years (p ;p : . ; . ) and median baseline hba c of . (p ;p : . ; . ). a cumulative incidence of % was seen during years of diabetes. there was no statistically significant difference in hr between the intervention groups or by sex but a significantly higher hr of . ( %ci: . ; . ) was seen for age (per year). the highest hr was found for a history of cardiovascular disease (myocardial infarction or stroke) up to ten years prior to the diabetes diagnosis with a hr of . ( . ; . ). weight, waist circumference, body-mass index and methylglyoxal (log transformed) showed modest but statistically significant associations with incident dpn with standardized hrs of . ( %ci: . ; . ), . ( %ci: . ; . ), . ( %ci: . ; . ) and . ( %ci: . ; . ) respectively. this study demonstrates a fairly low cumulative incidence of dpn in people with screen-detected t dm and provides evidence that macrovascular disease, obesity and oxidative stress are important risk factors for dpn even at the earliest stages of t dm. andermann syndrome, also known as agenesis of the corpus callosum and peripheral neuropathy (accpn), is an autosomal recessive disorder with a broad spectrum of mild to severe neuromuscular and psychiatric consequences. the gene variants causing disease were first identified in french-canadian families. in the present study, we intended to phenotype and genotype a series of non-french-canadian familial cases presenting with charcot marie tooth disorder associated with agenesis/dysgenesis of the corpus callosum. for this purpose, seven families, of consanguineous marriage, were studied. patients were clinically and para-clinically investigated using mri and electrophysiology (mncvs). for some, a sural nerve biopsy was taken. microsatellite markers around the accpn locus were used in two large families; followed by sanger sequencing of all the exons and intron-exons boundaries of the gene, in one patient from each of the families. the age at onset of the disease was at birth in the patients from the largest consanguineous family ( affected individuals). the biopsy from one patient showed a severe demyelinating neuropathy with many hypomyelinated fibres and mostly secondary axonal changes. these finding were compatible with electrophsiological data where the mncvs are of demyelinating range. dysgenesis of the corpus callosum in one patient and agenesis in another sib were revealed by mri. we identified one homozygous truncating mutation in this family. interestingly, no causative variant was found in a patient from another family and showing homozygous haplotype. two different heterozygous variants were identified at one hit in two patients from two non-consanguineous families. genetic investigations will be continued to identify the possible second hit. in the remaining families, no variant was found. the negative family cases will be subjected to ngs. at this stage, it is tempting to speculate on the genetic heterogeneity of accpn in our series. baba m , suzuki c , ogawa c , tomiyama m . department of neurology; diabetes center, aomori prefectural central hospital, aomori, japan. in we introduced a staging system of severity of diabetic polyneuropathy (dpn) by nerve conduction study (ncs) of the lower limb: sensory ncs of the sural nerve and motor ncs of the tibial nerve. the system consists of five stages; ncs- (normal): no abnormalities, ncs- (mildly abnormal): presence of delay of mcv, scv, minimal f-wave latency, or positive a-wave, ncs- (moderately abnormal): decrease in sural snap less than uv, ncs- (severely abnormal): decrease in plantar muscle-cmap to - mv, ncs- (ultimately abnormal): plantar muscle-cmap lost or less than mv with trace of sural-snap. to examine validity of the system, we conducted -year prospective observation on development of diabetic foot (df) by the ncs staging system. in addition, occurrence of ischemic heart disease (ihd) and stroke (is), and death of neuro-vascular events were also counted. n - , we carried out ncs in diabetics, and categorized them by the ncs staging system: % was ncs- , % was ncs- , another % was ncs- , % was ncs , and % was ncs- . we then followed them and prospectively counted the occurrence of df, ihd and is in patients (mean age ys). the occurrence of df during the following years was; ncs- : %, ncs- : %, ncs- : %, ncs- : %, ncs- : %. occurrence of any of df, ihd and/or is was as follows; ncs- : %, ncs- : %, ncs- : %, ncs- : %, ncs- %. there was no death from nsc- , − , and − groups (n= ), while two from ncs- group (n= ) were found dead in a bed or on a driver's seat, and other two from ncs- group (n= ) died of sudden cardiac arrest or infection after foot amputation. in summary, the present ncs grading system seems to work satisfactory not only for diagnosis of severity of the current dpn, but also for prognostic prediction of the dpn-related foot and vascular events. bacon c , feely sme , shy me . university of iowa hospital, iowa city, ia, usa. for patients with charcot-marie-tooth disease, also known as hereditary motor and sensory neuropathy, the rasch modified cmtnsv is a validated measurement of symptoms and impairment. the score is comprised of nine parameters of a clinical examination, including nerve conduction studies (ncs) . each parameter has an individual score ranging from zero to four, with the composite score having a maximum of prior to rasch modification. for patients who do not complete a ncs, the cmt exam score (cmtesv ) is used, with a maximum score of before rasch modification. one parameter of the cmtesv is the vibration sense. using a rydel tuning fork, a care-giver measures vibration sense in a patient's feet, ankles, and knees and a score is determined by the severity of reduced vibration sense. in cmt a patients, % received a score of out of , noted by a reduced vibration sense at the knee. in order to capture a more sensitive vibration measurement, we tested ways of taking the total raw score of the rydel tuning fork at each point of vibration sense, the toe, ankle, and knee bilaterally. scores range from zero to , with reflecting full vibration sense. in this modified measurement, vibration sense scores varied in wider distribution. for cmt a patients captured in this studied, a modified vibration sense score results in a potentially more sensitive total cmtesv score. bae dw , an jy . st. vincent's hospital, the catholic university of korea, suwon, korea. diabetic lumbosacral radiculoplexus neuropathies (dlrpn) are usually subacute painful, monophasic, asymmetrical lower limb neuropathies with incomplete recovery due to ischaemic injury and microvasculitis. the diagnosis relies mostly on clinical suspicion and characteristic electromyographic findings. however, in acute phase, neuroimaging has more important diagnostic significance than electrophysiological studies. here we describe mri and ultrasound findings in a -year-old woman with dlrpn who was diagnosed with diabetes three years ago, but has not received any other treatment. she had a -day history of acute-onset severe pain with weakness of muscles innervated by left femoral and obturator nerve and decreased sensation in left l -l dermatome. nerve conduction studies showed reduced amplitude in left femoral nerve and electromyography showed only increased insertional activity in left iliopsoas muscle and no volitional activity in muscles innervated by left femoral and obturator nerve. ultrasound revealed increased cross-sectional area (csa) of left femoral and lateral femoral cutaneous nerve. mri showed enhancement in left l , nerve roots and proximal femoral nerve and increased signal intensity in left iliacus and iliopsoas. we diagnosed her with dlrpn and started corticosteroid. nerve ultrasound has not been previously reported in a patient with cabin air in commercial airliners originates from aircraft engines or auxiliary power units. this bleed air may occasionally be contaminated with hydraulic fluids and engine oil that contains a number of potentially hazardous chemicals including tricresyl phosphate (tcp). over the last years reports are emerging about aircrew members that experience symptoms such as tingling or burning of extremities in addition to headache, and vertigo. this "aerotoxic syndrome" is controversially discussed in the literature and has been attributed to exposure to organophosphate contaminated cabin air. since tcp has been associated with peripheral neuropathy we aimed to determine the frequency of peripheral neuropathy among frequent flyers. civilian air crew members and frequent flying passengers (m:f = : , median age years, median exposition time in aircrafts of . hours) were examined at the university hospital of cologne or at the frankfurt airport (iata code fra) by a detailed questionnaire of past medical conditions, a standardized neurological examination and nerve conduction studies of sural, tibial, and ulnar nerves. we identified subjects with clinical and electrophysiological evidence for large fiber peripheral neuropathy. incidence of peripheral neuropathy was not correlated to exposition time in aircrafts. in addition subjects showed signs of ulnar neuropathy, subjects reported abnormal vibration sensation, subjects suffered from gait imbalance and individuals reported tingling of extremities. our study shows a . % prevalence of large fiber peripheral neuropathy among frequent flyers. comparison of these data with prevalence rate in an age-matched control group will reveal a possible association of chronic exposure to cabin air and risk for peripheral neuropathy. the high incidence of the symptom tingling in our cohort warrants further studies to determine the risk for small fiber neuropathy in this condition. intravenous immunoglobulin (ivig) are human igg derived from plasma pools of healthy donors. although there are studies in literature evaluating their effectiveness in different pathological animal models, there are no data about their possible role on bortezomib (btz)-induced peripheral neuropathies. female wistar rats were treated following a preventive schedule (btz and ivig co-treatment for and weeks) and a therapeutic schedule ( weeks of btz treatment followed by a -week ivig-btz co-treatment). caudal nerve conduction velocity (ncv), plantar and dynamic tests were performed at different time points. animals were sacrificed after ws (acute phase) or ws (chronic phase) and tissue samples (dorsal root ganglias -drg-, sciatic nerve, caudal nerve, skin) were collected for morphological, morphometrical and immunohistological analysis.in the preventive schedule, ivig was not able to rescue caudal ncv reduction caused by btz neither after nor after weeks of co-treatment. same results were observed in the therapeutic schedule. on the other hand, the evaluation of mechanical allodynia and cold hyperalgesia showed that ivig injection protected from btz effect in both treatment schedules. morphometric analysis evidenced that, even if not statistically significant only the preventive schedule has a tendency to protect the caudal nerve from btz damage. this result is consistent with the morphological evaluation of the nerve. also, intra-epidermal nerve fibers density was preserved in the preventive schedule but not in the therapeutic one. finally, sciatic nerve and drg macrophage infiltration levels tended to be reduced in the therapeutic schedule and were brought back to ctrl (rats not treated or injected with ivig alone) levels in the preventive one. in conclusion, we were able to demonstrate for the first time that ivig treatment especially used as preventive treatment option may reduce btz-induced neuropathic painful pointing out the possible role of inflammation in the pathogenesis of this invalidating pathology. this work was supported by kedrion spa. we studied the prevalence, the molecular cause and clinical presentation of hereditary motor neuropathies in a large cohort of patients from the north of england. detailed neurological and electrophysiological assessments and next generation panel testing or whole exome sequencing were performed in patients with clinical symptoms of distal hereditary motor neuropathy (dhmn, patients), axonal motor neuropathy (motor cmt , patients) or complex neurological disease predominantly affecting the motor nerves (hmn plus, patients) . the prevalence of dhmn is . affected individuals per . inhabitants ( % confidence interval: . - . ) in the north of england. causative mutations were identified in out of index patients ( . %). the diagnostic rate in the dhmn subgroup was . %, which is higher than previously reported ( %). we detected a defect of neuromuscular transmission in cases and identified potentially causative mutations in patients with demyelinating multifocal motor neuropathy. many of the genes were shared between dhmn and motor cmt , indicating identical disease mechanisms therefore we suggest changing the classification and include dhmn also as a subcategory of cmt. abnormal neuromuscular transmission in some genetic forms provides a treatable target to develop therapies. barohn rj , gajewski b , pasnoor m , brown l , herbelin l , kimminau k , jawdat o , parks c , shlemon p , dimachkie mm and the pain-controls study team . the university of kansas medical center, kansas city, ks, usa. cryptogenic sensory polyneuropathy (cspn) is a common slowly progressive neuropathy that affects adults and presents with significant neuropathic pain for which multiple medications have been tried including antiepileptics, antidepressants, topicals and narcotics. a web based survey among neuromuscular experts suggested pregabalin as being more effective than other medications, however there are presently no comparative studies to assess the most effective medication. the objective of this study was to determine which of the pharmaceutical therapies (pregabalin, duloxetine, nortriptyline or mexiletine) is most effective for neuropathic pain and best tolerated in cspn. to achieve this objective we performed a prospective randomized open labelled comparative effectiveness adaptive design study of cspn patients through the patient centered outcomes research institute (pcori). cspn patients who fulfilled the inclusion and exclusion criteria were enrolled into this study. patients underwent a baseline neurological evaluation and randomly assigned to one of the neuropathic medications for months. the primary outcome is the change in likert-like pain scale. the secondary outcomes included nih pain interference scale, nih fatigue interference scale, nih sleep disturbance scale, sf- and adverse events. the outcome measures are performed at baseline, month , and . statistical analysis using bayesian adaptive design developed by berry consultant software will be performed to determine winner and losers (winner = greater than point improvement in pain). total number of patients to be enrolled is . recruitment has been challenging and a number of recruitment techniques have been used. to date, there have been patients screened, patients randomized from us sites. anticipated completion of enrollment by june and end of final patient assessment by september . interim analysis performed after first patients completed their months as part of bayesian adaptive design analysis and occurs every weeks. the distribution of randomization of patients to the medications at last adaptive design randomization was . % to medication , % to medication , . % to medication and . % to the th medication. this study may give physicians and patients evidence for future management of cspn patients. transthyretin amyloidosis (attr), which encompasses a group of disorders with significant clinical variability, is caused by transthyretin (ttr) derived amyloid deposition. the clinical aspects of autonomic nervous system involvement in attr are only partially known. the ongoing, multinational, longitudinal, observational transthyretin amyloidosis outcomes survey (thaos) provides the opportunity to expand our understanding of dysautonomia in attr. data from all symptomatic subjects enrolled in the thaos registry with a diagnosis of attr (cut-off date: january , ) were assessed for the presence and temporal course of autonomic symptoms, genotype and phenotype associations, and clinical burden according to the frequency and severity of symptoms. of symptomatic subjects enrolled in thaos, ( . %) had autonomic symptoms at enrollment including: gastrointestinal ( subjects, . %), urinary ( , . %), erectile dysfunction ( , . %), orthostatic hypotension ( , . %), xerophthalmia ( , . %) and dyshydrosis ( , . %). subjects with autonomic manifestations, compared with those without, were younger (mean age [standard deviation, sd] of . [ . ] vs . [ . ] years), with a longer duration of attr symptoms ( . [ . ] vs . [ . ] years). autonomic dysfunction was less common with wild-type attr ( of subjects, . %) than in mutation groups: val met ( / , . %); non-val met/non-cardiac ( / , . %); and "cardiac mutations" (val ile, leu met, thr ala, or ile leu mutations; / , . %). similarly, time (mean [sd] , years) from first attr symptoms to onset of autonomic symptoms, was longest for wild-type attr ( . [ . ] ) followed by "cardiac mutations" ( . [ . ]), non-val met/non-cardiac ( . [ . ]), and val met ( . [ . ] ). autonomic symptoms were present at disease onset in over a third of subjects ( , . %) . autonomic dysfunction was less frequent in subjects with cardiac phenotype ( of subjects, . %), than with mixed ( / , . %) or neurologic ( / , . %) phenotypes. the burden of autonomic symptoms (mean [sd] ) varied by genotype, val met ( . [ . ] , non-val met/non-cardiac ( . [ . ], "cardiac mutations" ( . [ . ]), wild-type attr ( . [ . ]), and by phenotype, mixed ( . [ . ]), neurologic ( . [ . ]), cardiac ( . [ . ]). dysautonomia is common, and a significant burden, in subjects with hereditary forms of attr. its prevalence is higher in val met than in other genotypes, and in the neurologic or mixed phenotypes. the objective of this study is to assess the usefulness of motor unit number index (munix) technique in charcot-marie-tooth type a (cmt a) disease and to test correlation between munix and clinical impairment. munix technique was performed in abductor pollicis brevis (apb), abductor digiti minimi (adm) and tibialis anterior (ta) muscles in the non-dominant side. a munix sum score was calculated by adding munix of these muscles. muscle strength was measured using the mrc (medical research council) scale. disability was evaluated with several functional scales including cmt neuropathy score version (cmtnsv ) and overall neuropathy limitation scale (onls). cmt a patients with known pmp gene duplication were enrolled. the munix of the adm, apb and ta muscles were correlated with the mrc of the corresponding muscle (p< . ). munix sum score was correlated with clinical scales: cmtnsv (r=− . , p< . ), onls (r=− . , p< . ). in conclusion, munix correlates with muscle strength and clinical measurements of disability in cmt a patients. the munix technique evaluates motor axonal loss and correlates with disability. the munix sum score may be a useful outcome measure of disease progression in cmt a. the objective of this study was to assess the usefulness of mri in charcot-marie-tooth type a (cmt a) disease and to test correlation between muscle fat fraction and clinical impairment. mri was performed in the non-dominant lower limb of cmt a patients and healthy controls. fat fraction of tibialis anterior muscle, cross section area and volume of sciatic nerve were determined. muscle strength of dorsiflexion was measured using a dynamometer. disability was evaluated with cmt neuropathy score version (cmtnsv ). cmt a patients with known pmp gene duplication were enrolled. fat fraction of tibialis anterior muscle was significantly increased in patients compared to healthy controls. it was correlated with muscle strength (r=− . , p< . ) and cmtnsv score (r=− . , p< . ). cross section area and volume of sciatic nerve were significantly increased in patients compared to healthy controls. in conclusion mri fat fraction correlates with muscle strength and clinical measurement of disability in cmt a patients. it may thus be a useful outcome measure of disease progression in cmt a. basta i , peric s , cobeljic m , bjelica b , bozovic i , kacar a , nikolic a , rakocevic stojanovic v , stevic z , lavrnic d . neurology clinic, clinical center of serbia, school of medicine, university of belgrade, belgrade, serbia. there is a complete lack of data about epidemiological and clinical features of chronic inflammatory demyelinating polyneuropathy (cidp) in serbia and surrounding countries. furthermore, there is a striking scarcity of information about quality of life (qol) in cidp patients and all qol studies have been conducted in countries with high standards of health care. in august we have designed the iness (inflammatory neuropathy study of serbia) in order to comprise as many patients with cidp from serbia, republic of srpska (bosnia and herzegovina) and montenegro covering population of more than nine million people. our first aim is to analyze overall impact of cidp on physical, mental and social areas of life measured with generic, symptom specific and disease specific questionnaires -sf- , inqol and cap-pri, respectively. furthermore, we aim to analyze influence of the disease on patients' working status and presence of depressive mood measured by beck's inventory. following features of patients are included: sociodemographic data, clinical aspects of the disease, level of disability, severity of sensory symptoms, presence of comorbidities, electrophysiological characteristics, as well as fatigue, autonomic symptoms and neuropathic pain. we intend to define the most significant predictors of decreased qol in order to focus on patients with the highest risk and to improve care of cidp. we also want to see if cidp patients in complete remission as per clinical findings still have reduced quality of life. we have recruited patient so far and we expect to include around subjects overall. we will present the first data of the study at the pns meeting . beaudonnet g , prud'hon s , cauquil c , labeyrie c , not a , bouilleret v , adams d . neurophysiology chu bicêtre, le kremlin bicêtre, france; neurology chu bicêtre, le kremlin bicêtre, france. familial amyloid neuropathy (fap) is a life-threatening disease of autosomal dominant inheritance due to transthyretin (ttr) gene mutation, a liver-produced protein. current treatments slow down its natural course and are indicated from the very first objective symptoms. we aimed to evaluate two neurophysiological markers: sympathetic skin response (ssr) and heart rate variability (hrv) in the early detection of sympathetic damages due to fap. ssr and hrv were assessed in ttr gene mutated patients with neither clinic nor electroneuromyographic abnormalities and controls matched on gender and age. cases were recruited consecutively from current care in the french reference center for rare diseases of bicetre university hospital. ssr was recorded on the two palms and on the sole of the left foot with to stimulations between and ma. hrv was registered during three conditions of seconds each: normal breathing, deep breathing ( cycles of seconds of inspiration and seconds of expiration) and valsalva manoeuver during seconds. valsalva ratio, defined by the ratio between the longest and shortest rr intervals, was significantly higher in the control groups after bonferroni correction (means of . and . , respectively, p< . ). there was no significant difference between the two groups for any ssr parameter, although means of amplitudes were systematically higher in controls than among cases. our results confirm that autonomic nervous fibers are damaged early in both clinical and electroneuromyographic asymptomatic patients mutated on the ttr gene. valsalva ratio seemed to be the most discriminative marker. long-term follow-up with test repetition and confrontation with cardiologic assessment will help to precise how these tests could be used in current care. they might help to identify high risk patients to propose them an appropriate early treatment and could be used to follow treatment efficacy. familial amyloidotic polyneuropathy (fap) was originally characterized by andrade as an axonal neuropathy which subsequently was found to be associated with a number of mutations in the plasma protein transthyretin (previously named prealbumin). it is now recognized that cardiomyopathy may be a significant factor in a majority of patients with the hereditary form of transthyretin amyloidosis (fap) and many of the transthyretin (ttr) mutations are associated with cardiomyopathy with no or minimal signs of peripheral neuropathy. attr-wt also called senile cardiac amyloidosis and senile systemic amyloidosis is recognized as late-onset, usually in the th or th decade of life, and the fact that the majority of patients are males. transthyretin neuropathy proven by nerve biopsy has been rarely reported in this population. here we report our experience with patients having attr-wt characterized by cardiomyopathy but also with varying degrees of peripheral neuropathy. clinically, the neuropathy appears as typical axonal or mixed axonal/demyelinating neuropathy as is seen in fap. pathologically, two types of ttr deposition have been found, ( ) intraneural ttr amyloid deposits as seen in fap are present in some patients and ( ) other patients have extensive vascular deposition of amyloid in both perineural arteries and veins without deposits within nerve trunks. in conclusion, peripheral neuropathy may definitely be a part of the attr-wt clinical presentation and with the increase in numbers of attr-wt cardiomyopathy patients being identified, it is important to ascertain whether any evidence of peripheral neuropathy is due to the amyloidosis and not to compounding syndromes such as diabetes mellitus type ii. besora s , santos c , izquierdo c , martinez-villacampa m , simó m , bruna j , , velasco r , . neuro-oncology unit, hospital universitari de bellvitge-institut català d́oncologia, ĺhospitalet, barcelona, spain; medical oncology department, institut català d́oncologia, ĺhospitalet, barcelona, spain; department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. oxaliplatin (oxa) is the first-line chemotherapy agent in the treatment of colorectal cancer (crc). oxa-induced peripheral neuropathy is the most frequent long-term side-effect. retreatment with oxa is frequently considered in patients as salvage treatment. patients receiving oxa-based chemotherapy regimen at least twice at our institution between and were reviewed. the aim of this study was to investigate whether retreatment with oxa increases the risk of developing or worsening previous oxa-induced peripheral neuropathy. the severity of neuropathy was measured by national cancer institute-common toxicity criteria (nci), total neuropathy score (tns) © and nerve conduction studies. one hundred twenty-five crc patients were included. median age was years. after first-line oxa-based chemotherapy, . % of patients developed neuropathy according nci, after a median of [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. severity of neuropathy was grade ( . %), grade ( %), and grade ( . %). median time to retreatment with oxa was months. frequencies of neuropathy before retreatment were as follows: . % grade , . % grade , and . % grade . after retreatment, severities of neuropathy were . % grade and . % grade . no patient developed grade . . % of patients did not develop neuropathy. peripheral neuropathy was the reason for stopping prematurely treatment after first-line and retreatment in . % and . % of patients, respectively. worsening of previous nci score was observed in . % of patients. the great majority of patients ( . %) remained within the same nci score than before retreatment after median [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. among those patients that did not develop neuropathy after first treatment (n= ), only and patients developed grade and , respectively, after a median of [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. among those patients who initially developed grade and neuropathy, no differences in tnsc © scores just before and after finishing retreatment with oxa were identified ( [ - ] vs [ - ], p= . ). retreatment with oxa in crc patients is a feasible option even in patients who developed moderate or severe neuropathy previously. lack of worsening of previous neuropathy is observed in the great majority of patients. neurological monitoring of patients candidates to retreatment with oxa should be considered. bessaguet f , sturtz f , magy l , , desmouliere a , bourthoumieu s , demiot c . ea -myelin maintenance & peripheral neuropathy, faculties of medicine and pharmacy, university of limoges, limoges, france; department of neurology, reference center for rare peripheral neuropathies, university hospital of limoges, limoges, france. prolonged pressure and the resulting local ischemia are widely accepted as the primary etiology of skin pressure ulcers (pus) but precise mechanisms of their formation remain unclear. in this study, we wanted to study the potential role of sensory small nerve fibers in regulation of inflammation during pus formation. to achieve this goal, we developed a mouse model of a purely sensory neuropathy and this was induced by resiniferatoxin (rtx). in this model, seven days after a single injection of rtx ( g/kg; ip), mice present a thermal and mechanical hypoalgesia associated with large substance p (sp) and calcitonin gene-related peptide (cgrp) depletion without neurodegeneration. this model mimic quite well what is observed in early stages of sensory nerve fiber defect. studies have shown that sp and cgrp are involved in cutaneous inflammation regulation. in fact, these neuropeptides are released by sensory fibers and are pro-inflammatory mainly through recruitment of immune cells and vasodilation. thus, we studied gene expression of pro and anti-inflammatory cytokines by a rna array approach during pus formation in control and rtx mice. seven days after a single injection of rtx, epidermis, dermis and subcutaneous tissue layer were pinched with magnetic plates during hours. pressure induced a stage pus. gene expression was evaluated in each compressed area h after pressure. results showed mainly a down-regulation of gene expression in pus of rtx mice compared to control mice. a decrease of cgrp/sp in skin sensory small fiber increased pus formation associated with an increase of interleukins (il)- , il- , il- and il- expression and a decrease of il- expression in rtx mice. supplementary experiments with rt-qpcr for each cytokine will be necessary to confirm these preliminary results. these observations suggest a cgrp/sp role in regulation of cytokines expression during pus formation. the new inflammatory profile exhibited in this study might help in the design of new treatments improving the quality of life of neuropathic patients prone to developing bedsores. bhattacharyya bj , jayaraj nd , belmadani a , ren d , rathwell ca , hackelberg s , miller rj , menichella dm . department of neurology, northwestern university, chicago, il, usa; department of pharmacology, northwestern university, chicago, il, usa. painful diabetic neuropathy (pdn) is one of the most common and intractable symptoms of diabetes, affecting % of diabetic patients. the hallmarks of pdn are neuropathic pain and small fiber degeneration, manifested by the loss of dorsal root ganglion (drg) nociceptor axons. neuropathic pain is associated with nociceptor hyper-excitability in the absence of physiologically appropriate stimuli. in states of neuropathic pain, drg nociceptors become increasingly responsive to a variety of excitatory influences, including inflammatory cytokines. in particular, we have shown that stromal cell derived factor- (sdf- ) and its receptor cxcr are necessary for the generation of neuropathic pain in mouse models of pdn. however, the molecular mechanisms leading to the hyper-excitability of drg nociceptors in pdn are unknown, as are the mechanisms leading to small fiber degeneration. this fundamental gap in our knowledge represents a critical barrier to progress in developing novel therapeutic approaches for pdn. the objective of this study is to identify the molecular cascade linking cxcr /sdf- chemokine signaling to drg nociceptor hyper-excitability, neuropathic pain, and small fiber degeneration in pdn. drg nociceptors can be identified by a series of molecular markers, including expression of the sodium channel na v . . indeed, > % of na v . -expressing drg neurons are nociceptors. feeding mice a high fat diet (hfd) for several weeks induces glucose intolerance, obesity, and mechanical allodynia, a particular pain hypersensitivity associated with pdn. using the hfd model combined with dreadd receptor technology, we have shown that reducing excitability of na v . -expressing neurons prevents and reverse neuropathic pain, neuronal calcium overload, mitochondrial dysfunction, and small fiber degeneration. furthermore, we have shown that cxcr receptors are necessary for neuropathic pain and small fiber degeneration in pdn. taken together these data demonstrate that na v . nociceptor hyperexcitability in pdn is driven through the activation of cxcr receptors. inhibition of hyperexcitability can prevent and reverse the development of pdn. furthermore, these observations will advance our understanding as to how changes in excitability, calcium influx, and mitochondrial dysfunction in nociceptors contribute to neuropathic pain and small fiber degeneration in pdn, which is a critical barrier to progression for effective and disease modifying treatment for pdn. bianco m , terenghi f , gallia f , nozza a , scarale a , fayoumi mz , giannotta c , morenghi e , nobile-orazio e . poems (polyneuropathy, organomegaly, endocrinopathy, m protein and skin changes) syndrome is an unusual multisystem disease with neurological disability due to a severe disabling polyneuropathy, with high mortality by multiorgan failure. peripheral blood stem cell transplantation (pbsct) is considered the treatment of choice for poems while lenalidomide is the most promising therapy for patients not eligible for pbsct. the aim of the present study was to compare the long-term effects on clinical, biological and neurophysiologic parameters in patients with poems treated with lenalidomide or pbsct. the clinical, biological and neurophysiologic data were reviewed in poems patients treated with pbsct (n: ) or lenalidomide (n: ). the mrc sumscore on muscles, onls scale, vegf serum levels and nerve conduction studies were assessed before (t ) and after (t ) and years (t ) of treatment and the differences were compared using anova. combining the two groups of patients, there was a significant improvement after treatment in the mean mrc sumscore (t = ± ; t = ± ; t = ± ; p = . ), in the mean onls score (t = . + . ; t = . + . ; t = . + ; p = . ), in the ulnar mean distal motor latency (t = . ± . msec; t = . ± . msec; t = . ± . msec; p = . ), distal compound muscle action potentials amplitude (t = . ± . mv; t = . ± . mv; t = . ± . mv; p = . ), motor conduction velocity (t = . ± . m/sec; t = ± m/sec; t = . ± . m/sec; p= . ) and serum vegf levels (t vs t : p = . ; t vs t : p = . ). the difference was also significant when we separately analyzed patients treated with lenalidomide and pbsct and there was no difference between the two groups in any of the analyzed parameters. treatment with pbsct and lenalidomide significantly and similarly improved clinical, biological and neurophysiologic parameters in patients with poems syndrome up to two years. since pbsct may not be suitable for all patients, lenalidomide may represent an effective and a valuable alternative in these patients or in those relapsing after pbsct inducing a prolonged clinical, biological and neurophysiologic improvement. bis d , tao f , abreu l , sleiman p , hakonarson h , zuchner s and inherited neuropathy consortium. dr. j.t. macdonald department for human genetics, hussman institute for human genomics, university of miami, miami, fl, usa; center for applied genomics, the children's hospital of philadelphia, philadelphia, pa, usa. inherited peripheral neuropathies are clinically and genetically heterogeneous diseases that can cause distal muscular atrophy and sensory loss. alleles in over one hundred different genes have been shown to cause peripheral neuropathies; yet, greater than % of axonal neuropathy patients do not receive a genetic diagnosis. large scale exome studies are now beginning to be sufficiently powered to perform mutational burden analysis. this approach compares damaging allele frequencies of cmt cases with a control group to identify additional causes for neuropathies. this approach will also identify genes that require an oligogenic inheritance to cause a phenotype. in a deviation from the classic linkage-based and heuristic variant filtering approaches to gene identification, we are performing burden analyses in a large cohort of cmt families. in known neuropathy genes, we saw that neuropathy cases carried on average . rare, non-synonymous variants, while unrelated non-neuropathy controls harbored . variants (p= . e- , mann-whiney u-test). enrichment of rare, non-synonymous variants in cmt disease genes within inherited peripheral neuropathy cases suggests the presence of multiple weaker alleles in individual patients. we also performed an unbiased exome-wide gene-based burden analysis and ranked genes after multiple testing correction. several new candidate genes were identified that need further follow up conformational studies. a number of known cmt and related genes were observed in the list of top candidates. we are currently analyzing additional aspects of this sample and are actively seeking more cmt exomes to enlarge our study. in summary, statistical methods traditionally reserved for more 'common' phenotypes' increasingly are becoming available for rare disease genetics. c bl/ mice were treated with bortezomib alone or in combination with monastrol. neuropathic changes were assessed by nerve conduction studies and histological analysis. analysis of axonal morphology was performed with light and electron microscopy. anti-neoplastic properties of monastrol alone and in combination with bortezomib were assessed in different blood cancer cell lines. prolonged treatment with bortezomib induced a sensory neuropathy in mice. significant changes in axonal morphology correlated with reduced function of peripheral nerves. the administration of monastrol substantially ameliorated morphological features of axonal alterations and sensory neuropathy. cytotoxicity studies in blood cancer cell lines showed no interference of monastrol with the cytostatic effects of bortezomib. our data indicate that monastrol may alleviate bortezomib induced neuropathy. the favorable cytotoxic profile of monastrol makes it an interesting candidate as neuroprotective agent to prevent bortezomib-induced neuropathy. boczonadi v , meyer k , gonczarowska-jorge h , , bartsakoulia m , roos a , , bansagi b , zahedi rp , talim b , bruni f , kaspar b , , lochmüller h , boycott km , müller js , horvath r . jwmdrc, wtcmr, igm, newcastle university, newcastle upon tyne, uk; rinch, columbus, oh, usa; leibniz-institute für analytische wissenschaften-isas-e.v., dortmund, germany; capes foundation, brazil; department of pediatrics, hacettepe university children's hospital, ankara, turkey; dbbb,university of bari aldo moro, bari, italy; department of neuroscience, the ohio state university, columbus, oh, usa; department of genetics, cheo, university of ottawa, ottawa, canada. while autosomal dominant mutations in gars, encoding the glycyl-trna synthetase, have been identified in patients with charcot-marie-tooth peripheral neuropathy (cmt d) and distal spinal muscular atrophy type v (dsma-v), autosomal recessive mutations cause mitochondrial disease affecting skeletal muscle and heart. gars is a bi-functional enzyme and it is responsible for normal protein translation both in mitochondria and the cytoplasm. in this study we have focused on the mitochondrial function of the gars by investigating a mouse model (gars c r) , human fibroblasts and induced neuronal progenitor cell lines (inpcs). mild mitochondrial abnormalities were detected in skeletal muscle of the gars c r mice while no other tissues were affected. control and patient fibroblasts harboring gars mutation were directly converted into inpcs. we identified tissue specific impairment of mitochondrial function in neuronal cells carrying not only recessive but also dominant gars mutations, suggesting neuron-specific effects of mitochondrial alterations.comparative proteomic analysis of inpcs showed significant changes in mitochondrial proteins. furthermore, the reduction of the vesicle-associated membrane protein-associated protein b (vapb) and its downstream pathways in gars-deficient inpcs suggests that altered mitochondria-associated endoplasmic reticulum (er) membranes (mam) may also contribute to the motor neuropathy. boczonadi v , king ms , bansagi b , roos a , , eyassu f , borchers c , lane m , ramesh v , lochmüller h , pyle a , griffin h , smith ac , chinnery pf , , alan j robinson aj , edmund rs kunji ers , horvath r . jwmdrc, wtcmr, igm, newcastle university, newcastle upon tyne, uk; mitochondrial biology unit, medical research council, cambridge; leibniz institute of analytic sciences (isas), dortmund, germany; uvic-genome bc proteomics centre, vancouver, canada; paediatric neurology, royal victoria infirmary, newcastle upon tyne foundation hospitals nhs trust, newcastle upon tyne, uk. members of the mitochondrial carrier family (slc ) transport nucleotides, keto acids, amino acids, fatty acids, co-factors and inorganic ions across the mitochondrial inner membrane. several inherited diseases with very variable clinical presentations are associated with dysfunctional mitochondrial carriers. we report a patient with childhood-onset spinal muscular atrophy and mitochondrial myopathy caused by a homozygous mutation in slc a , encoding the mitochondrial oxodicarboxylate carrier (odc). the mutation renders the carrier dysfunctional and, consequently, -oxoadipate cannot be imported into the mitochondrial matrix. computer modelling of the metabolic defect caused by the mutation predicted that the impaired transport leads to accumulation of -oxoadipate, pipecolic acid and the known neurotoxin quinolinic acid, which were precisely confirmed by targeted metabolomics in serum and urine. exposure of -oxoadipate and quinolinic acid reduced the level of mitochondrial complexes in sh-sy y cells in-vitro suggesting a possible pathomechanism. here we demonstrate that -oxoadipate and quinolinic acid are toxic for spinal motor neurons and their increased levels may contribute to neuropathy. and families have been reported. interestingly, most mutations target the same amino acid residue (k e, k m, k n, k t) in the highly conserved -crystallin domain of the hspb protein. the spectrum of diseases caused by mutations in the hspb gene was recently expanded to distal myopathy. hspb is ubiquitously expressed, but is highly expressed in motor neurons and muscles. the hspb is a chaperone that participates in clearing misfolded poly-q containing proteins such as mutant huntingtin and ataxin- involved in respectively huntington's disease and spino-cerebellar ataxia. hspb directly interacts with the co-chaperone bag and their role in chaperone-assisted selective autophagy is well described. to delineate the molecular deficits and functional consequences of hspb mutations we generated a knock-in (ki) mouse model for the k n missense mutation mimicking the neuropathy phenotype. we observed that homozygous mutant mice (hspb k n/k n ) develop a progressive axonopathy, with decreased compound motor action potential amplitudes, and loss of large and medium myelinated axons. this results in locomotor deficits with an impaired performance at the rotarod and grip strength tests. at the ultrastructural level, the hspb -ki model displays severe signs of axon degeneration and a clear myofibrillar myopathy, as observed in some patients with hspb mutations. interestingly, hspb positive aggregates were found in the sciatic nerve and gastrocnemius muscle of our mutant mice. additionally, our model allowed us to generate hspb knock-out (ko) mice using the same targeting vector. strikingly, the homozygous hspb -ko animals do not show any sign of axonopathy and display a much milder myopathy than the hspb -ki animals. these data suggest that part of the pathomechanisms is due to toxic gain-of-function of the mutant protein. boutahar n , reynaud e , nasser y , camdessanché jp , antoine jc . university hospital, saint-etienne, france. dysimmune sensory neuronopathies (snn) depend on neuron cell death induced by an inflammatory reaction in dorsal root ganglia. we have recently identified the intracellular tyrosine kinase (trk) domain of the fibroblast growth factor receptor- (fgfr ) as the target of antibodies in a subset of patients with non paraneoplastic snn. fgfr is one of the four fgfrs and has been involved in sensory neurons maintenance during development and cell death induction after axotomy. fgfrs ligand fixation results in the activation of several intracellular pathways through adaptator protein interactions with the trk domain. in particular ras activation may lead to cell proliferation or apoptosis through erk / or p map kinase signaling. the p mapk pathway is also involved in neuronal cell death induced by nmda and ampa receptor activation. as fgfr is a cell surface protein, human antibodies may interfere with the receptor functioning as a growing number of evidence has showed with other cell surface antibodies in neurological diseases. to test this hypothesis we developed an in vitro model using fvbn mice cortical neurons culture exposed to a rabbit polyclonal antibody reacting with the trk domain of fgfr . comparatively to normal rabbit iggs, the fgfr antibody induced neuron cell death in a dose dependent manner. neuron cells were exposed to fgfr antibody concentration leading to - % cell death in absence or presence of the p mapk inhibitor sb and the expression of fgfr , glur subunit of ampa receptors and nr subunit of nmda receptor was measured by quantitative rt-qpcr. the fgfr antibody induced an upregulation of fgfr while the glur and nr subunits were modulated. these changes were prevented in presence of sb . these preliminary results indicate that anti-fgfr iggs may interfere with the functioning of the intracellular domain of the protein and the expression of nmda and ampa receptors through the p map kinase pathway. this model may be used to test the effect of human anti-fgfr iggs in vitro. braathen gj , tveten k , holla Øl , busk Øl , hilmarsen ht , svendsen m , høyer h . section of medical genetics, department of laboratory medicine, telemark hospital, skien, norway. next-generation sequencing (ngs) has during the last years entered the clinical diagnostics. ngs has proven to be very efficient in the diagnostics of disorders where multiple genes can be involved. our ngs-based targeted gene panel consists of hereditary neuropathy genes, i.e. mostly charcot-marie-tooth genes. this study is a retrospective study of clinic samples received between may and february . we describe the identified novel likely pathogenic sequence variants, according to international guidelines. in this period we identified novel, not previously described, likely pathogenic sequence variants in the following genes: aars, fgd , gan, hint , litaf, lrsam , mme, mpz, nefl, pmp , sbf , sh tc and yars. there is now a large range of genes causing hereditary peripheral neuropathies and many likely pathogenic sequence variants. likely pathogenic sequence variants are not only identified in old well established neuropathy genes but also in the newer genes like mme. modelling brown-vialetto-van laere syndrome in c. elegans mutations in slc a and slc a , which encode the riboflavin transporters rfvt and rfvt . patients with rfvt deficiency exhibit proximal and distal limb weakness, sensory ataxia, diaphragmatic paralysis, optic atrophy, sensorineural deafness and bulbar palsy. riboflavin is critical for the biosynthesis of flavin mononucleotide and flavin adenine dinucleotide, essential cofactors for carbohydrate, amino-acid and lipid metabolism. mutations in slc a reduce or abolish rfvt expression resulting in impaired riboflavin uptake into sensory and motor neurons. high-dose riboflavin treatment can improve or stabilise a patient's condition, however the optimum dose and long term effects of riboflavin treatment, and disease pathomechanisms remains poorly understood. to further understand the pathophysiological consequences of slc a mutations, we propose developing an animal model for bvvl. caenorhabditis elegans (c. elegans) are small round transparent nematodes extensively used for studying the genetics and molecular biology of neurodegenerative diseases. there are two c. elegans riboflavin transporter genes, rft- and rft- . based on protein sequence homologies and expression profiles for both genes, rft- is the ortholog of rfvt . the expression of rft- is regulated by riboflavin availability and knock-down of the rft- gene by sirna perturbs c. elegans development. our aim is to develop a knock-in c. elegans model of bvvl. human rfvt and c. elegans rft- protein sequences were aligned with clustal omega to identify conserved amino acid residues associated with bvvl mutations. the amino acid involving the l p rfvt mutation is conserved in rft- (residue l ). to create our model, we will introduce the l p rft- mutation into the rft- locus in c. elegans genomic dna using crispr/cas- technology. this bvvl c. elegans model will allow us to explore the pathogenic consequences of rfvt deficiency underlying motor nerve degeneration and to evaluate drug therapy regimes by determining the optimal riboflavin dose and treatment initiation, and trialing other compounds that may improve benefits seen with riboflavin supplementation. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune-mediated inflammatory disease of the peripheral nervous system. cidp can present chronic progressive or relapsing-remitting courses and can predominantly affect motor but also sensory nerve fibers causing weakness of proximal and distal muscles. it represents the most common chronic autoimmune neuropathy and is pathologically characterized by focal inflammatory-mediated demyelination followed by axonal degeneration. recently, we have developed a new animal model for cidp, the chronic-ean, induced in lewis rats by active immunization with s-palmitoylated p ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) peptide. this model fulfills electrophysiological criteria of demyelination with axonal degeneration, a result confirmed by immunohistopathology. the late phase of the chronic disease is characterized by an accumulation of il- + cells and macrophages in sciatic nerves and as well as high serum il- levels. it is a reliable and reproducible animal model for cidp that can now be used for translational drug studies for chronic human autoimmune-mediated inflammatory diseases of the peripheral nervous system, particularly cidp, for which, there is a real need for new immunotherapies. the aim of this study was to test the therapeutic efficacy of ivig and a recombinant fc fragment (fcrec) in this new cidp animal model. treatments with ivig and fcrec proved effective in preventing further progression of cidp in rats. the therapeutic treatments not only decreased the maximal clinical scores of the cidp rats compared to albumin treatment but also abolished the disease chronicity. interestingly, a better efficacy of fcrec treatment compared to ivig was demonstrated at histological level, with the myelinated fibers well preserved and the greatly reduced accumulation of macrophages and il- + cells in sciatic nerves. ivig and fcrec therapeutic activities in this model can also be followed by measurement of antibodies in the serum and could therefore be used as biological markers. the current study provides for the first time direct evidence that ivig is effective in the treatment of cidp rats and suggests that a novel fcrec compound is more effective than ivig. it will contribute to the development of more effective and safer drugs for the treatment of autoimmune peripheral neuropathies like cidp. each time, one measurement with supramaximal stimulation according to the conventional ncs and another measurement with % enhanced stimulation intensity were conducted. we analyzed the results with special consideration of minimal and maximal fiber velocities and of the velocity with maximal fiber proportion. as a result, we detected statistically significant differences between cidp patients and controls in all these parameters. compared with controls, cidp patients had slower minimal and maximal fiber velocities and also the spectrum of motor conduction velocities was definitively shifted to slower velocities. slower minimal conduction velocities could be detected in some cidp patients by using the enhanced stimulation intensity. interestingly, in some cidp patients the conventional ulnar ncs were normal, but the collision technique showed fibers with a conduction velocity of less than m/s, indicating demyelination. to our knowledge, this is the first study using hopf's collision technique systematically in cidp patients. significant differences between the variation of ulnar motor nerve conduction velocity of cidp patients and controls could be detected. this method also showed signs of demyelination in some cidp patients with normal ulnar ncv. by enhancing stimulation intensity above the threshold of supramaximal stimulation in conventional ncs, the collision technique may be even more valuable. the clinical course of guillain-barré syndrome (gbs) is highly variable and some patients may develop treatment-related fluctuations (trfs) as an indication of ongoing disease activity and temporary treatment effect. other patients present as gbs, but subsequently develop repetitive relapses as an indication of acute-onset chronic inflammatory demyelinating polyneuropathy (a-cidp). this distinction is important because treatment may differ. we determined the frequency and clinical presentation of patients with gbs-trf and a-cidp in the first patients included in the international gbs outcome study (igos) with a follow-up of at least months. thirty-eight patients ( %) were excluded because of alternative diagnoses. of the remaining patients, ( %) had at least one trf, ( %) had a-cidp. preliminary analysis showed no significant differences between the groups (total-gbs, gbs-trf and a-cidp) for sex, age, sensory symptoms, cerebrospinal fluid results and mechanical ventilation. a-cidp patients had a median age of years (iqr - ), % was male, and all patients were treated in the acute phase of the disease (intravenous immunoglobulins (ivig) ( ), plasma-exchange (pe) ( ) or methylprednisolone (mp) ( )). gbs-trf patients has a median age of years (iqr - ), % was male and all patients received treatment (ivig ( ) and pe ( ). gbs-trf patients showed more antecedent events ( % versus %, p= . ), a higher gbs disability score (≥ ) at nadir ( % versus %, p= . ) and less frequently developed ataxia ( % versus %, p= . ) than patients with a-cidp. onset to nadir was longer in a-cidp than in gbs-trf ( days (iqr - ) versus days (iqr - ), p= . ) and the total gbs group ( days (iqr - ), p= . ). the time until the first clinical deterioration tends to be longer in the a-cidp patients (median days (iqr - ) versus median days in the gbs-trf group, not significant). the diagnosis a-cidp was made after a median of days . in conclusion, this first analysis identified distinctive characteristics of gbs-trf and a-cidp in support of a different pathogenesis that may help with early identification of these disorders in clinical practice. additional results will be presented at the conference. chronic inflammatory demyelinating polyneuropathy (cidp) is a disorder with a highly diverse clinical presentation, electrophysiological phenotype, response to treatment and outcome. this heterogeneity may indicate the presence of distinct subtypes of cidp, which may have a different pathogenesis and require more personalized treatment. the international cidp outcome study (icos) is a prospective, observational, international multi-center study that aims to describe this variation in clinical and electrophysiological subtypes and to define the clinical and biological determinants of these subtypes, disease activity, treatment response and outcome. in addition icos aims to provide an infrastructure for conducting new (therapeutic) studies in cidp, similar to the international gbs outcome study (igos). all patients fulfilling the efns/pns ( ) diagnostic criteria for cidp can be included in icos, independent of age, duration and severity of disease or treatment. we collect information on neurological deficits, diagnostic characteristics, various validated clinical outcome measures, previous and current treatment and we collect biomaterials (dna, cerebrospinal fluid, nerve biopsies and repeated serum samples). icos was started as a pilot study in dutch university centers. by february , patients were included in icos, patients recently diagnosed with cidp and previously diagnosed patients. included were ( %) males and ( %) females with a median age of years (iqr - ). the current cohort consists of classic (sensory-motor) cidp, madsam and pure motor variants. of the patients diagnosed in the past, patients ( %) were treated (intravenous immunoglobulins (ivig) ( ), prednisolone ( ), subcutaneous immunoglobulins (scig) ( ), dexamethasone ( ), plasma-exchange ( ) and ivig with methylprednisolone (mp) ( )). the recently diagnosed patients all received treatment (ivig ( ), dexamethasone ( ), plasma-exchange ( ) and patients were treated in a pilot study with ivig with methylprednisolone (mp)). the protocol has been evaluated and adjusted and will be shared with other researchers via the inflammatory neuropathy consortium and igos consortium. our aim is to include at least cidp patients worldwide with a minimum follow-up period of years. dominant mutations in glycyl trna synthetase (gars) cause inherited axonal neuropathy (charcot-marie-tooth type d). mutations in the mouse gars gene cause a similar phenotype, and represent valid disease models. we routinely use two mouse strains, one with a severe neuropathy, and one with a milder, later onset neuropathy. using these mouse models, we are exploring the mechanisms through which gars mutations cause peripheral axon degeneration. efforts include ribosome tagging to isolate ribosome-associated mrnas specifically from motor neurons, and non-canonical amino acid tagging to visualize and isolate newly synthesized proteins. taking advantage of the anatomy of motor neurons, we are able to analyze cell bodies separately from peripheral axons in both of these approaches. in addition, we are using these mouse models for preclinical studies testing gene therapy approaches to treat cmt d. consistent with our previous genetic studies in mice, knockdown of the mutant transcript, while preserving sufficient levels of the wild type, is a very successful approach when administered before the onset of neuropathy. we are now testing this approach in mice after the onset of symptoms, and in mice carrying a mutant gars allele associated with human disease. these studies are potentially translational, and also address mechanistic questions such as the timing and cell autonomy of the pathophysiology. burnor e , yang l , hao z , patterson k , quinn c , scherer ss , lancaster e . the university of pennsylvania, philadelphia, pa, usa; the second xiangya hospital, central south university, hunan, china. autoantibodies to two isoforms of neurofascin (nf and nf ) have been reported in patients with guillain-barre syndrome (gbs) or chronic inflammatory demyelinating neuropathy (cidp). it is not clear which of these responses reliably distinguish autoimmune neuropathies from other severe neuropathies, which responses are transient versus persistent, or which responses may target multiple isoforms of neurofascin. in addition, approximately % of neuropathy patients have no known cause, and it is unknown whether a subset of these patients may have autoantibodies to neurofascins. we have studied cohorts of patients with autoimmune neuropathy (n= ), genetic neuropathy (n= ), and idiopathic neuropathy (n= ) for igg and igm responses to neurofascins. neurofascin antibodies were found in ( %) of patients with autoimmune neuropathy, and ( . %) idiopathic neuropathy patients, but only % ( of ) in patients with genetic neuropathy. follow-up serum samples were available for positive cases. persistent responses were associated with chronic neuropathy while transient responses were seen in gbs or with remission of cidp. most patients had responses specific to either nf of nf . however, a particularly severe, treatment-resistant form of cidp, approaching a locked-in state, was seen in a patient with a unique response to all three isoforms of neurofascin (nf , nf , nf ). treatment of this patient with rituximab resulted in clinical improvement and resolution of the neurofascin antibody response. in conclusion, autoantibodies to neurofascins distinguish autoimmune neuropathies from severe genetic neuropathies, but the clinical phenotype may depend on the persistence and isoform specificity of the immune response. antibodies to the common domains shared by nf and nf may portend a severe but treatable neuropathy. a subset of idiopathic neuropathy patients may have an autoimmune mechanism. burns j , , sman ad , cornett kmd , wojciechowski e , , walker t , menezes mp , , mandarakas mr , rose kj , , bray p , , sampaio h , farrar m , , refshauge km , raymond j and the fast study group. university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia; university of new south wales, sydney, australia. exercise has undisputed benefits for human health and potentially as a treatment for neuromuscular disease. but there is also a risk of harm due to overwork weakness. we report the results of a -month randomised, double-blind, sham-controlled trial evaluating progressive resistance exercise of foot dorsiflexor muscles in pediatric cmt. sixty patients ( cmt a, cmt e, cmt f, cmt a, cmt c, cmtx , cmtx ) aged - years were randomly assigned to undergo -weeks ( sessions) of moderate-intensity progressive resistance exercise or sham exercise. the primary endpoint was change in isometric dorsiflexion strength between groups assessed by hand-held dynamometry (expressed as a z-score based on age-and sex-matched normative reference values, positive values indicate an improvement in strength). the primary safety endpoint was change in muscle and fat volume of the muscles responsible for dorsiflexion by mri between groups. secondary outcomes were function (balance, long jump, -min walk test), walking ability ( d gait analysis), self-reported ankle instability, parent-reported quality of life (physical summary, psychosocial summary and global impression of change scores), and adverse events. fifty-five ( %) children completed the trial. adherence was comparable between exercise ( %) and sham ( %) groups. while patients experienced muscle soreness during training in the exercise group, adverse events did not differ between groups. the mean z-score for dorsiflexion strength increased in the exercise group by % at -months (from − . ± . to − . ± . ) and decreased in the sham group by %, mirroring the natural history of cmt (from − . ± . to − . ± . ). between-group ancova-adjusted difference at -months was . ( %ci, . to . ; p= . ). the mean scaled scores for mri muscle volume and fat volume were comparable between groups at months (p> . ). the global impression of change scores favoured the exercise group at -months (p= . ) and -months (p= . ). there was no other measurable effect of exercise. pre-specified subgroup analyses according to age ( - years vs. - years) showed a larger treatment effect with exercise in adolescents. targeted progressive resistance exercise was effective at halting progression of dorsiflexion weakness without detrimental effect on muscle morphology or other signs of overwork weakness in children with cmt. byung-nam y , yoon-ho h , suk-won a , seok-jin c , jung-joon s . inha university hospital, incheon, south korea; seoul metropolitan government boramae medical center, seoul national university, seoul, south korea; joong ang university hospital, seoul, south korea; seoul national university hospital, seoul, south korea. a years old female patient visited the hospital with weakness in both upper limbs from months under the suspicion of motor neuron disease with the finding of extensive denervation changes on electromyography. whole spine mri showed ventral nerve rootlet enhancement in the c and t bundles. the emg had confirmed active denervation changes in the muscles innervated by bilateral c ∼c roots. the cerebrospinal fluid culture test showed a protein (csf) elevation to mg / dl. she got a steroid pulse therapy. she had the symptoms of dry mouth and dry eyes during the recent years. a salivary gland scan test was performed for the possibility of sjögren's syndrome, and as a result, absorption of the contrast agent in both parotid and submandibular glands was decreased. the shammer test showed mm on the right side and mm on the left side. she was diagnosed of sjögren's syndrome. after weeks, overall strength improvement was observed, and bilateral shoulder abduction was improved to mrc grade or higher. the follow-up spine mri also showed that the initially seen ventral nerve root enhancement was disappeared. the case had visited the hospital with major symptoms of weakness and atrophy of the muscles, showing similar pattern to motor neuron disease, and was diagnosed as inflammatory polyradiculopathy and confirmed as primary ss during differential diagnosis. this case suggests that primary ss may induce inflammatory polyradiculopathy, which shows motor symptoms as major symptoms rather than sensory symptoms, and that a fast and accurate diagnosis is needed in terms that it can be treated with steroids and appropriate immune suppressive agents. traumatic injuries to peripheral nerves are frequent. however, effective pharmacological treatments are lacking. curcumin, a polyphenol found in rhizomes of curcuma longa, has been shown to develop antioxidant, anti-inflammatory, and neuroprotective properties. however, due to its poor hydrosolubility and its extensive metabolism, the use of large curcumin doses is required for therapeutic purpose. the aim of the present study was to investigate the effects of a local infusion of a low curcumin dose on nerve regeneration and functional recovery after sciatic nerve injury in rats. the experiments were conducted in g sd male rats submitted to unilateral sciatic nerve crush at d . curcumin was solubilized in polyethylene glycol and continuously administered using osmotic pumps ( . mg/day until d ) with a catheter delivering the drug near the lesion site. functional analyses using von frey, beam walking, static sciatic index (ssi) and grip strength tests were carried out at d (reference test) and every week after injury (d , d , d , d and d ). in addition, an evoked electromyogram was performed at d and d . after euthanasia (d ), nerve and muscle samples were collected and analyzed by light and electron microscopy. functionally, a significant improvement of the mechanical sensitivity (+ %) was observed at d in the curcumin-treated group (n= ) vs. vehicle group (n= ). in curcumin-treated group, skillful walking and finger spacing of the ipsilateral paw (ssi) were fully restored respectively at d and d contrary to vehicle group. furthermore, curcumin treatment improved the grip strength recovery (+ % at d ). the electrophysiological results indicated a full recovery of motor nerve conduction velocities (mncv) after days of curcumin treatment, while mncv remained altered in vehicle group ( % of the mncv at d ). morphometric analysis of nerve sections using g-ratio showed an improvement in the thickness of the myelin sheath in curcumin treated animals (+ % vs. vehicle group). histological investigation of gastrocnemius muscle indicated decreased neurogenic lesions in curcumin group. proteomic analysis is currently under investigation to understand the mechanisms involved in curcumin effects. our data could lead to the development of new therapeutic strategies in peripheral nerve regeneration using low doses of curcumin. previous studies suggest that the metabolic syndrome (mets) is associated with distal symmetrical polyneuropathy (dsp), and that diabetes, pre-diabetes, and obesity are the main metabolic drivers. the aim of this study is to investigate the association of mets components with retinal and cognitive function in a bariatric surgery cohort prior to surgery. patients were recruited from the adult bariatric surgery clinic at the university of michigan and lean controls from a research website (no mets components based on ncep/atpiii definition). participants underwent extensive metabolic phenotyping including a glucose tolerance test and fasting lipid profile. dsp was defined using the toronto consensus definition of probable clinical neuropathy. retinal function was measured with frequency doubling technology perimetry (average mean deviation), and cognitive function with the nih toolbox (composite score). univariate linear regression models were used to evaluate the association between mets components and retinal and cognitive function. to date, we have recruited bariatric surgery participants and lean controls. in the bariatric population, the mean (sd) age was ( . ) with % female compared with a mean age of . ( . ) with % female in the lean group. in the bariatric group, . % had diabetes, . % pre-diabetes, and . % normoglycemia. the dsp prevalence was % in lean controls, . % in normoglycemic, . % in pre-diabetic, and . % in diabetic bariatric participants (p< . for trend). retinal function was . ( . ), − . ( . ), − . ( . ), and − . ( . ) (p= . for trend), and cognitive function was . ( . ), . ( . ), . ( . ), . ( . ) (p= . for trend) in these same groups for lean controls, normoglycemics, pre-diabetics and diabetics, respectfully. pre-diabetes (− . , %ci: − . ,- . ) was the only mets component associated with retinal function, and waist circumference was the only one associated with cognitive function (− . , %ci − . ,- . ). dsp and retinal function, but not cognitive function decline with worsening glycemic status. similar to previous data for dsp, pre-diabetes and obesity are associated with retinal and cognitive function respectively. interestingly, while clinical dsp is common in this population, clinical retinopathy and dementia are not, indicating that dsp may be the first metabolic complication in the morbidly obese. and anti-cntn antibody-positive cases were confirmed by cba and were of igg subclass in half of them. by cba we identified additional / ( %) anti-caspr seropositive patients, whose isotype is currently being tested. sera of anti-nfasc and anti-cntn igg seropositive patients and patients with anti-caspr antibodies stained paranodes by indirect immunofluorescence on mouse teased nerve fibers. of note, seronegative patients for known antibodies showed reactivity against node and/or paranodes. compared to other seronegative cipd patients, seropositive patients had more frequently subacute onset of the neuropathy and a younger age at onset, particularly for nfasc or caspr antibodies. weakness was more severe and was often associated with proprioceptive loss, sensory ataxia and tremor. neuropathic pain was not a feature of caspr -seropositive patients. frequent findings were increased distal motor latencies and temporal dispersions on nerve conduction study and a higher protein level in csf. finally seropositive patients tended to have a higher disability and showed worst response to ivig. rituximab was effective in one patient with anti-nfasc antibodies and two patients with anti-cntn antibodies showed good and persistent recovery after cyclophosphamide. prevalence of antibodies was % in italian cidp patients and their presence was associated with distinctive clinical features. their determination, followed by characterization of igg subclass in positive cases, has clear clinical impact, by helping to guide therapeutic choices. the reactivity against nodal and paranodal components in sera from patients without known antibodies suggests that other targets could play a role in the autoimmune response in cidp and they still need to be identified. mutations in amynoacyl trna synthetases (arss), enzymes that catalyse the covalent attachment of amino acids to their cognate trna, are responsible for autosomal dominant cmt , intermediate cmt (cmt-i) and dhmn. we report the case of a male of italian ancestry who first presented with bilateral ankle clonus at three months, followed by toe walking and ankle instability. the ankle clonus subsided during adolescence. in the third decade he developed progressive walking difficulties followed by distal sensory loss. neurological examination at the age of revealed a steppage gait, distal lower limb weakness, decreased pinprick to the ankles, and reduced vibration sensation at the knees. reflexes were brisk in the upper limbs, reduced at the knees and absent at the ankles. muscle tone was increased in the lower limbs and plantar responses were extensor. nerve conduction studies revealed an axonal neuropathy. brain and spinal cord mri were normal. sanger sequencing of pmp , gjb , mpz , gdap and mfn were negative. sureselect focused exome sequencing (agilent technologies, santa clara ca, usa) demonstrated a c. g>a, p.arg his mutation in aars. the p.arg his mutation in aars has previously been reported in families with intermediate or axonal motor-sensory neuropathy, and in one case was associated with sensory-neuronal deafness. cns involvement has not previously been described with this mutation. mutations in aars have been associated with a range of phenotypes including cmti, cmt and dhmn with variable age on onset ranging from to years (mean years). of note, the aars p.gly arg mutation has been reported in a family with cmt and pyramidal signs. this study provides further evidence that pyramidal tract involvement can be an early feature of cmt n due to mutations in aars, further expanding the spectrum of arss-associated phenotypes. small fibres in the skin are vulnerable to damage in metabolic or toxic conditions such as diabetes mellitus or chemotherapy resulting in small fibre neuropathy and associated neuropathic pain (np). whether injury to the most distal portion of sensory small fibres due to a primary dermatological disorder can cause np is still unclear. recessive dystrophic epidermolysis bullosa, (rdeb) is a rare condition in which mutations of proteins of the dermo-epidermal junction lead to cycles of blistering followed by regeneration of the skin. damage is exclusive to the skin and mucous membranes, with no known direct compromise of the nervous system. it is increasingly recognised that most rdeb patients experience daily pain, the aetiology of which is unclear but may include inflammation (in the wounds), musculoskeletal (due to atrophy and retraction scars limiting movement) or np. in this study we investigated the incidence of np and examined the presence of nerve dysfunction in rdeb patients. around three quarters of patients presented with pain of neuropathic characteristics which had a length dependent distribution. quantitative sensory testing of the foot revealed striking impairments in thermal detection thresholds combined with an increased mechanical pain sensitivity and wind up ratio (temporal summation of noxious mechanical stimuli). nerve conduction studies showed normal large fibre sensory and motor nerve conduction however skin biopsy showed a significant decrease in intraepidermal nerve fibre density. autonomic nervous system testing revealed no abnormalities in heart rate and blood pressure variability however the sympathetic skin response of the foot was impaired and sweat gland innervation was reduced. we conclude that chronic cutaneous injury can lead to injury and dysfunction of the most distal part of small sensory fibres in a length dependant distribution resulting in disabling np. these neuropathic pain (np) following peripheral nerve injury is associated with hyperexcitability in damaged myelinated sensory axons which begins to normalise over time. we investigated the composition and distribution of shaker type potassium channels (kv channels) within the nodal complex of myelinated axons following injury. at the neuroma that forms after damage, expression of kv . and . (normally localised to the juxtaparanode) was markedly decreased. in contrast kv . and . , which were hardly detectable in the naïve state, showed increased expression within juxtaparanodes and paranodes following injury, both in the rat and in humans. within the dorsal root (a site remote from injury) we also noted a redistribution of kv channels towards the paranode. blockade of kv channels with dtx after injury reinstated hyperexcitability of a-fibre axons and enhanced mechanosensitivity. changes in the molecular composition and distribution of axonal kv channels, therefore represents a protective mechanism to suppress the hyperexcitability of myelinated sensory axons that follows nerve injury. members of the francophone anti-mag cohort group are listed in "appendix". appendix polyneuropathy with anti-mag igm antibodies is classically progressive, predominantly sensory, and distal with ataxia and sometimes postural-intention tremor. we assessed clinical, biological, electrophysiological, and histopathological features in patients with igm gammopathy and anti-mag antibody titres higher than btu. we focused on characteristics of patients according to the anti-mag antibody titres at diagnosis. we retrospectively and prospectively analysed standardized report forms of patients from fourteen french-speaking neuromuscular centres. mean age at onset was . years (range - . ). mean time between symptoms onset and last follow-up was . years ( . - . ). about . % of patients presented with a "variant" clinical phenotype independently of anti-mag titres (< or ≥ btu). this included acute or chronic sensorimotor polyradiculoneuropathies, paucisymptomatic sensory polyneuropathy and multifocal neuropathy. at the most severe disease stage, . % of patients were significantly disabled. anti-mag antibody titres at diagnosis were low (< btu), medium ( - ) or high (≥ ) in respectively , and % of patients. patients presented with mgus or waldenström's macroglobulinemia in respectively % and % of cases. sixteen percent of patients did not meet the electrodiagnostic criteria of definite demyelinating neuropathy, independently of anti-mag titres (< or ≥ btu). nerve biopsy, performed in nineteen patients, provided support to the diagnosis of anti-mag neuropathy in some particular issues (low titres of anti-mag, unusual clinical or electrophysiological phenotype). we assessed the degree of probability (probable, possible or uncertain) that patient neuropathies are directly related to anti-mag antibodies, according to anti-mag titre, electrophysiological data and nerve biopsy characteristics if available. it appears uncertain in patients with low anti-mag titres ( . % of the whole population). the clinical phenotype didn't appear to be different according to anti-mag antibody titre. many of the patients with low anti-mag titres presented "genuine" anti-mag neuropathy as demonstrated by edx studies, clinical presentation and sometimes nerve biopsies. for a small proportion of these patients, a direct relation between neuropathy and anti-mag antibodies is uncertain due to atypical clinical presentation, axonal neuropathy pattern or nerve biopsies, and positivity of antigangliosides antibodies. we assessed therapeutic management, response to immunotherapies and adverse events in a cohort of patients presenting with igm gammopathy and anti-mag antibody titres higher than btu. we retrospectively and prospectively analysed standardized report forms of patients from french speaking neuromuscular centres. mean age at onset was . years (range - . ). mean time between symptoms onset to diagnosis and last follow-up were respectively . ( - ) and . years ( . - . ). anti-mag antibody titres at diagnosis were low (< btu), medium ( - ) or high (≥ ) in , and % of patients. patients presented with mgus or waldenström's macroglobulinemia in respectively % and % of cases. seventy eight percent (n = ) of patients received immunotherapies. transient response to ivig or plasma exchanges at six month was observed in less than and % of patients respectively. chemoimmunotherapies and rituximab were more frequently administered in the group of patients with malignant hemopathies (n = ) compared to mgus (n = ) (mean lines of therapy = . , range - , sd . versus . , range - , sd . , p = . ). more than % of patients (n = ) received rituximab monotherapy. clinical worsening, mostly transient and reversible, was observed in / patients after rituximab. clinical response to rituximab at months and/or during - months follow-up period was observed in . % of patients, and correlated with anti-mag titre ≥ btu ( / responder versus / non-responder patients, p = . ). at - months follow-up, responder patients presented shorter symptom duration compared to non-responders, though not significant after logistic regression ( . years, range . - . , sd . in responder patients versus . years, range . - . , sd . , in non responder patients, p = . ). in some cases, electrodiagnostic studies were recorded during rituximab treatment follow-up and showed in responder patients a clear improvement of motor conduction velocities. these data may support another clinical trial to study benefice of rituximab in anti-mag neuropathies early in the disease. it raises issues about the value of incorporating electrodiagnostic parameters as end-points. schwann cells (scs) are essential for axon integrity and myelination in the uninjured pns. after pns injury, scs function as "first responders" , undergoing phenotypic re-programming and orchestrating many processes that lead to functional nerve repair. receptors in scs that contribute to sc repair programs remain incompletely understood. we identified a member of the ionotropic glutamate receptor (igr) family, n-methyl-d-aspartate receptor (nmda-r), in scs that is upregulated after nerve injury and acts as a co-receptor with ldl-receptor related protein (lrp ). lrp is a well-known regulator of the sc response to pns injury. herein, we used pcr to profile igr expression in cultured rat scs. obligate receptor subunits required for assembly of nmda-rs, ampa-rs, and kainate receptors were identified. treatment of rat scs with - microm glutamate or . - . microm nmda robustly activated akt and erk / . the response was transient and bimodal; glutamate concentrations greater than microm failed to activate cell-signaling. phosphoprotein profiling demonstrated other cell-signaling and transcription factors regulated by glutamate in rat and human scs, including p s -kinase, glycogen synthase kinase- , ribosomal s kinase, c-jun, and creb. activation of cell-signaling by glutamate in scs was blocked by eliminating calcium from the medium, by the selective nmda-r antagonist, mk , and by genetic silencing expression of the obligate nmda-r nr subunit. phosphoinositide -kinase/pi k functioned as an essential upstream activator of both akt and erk / in glutamate-treated scs. by activating pi k and erk / , glutamate promoted sc migration. glutamate ( microm) or nmda ( microm) injected into crush-injured sciatic nerves robustly activated p-erk / in both myelinating and non-myelinating scs in vivo. these results identify igrs as potentially important cell-signaling receptors in scs that may promote axon-glial interactions. understanding the function of sc nmda-r is important given current efforts to develop nmda-r-targeting drugs for patients with pain, depression, and alzheimer's disease. while frequently overlooked in a therapeutic context, scs are extremely important in the pathogenesis of chronic neuropathic pain. if these drugs modulate the activity of sc nmda-r and sc physiology, the response to pns injury may be altered and the possibility that neuropathic pain develops increased. candayan a , atkinson d , durmus tekce h , parman y , jordanova a , battaloglu e . department of molecular biology and genetics, bogazici university, istanbul, turkey; center for molecular neurology, antwerp university, antwerp, belgium; istanbul medical school, istanbul university, istanbul, turkey. charcot-marie-tooth (cmt) disease is a group of inherited peripheral neuropathies affecting one in individuals worldwide. the disease presents both clinical and genetic heterogeneity. so far, mutations in genes and loci are associated with cmt with autosomal dominant, autosomal recessive, x-linked and mitochondrial inheritance. despite the advances in genetic testing, approximately % of all cmt patients worldwide remain without a molecular diagnosis. we have investigated unrelated cmt patients of turkish descent, in all of which pmp duplication has been excluded previously. we used multiplex amplification of specific targets for resequencing (mastr) assay to sequence exonic regions of common cmt genes. recurrent mutations were identified in cases in mfn , gjb , mpz and hint genes. we have also identified novel variants in cases in mfn , pmp , gars, aars, ighmbp and gdap genes, all of which are very rare or not present in the variation databases and are predicted to be pathogenic by in silico tools. familial segregation analyses are ongoing for novel variations. mfn and gjb genes were the most commonly mutated causative genes in this cohort. cases without molecular diagnosis after the mastr testing are candidates for further analyses such as whole exome sequencing or whole genome sequencing. outcomes of the current study and our previous experience with turkish cmt patients suggest a high genetic heterogeneity. our insight is that different genetic strategies or larger panels are essential to determine the causes underlying cmt especially in regions where rare recessive types of the disease can be observed due to high frequency of consanguineous marriages. capoccitti g , giannini f , ginanneschi f , casali s , insana l , rossi a . university of siena, siena, italy. several efforts have been made to elaborate new electrophysiological criteria for early diagnosis of guillain-barré syndrome (gbs) and to differentiate demyelinating (aidp) from axonal (aman/amsan) forms. the aims were to verify the diagnostic power of total cmap (tcmap) duration, firstly applied in gbs field. this parameter was compared with commonly used neurophysiological measures, including negative phase cmap duration (ncmap), and was added to modified rajabally criteria. we reviewed the clinical and electrophysiological data of patients with gbs (level or of brighton clinical criteria). each patient underwent at least two neurophysiological studies, the first within weeks, the second between and weeks from symptom onset. at least four motor and three sensory nerve conduction studies were recorded for each test. regarding early diagnosis, the binary logistic regression model with multiple variables, including ncmap duration, showed that the features of predictive model presenting greater significance (p < . ) were tcmap duration, sural sparing and a-waves. among these, tcmap duration showed greater significance (p = . ). the tcmap was diffusely prolonged in aidp compared to aman/amsan, already in first examination and confirmed in the second one. roc analysis for tcmap duration in aidp vs. aman/amsan showed: cut-off . ms, auc . , ppv . %. we propose the tcmap duration as a new useful electrophysiological measure for early diagnosis of "generic" gbs and for early differentiation between aidp and aman/amsan. moreover, the prolongation of tcmap, the presence of a-waves and sural sparing represent a strongly diagnostic predictive triad of aidp. aifm (apoptosis-inducing factor, mitochondrionassociated- ) has captured great attention from biomedical researchers due its critical role in the regulation of cell apoptosis. this flavoprotein is typically located in the mitochondrial intermembrane space where it is associated with respiratory chain complex-i. upon a cell-death insult, aifm is cleaved into a kd protein that is released into the cytosol. the kd peptide may enter the nucleus to trigger chromosome condensation and fragmentation, initiating a caspase-independent pathway of apoptosis. however, this nuclear translocation may be blocked by cytosolic heat-shock protein- (hsp ) that binds with the fad domain (aa - ) of aifm . mutations in aifm gene have resulted in several clinical phenotypes, including a family with cmtx (glu val). clinical deficits in these patients usually involve multiple organs. in this study however, we identified a family with a novel missense mutation (phe leu) in aifm that developed a late-onset sensory motor axonal polyneuropathy by nerve conduction criteria. the proband and affected siblings exhibited distal muscle weakness and atrophy with normal cognitive and cranial nerve functions. there was no obvious phenotype from other organs. interestingly, this phe leu mutation affects a highly conserved amino acid at the center of the fad domain. we hypothesize that this mutation impairs the binding between aifm and hsp , leading to an enhancement of cell-death signaling. this family therefore provides a unique opportunity to explore how altered apoptotic signaling affects peripheral nerve system. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr chronic inflammatory demyelinating polyneuropathy (cidp) can be the key symptom leading to diagnosis of associated lymphoma. patients with diagnosis of cidp according to efns/pns criteria associated with b cell lymphoproliferative disorders (bld), in one center, between november and november were included. demographical, clinical, nerve conduction, immunological, histological data and response to treatment were recorded retrospectively. eight patients ( men), median age yo were included. onset of polyradiculoneuropathy was either chronic (n= ) or acute (n= ). neurological condition led to diagnosis of bld in all but one case, because of onset (n= ) or worsening of neuropathy (n= ). clinical presentation was that of cidp in patients or pseudo-canomad in and plexopathy in one. lymphoma type was: lymphoplasmacytic (n= ), diffuse large b cell (n= ), small lymphocytic (n= ), marginal zone (n= ), unclassified small b cell (n= ). only patients presented with lymphadenopathies. bld was diagnosed in all patients on myelogram or bone marrow biopsy, performed because of cytopenia (n= ), atypical (n= ) or severe (n= ) neuropathy. monoclonal gammapathy was identified in / patients (igm n= , igg n= ). neuromuscular biopsy was performed in patients and disclosed endoneural infiltration in . anti-neuronal or antigangliosides antibodies were positive in patients. none of the patients presented a systemic autoimmune disease, hemolytic anemia associated with bld (n= ). immunomodulating treatment was administered in all patients (ivig n= , plasma exchange n= , steroids n= ) and immunosuppressants (n= ). immunochemotherapy for lymphoma was initiated because of lymphoma type or severity in cases, in cases because of the associated neuropathy. median follow-up was of months after treatment initiation. four out of patients treated within months of neurological onset improved as well as one out of patients whose preexisting neurological condition had worsened. two patients presented neurological relapse during progression of lymphoma. two patients died. unusual presentation of cidp -i.e., rapid progression or treatment failure -should lead to further testing for associated lymphoma. because general symptoms and lymphadenopathy often lack, diagnosis requires analysis of bone marrow with lymphocyte phenotyping. early treatment with immunochemotherapy was associated with better prognosis in our series. cardiac scintigraphy is a useful tool for the diagnosis, prognosis and pre-symptomatic early detection of familial amyloidosis-associated neuropathies three main types of familiar amyloidosis: transthyretin (ttr), apolipoprotein a and gelsolin. cardiac involvement is a leading cause of morbidity and mortality; one new described mutation strongly related with isolated cardiac amyloidosis is the ttr val ile. the discovery of tests that allow early diagnosis of cardiac involvement in amyloidosis and to infer about the etiology of the disease is of major importance. in a cohort of patients with different types of familiar ttr amyloidosis, we aimed to assess the role of mtc- , -diphosphono- , -propanodicarboxylic acid ( mtc-dpd) in detecting myocardial amyloid infiltration. we enrolled four patients diagnosed with late familiar amyloidosis, which mutations were documented at deoxyribonucleic acid analysis: three patients with ttr val met mutation and one patient with ttr val ile mutation. three patients were asymptomatic for cardiac involvement and one patient (val ile mutation) had a previous diagnosis of heart failure. myocardial uptake of mtc-dpd scintigraphy was semiquantitatively and visually assessed at five minutes and three hours.the uptake of mtc-dpd highly demonstrated amyloid in cardiac area in two out the three cases of ttr val met and in ttr val ile. ttr val ile case presented the highest uptake due to the exclusive deposition of amyloid in cardiac area resulting in severe heart failure. in hereditary transthyretin-related amyloidosis, including the mutations ttr val met and val ile, mtc-dpd cardiac scintigraphy can identify infiltration even in asymptomatic individuals, allowing an early diagnosis of cardiac compromise in this group of diseases. we can consider that this non-invasive test would be a tool with potential importance in the diagnosis, prognosis and pre-symptomatic early detection of cardiac amyloidosis, giving emphasis on its applicability in familial forms of amyloidosis. cervellini i , galino j , zhu n , birchmeier c , bennett dl . ndcn university of oxford, oxford, uk; max-delbrück-center for molecular medicine, berlin, germany. erk/mapk pathway has a critical role in pns development since its involvement in many physiological processes. sustained erk / mapk activation in schwann cells enhances myelin growth during development and overcomes signals ending myelination leading to a continuous myelin production. however, strong activation of erk has also been shown to cause schwann cells dedifferentiation and demyelination in vivo. our aim was to investigate whether a mild activation of this signalling pathway in adult schwann cells (scs), by expression of gain of function mek dd allele, could have a beneficial role in remyelination and regeneration after injury. erk/mapk activation in adult scs in plpcreer t ;mek dd mutant mice, did not affect myelination during development. following sciatic nerve injury, wallerian degeneration was enhanced in mutants pushing towards a dedifferentiation stage of scs as previously described. however, mapk activation was detrimental during regeneration with a delay in functional recovery and a negative impact in both myelinated and non-myelinated fibres compared to controls. one month after injury the total number of axons in mutant sciatic nerves was half of the controls. although no differences in g-ratio have been found in the two groups, mutants presented a higher number of myelinated axons showing myelin disruption with start of myelin decompaction, lack of cajal bands, abundant sc processes surrounding axons and a shorter sc elongation, as seen by decreased internodal distance. in addition, we found a negative effect of mapk activation also in small diameter axons with the presence of abnormal remak bundle structures, reduced number of c-fibres/remak bundle and a significant decrease in intra epidermal nerve fibres density in the skin. we concluded that mild mapk activation has a different role in development and remyelination where negatively affects axon survival, myelin stability, remak bundle formation and small fibres regeneration. cervellini i , galino j , zhu n , bao lu , bennett dl . ndcn, university of oxford, oxford, uk; harvard medical school, boston, ma, usa. neprilysin (nep) is an endopeptidase which has been of interest due to its potential role in neurodegeneration and pain as a consequence of its ability to degrade amyloid and substance-p respectively. nep expression is not limited to cns and it has been reported to be expressed in schwann cells, nodes and schmidt-lanterman incisures. our interest in this gene was related to recent findings that have associated homozygous and heterozygous nep mutations with charcot-marie-tooth type- . in old mice lacking nep subtle morphological changes have been reported. our aim was to determine whether nep expression was modulated by nerve injury and to investigate its role in axon regeneration and re-myelination. we find that nep gene expression was decreased after nerve crush and furthermore was dependent on the growth factor (and pro-myelin signal) neuregulin- . in control mice nep expression was transiently reduced and returned to baseline at day after injury, in neuregulin- knock-out (ko) mice, in which re-myelination was impaired, the expression was still decreased at day . in assessing behavioural measures of locomotor and sensory function one month after sciatic nerve crush, nep ko mice showed a functional regeneration comparable to wt, as seen by sciatic functional index measurement, beam and toe spreading tests. the only significant difference we observed between wt and ko was in the sensorial test, showing ko mice recovering faster in the pinch test by days after crush. the results for all the tests at baseline did not differ between the two groups. detailed histological analysis of nerve repair was undertaken using electron microscopy. there was no difference between wt and ko in total axon number, g-ratio, axon diameters and other myelin features one month post crush. in summary, although nep expression is regulated by nerve injury in a neuregulin- dependent fashion this endopeptidase is dispensable for axon regeneration and re-myelination after nerve injury in the rodent. diabetes neuropathy is a common complication of diabetes, and neuropathic pain has a detrimental impact on quality of life. this study investigated sensory nerve excitability properties to elucidate the axonal changes of diabetic neuropathy. a total of diabetes patients ( type ii, and type i) were enrolled in this study. clinical assessment, nerve conduction studies, and nerve excitability testing data were analyzed to determine axonal dysfunction in diabetic neuropathy. among those patients, seventeen subjects had complained of spontaneous painful sensation over feet or hands (painful cohort), and seventy-eight patients had no sensory symptoms or decreased the sensation over foot (non-painful cohort). sensory nerve excitability of the painful cohort showed reduced late subexcitability (p= . ), increased superexcitability (p= . ) in compared to the non-painful cohort. there is no difference in disease duration, blood glucose levels (hba c) between these two cohorts. these findings suggested the possible pathogenesis of painful sensory axons might be hyperpolarized or slow potassium channels dysfunction. these insights our further understanding of painful diabetic neuropathy, and may provide a basis for neuroprotective or therapeutic approaches for painful polyneuropathy. the main purpose of this study was to assess the clinical feasibility of diffusion tensor imaging (dti) for the diagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (cidp). between march and december , we prospectively enrolled patients with definite cidp according to the efns criteria and two control groups: healthy volunteers matched on age and sex and patients with cmt- a. using a -t magnetic resonance imaging scanner, we obtained dti scans of brachial plexus of these groups and prepared fractional anisotropy (fa) maps, and compared these values between groups. adc values and cervical nerve roots diameters on stir sequences were evaluated too. two neuroradiologists, blinded to clinical informations, reviewed mri studies independently. in all patients with cidp, we also performed clinical evaluation and electroneuromyography. significantly decreased fa values (p< . ) and increased adc values were observed in cidp patients compared with healthy subjects. there is no significant difference between cidp and cmt group. inter-observer concordance was excellent for fa values ( c= . ; p< . ) and moderate for adc values ( c= . ; p< . ) and cervical nerve root diameters ( c= . ; p< . ). there is a significant correlation between fa and disease duration (r = − . , p < . ), inclusion mrc score (r = . , p < . ) and between fa mesured on c c and incat score at inclusion (r = − . , p < . ). no significant correlation is observed between fa and electrophysiological indices. compared with healthy subjects, cervical nerve root diameters were significantly increased (p< . ) in patients with cmt and cidp. contrary to fa values, moderate level of concordance was found between inter-observers measurements of diameters (cclin = . ). our preliminary data prove the clinical feasibility and reproductibility of dti for the evaluation of plexus and cervical nerve roots in patients with cidp. cheng yj , teng a , goh ejh , umapathi t . yong loo lin school of medicine, national university of singapore, singapore; department of neurology, national neuroscience institute, singapore. the sural-sparing pattern of the sensory nerve action potentials (snap) of guillain-barré syndrome (gbs) has been attributed to greater immunological injury of the blood-nerve barrier at its most vulnerable regions. we asked if entrapment sites, such as median nerve at the wrist, are more predisposed than the distal nerve endings to such injury. we compared the median snap with radial snap measured antidromically at digit in gbs patients whose nerve conduction study showed the sural-sparing pattern. the terminal nerves at digit are of similar length, but those of median nerve are prone to compression, often subclinically, at the carpal tunnel while those of radial nerve are not. we defined the sural-sparing pattern as a greater decrease in median and or ulnar snap than that of the sural, compared to age and height-matched normal controls. a total of gbs and miller fisher patients from our institution's database were studied. patients had the sural-sparing pattern, of whom had pre-existing carpal tunnel syndrome. of the remaining patients with sural-sparing, had abnormal median snap at digit , while had both abnormal median and radial snaps at digit . none had isolated abnormality of the radial digit snap. among the cases that had abnormal median and radial snaps at digit , the mean percentage decrease when compared to age and height matched norms was greater in median nerve compared to radial nerve ( % and % respectively). of the patients without sural-sparing pattern, had normal snaps; patient had inexcitable sensory nerves while the other had a length-dependent decrease in snap. in the latter patient, unlike those with sural-sparing, there was no differential decrease of median snap over radial snap at digit . our findings suggest that the disruption of blood nerve barrier at entrapment sites rather than the distal nerve endings may underlie the pathophysiology of the sural-sparing pattern seen in gbs. chiba a , uchibori a , gyohda a . kyorin university, tokyo, japan. serum igg anti-gq b antibody is the most specific biomarker for fisher syndrome and its related disorders (fs-rd), but approximately - % of the patients are seronegative for it in conventional assays (gq b-seronegative). some molecules need ca + cation to interact with their ligands, and antibodies with such a property (ca + -dependent antibodies) are reported. we have found that such a ca + -dependency is also present in igg anti-gq b antibody, and majority of gq b-seronegative patients with fs-rd have this type of antibodies. in patients with final clinical diagnoses as fs-rd (fisher syndrome, guillain-barré syndrome with ophthalmoplegia, bickerstaff brainstem encephalitis, and acute ophthalmoplegia), were seropositive for igg antibodies against gq b-related antigens (isolated gq b in , and gq b-conatinig complexes in two) in conventional elisa using phosphate-buffered saline. in the remaining patients, eight ( %) turned positive for igg antibody against gq b-related antigens (isolated gq b in seven and gq b-conatinig complexes in one) in elisa using ca + -added tris-buffered saline. the reaction strengths increased depending on ca + concentration, and reached to nearly maximum level in the physiological concentration. all the patients with the ca + -dependent antibodies were also positive for igg antibody against gt a-related antigens, suggesting that the terminal disialo residue common to both the gangliosides would be important as an epitope also for the ca + -dependent antibodies. in the patients with ca + -non-dependent antibodies, only two showed increased titers of igg anti-gq b antibody by adding ca + , and showed significantly decreased titers. this difference in the effect of ca + -addition between ca + -dependent and ca + -non-dependent antibodies suggests that ca + would not be just an enhancer of the antigen-antibody reaction. there are four single bonds between the two pyranose rings in the terminal disialo, and those rotatable bonds make it possible for the disialo structure to take various conformations. a molecular model shows that the distance between two minus-charged carboxy groups in the disialo could vary from nearly zero to approximately , pm and that the disialo would take specific conformations, if divalent ca + cation, which size is approximately pm in diameter, interacts with these two minus-charged groups. the ca + -dependent antibodies might recognize such particular conformations of gq b. charcot-marie-tooth disease (cmt) is a genetically and clinically heterogeneous disorder with variable inheritance modes. it is characterized by loss of muscle tissue and touch sensation, predominantly in the feet and legs but also in the hands and arms in the advanced stages of disease. as several molecules have been reported to have therapeutic effects on cmt, depending on the underlying genetic causes, exact genetic diagnostics have become important for executing personalized therapy. aminoacyl-trna synthetase (arss) genes encode enzymes responsible for charging trna with corresponding amino acids. arss are ubiquitously expressed, essential enzymes responsible for performing the first step of protein synthesis. specifically, arss attach amino acids to their cognate trna molecules in the cytoplasm and mitochondria. recent studies have demonstrated that mutations in genes encoding arss can result in neurodegeneration, raising many questions about the role of these enzymes in neuronal function. mutations in six cytoplasmic ars genes have been reported as the cause of cmt. this study was performed the whole exome sequencing to identify genetic defects in korean cmt patients from unrelated families. variants were sorted with cmt gene list that includes almost genes were related cmt neuropathy, and additionally sorted wes data as ars genes. capillary sequencing for family members and more than controls revealed five novel mutations, c. g>a (p.d n), c. c>t (p.s f), and c. c>a (p.p h) in gars; c. c>a (p.p t) in mars; _ ga>at (p.d i) in yars gene in each family. the mutation sites were well conserved between different species and each mutation were located in the well-conserved catalytic domain or between two catalytic domains or anticodon-binding domain. in silico analysis predicted all mutations may affect protein function. clinical features were similar to those reported in other countries, but differed in terms of age at onset and degree of disability. we believe that those novel ars mutations are the underlying causes of the each family. a -year-old man presented with a -year-history of weakness in biltearal upper limbs. he was complaining of intermittent fasciculation of upper and lower limbs with gradually worsening of paresthesia for years. dysphagia and dysarthria were also presented years ago. there was no patient affected muscle weakness and bulbar symptoms in his family members. in neurological examination, the patient had weakness in biltereal upper and lower limbs (mrc grade ) and prominent distal sensory loss were combined in length dependant pattern. deep tendon reflexes were absent on bilataral biceps and knee joints. in nerve conduction study, there was consistent with demyelinating sensorimotor polyneuropathy. molecular diagnostic analyses those spinobulbar muscular atrophy (sbma) and mutation related to peripheral myelin protein (pmp ) gene were performed and confirmed expansion of expansion of a polymorphic cag in androgen-receptor (ar) gene and deletion of pmp gene. smba, also known as kennedy disease, is an adult-onset, x-linked recessive trinucleotide, polyglutamine (poly-g) disorder caused by expansion of a polymorphic cag tandem-repeat in exon of ar gene on chromosome xq - . charcot-marie-tooth disease (cmt) is the most common hereditary neuropathies and cmt cases with motor conduction velocities(mcvs) of upper limb below m/s are defined as demyelinating (cmt ) and those with mcvs above m/s are defined as axonal (cmt ). most families with cmt linked to duplication of pmp gene on the short arm of chromosome ( p . ), called cmt a. the reciprocal deletion of pmp gene is a responsible genetic defect in % of hereditary neuropathy with liability to pressure palsy (hnpp). these "classical" phenotypes of cmt a and hnpp have been considered which are determined by different mutation mechanism of the same gene. however, an overlap of cmt a and hnpp due to pmp gene deletion was reported that suggestion the phenotype of hereditary neuropathies may differ variably. herein, we report a patient who simultaneously presented clinical and electrophysiologic features of smba and cmt a with genetical confirmation of cag expasion and deletion of pmp gene. charcot-marie-tooth type a disease (cmt a) is an inherited peripheral neuropathy stemming from overexpression of pmp protein in schwann cells due to the duplication of the pmp gene. this leads to abnormal schwann cell differentiation and dysmyelination, eventually leading to axonal loss and muscle wasting. no approved treatment is currently available for cmt a. we conducted a systems biology level analysis of the signaling network putatively underlying the processes driving cmt a pathology. based upon this, we identified and tested three repurposed drugs -baclofen, naltrexone and sorbitol -alone and in combination to determine their ability to rescue aberrant myelination in cultures derived from cmt a transgenic rats overexpressing pmp gene. to this end, we studied a validated in vitro co-culture model of sensory neurons and schwann cells adapted to -well culture plates. this model allows measurement of the appearance of myelin proteins as an index of the physiological process of in vivo myelination. total myelin length was quantified with an automatic image analyzer following pmp immunostaining. we first determined the full dose-response curves of single drugs, emphasizing their promyelinating activities. we then tested binary combinations of very low and inactive doses of each drug and compared these to the activity of the combination of the three, namely pxt . whereas combination of any two drugs was not significantly active at the doses tested, combination of all three produced a synergistic improvement in myelination. these findings clearly demonstrate the necessity of using pxt over its single components and highlight the value of pleiotropic combinational repurposing of drugs at low doses as a novel approach for rapid drug development in cmt a and other disorders. autonomic dysfunction is frequently observed in guillain-barré syndrome (gbs) and affects approximately % of the patients. it has been shown that the sweating function can be impaired in gbs. the aim of the present investigation is to summarize the current knowledge on sweating disturbances in gbs patients. we have used appropriate terms to systematically search for references published until and indexed in the following databases: medline, embase, lilacs and cochrane. the inclusion criteria were a diagnosis of gbs and a description of the methods used to test the sudomotor function. the search was limited to the english language. relevant information about study design, methods of assessing the deficit of sweating, patient's characteristics and main results were collected. we selected original references for the final analyses. the majority of the studies were cross-sectional in nature and there were two longitudinal studies. the severity of sweating impairment varied according to the applied method, ranging from normal to almost sympathetic nervous system failure. in seven research papers, the sympathetic skin response was used to evaluate the sudomotor function in patients, and approximately % demonstrated abnormal results. however, researchers used different stimulation protocols and parameters to interpret their results. regarding whole-body sweating test, four research papers applied the thermoregulatory sweating test in patients and they showed areas of anhidrosis on the lower limbs in all of them. eight patients presented sweating impairment on the upper limbs and abdominal wall. results on the sudomotor axon reflex test suggested a length-dependent pattern of sweating loss according to one case report. in another study, eight gbs patients were tested only on the distal leg and foot dorsum and the authors proposed an association between post-ganglionic sudomotor function and antiganglioside antibodies. the present literature review showed that the studies of sweating disturbances in gbs patients included only small cohorts. future studies with larger patients sample sizes are necessary to investigate the patterns of sweating loss in gbs and their changes along the follow up. funding: grants # / - and # / - , são paulo research foundation (fapesp). sensory neuronopathy (sn) represents a rare subgroup of peripheral neuropathies characterized by degeneration of primary sensory neurons at the dorsal root ganglia on the spinal cord. depending on the neuronal population affected, its clinical presentation may manifest as gait ataxia, proprioceptive sensory loss and positive and negative sensory symptoms. although a few reports have mentioned areas of anhidrosis in sn, we were not been able to find previous case series studies on the sweating function in sensory neuronopathies. the aim of the present investigation was to study the whole-body distribution of sweating on both anterior and posterior surfaces in patients diagnosed with sn. quantitative sensory testing for cold and warm sensation threshold (method of levels) was performed on the dorsum of the hands and feet in a randomized order. we tested the thermoregulatory sweating using a sweat chamber ( to ∘ c air temperature and % relative humidity). the oral and skin temperature was monitored and the test time did not exceed min. in order to study the sudomotor axon reflex we employed the q-sweat device on standardized body sites. the test was performed on both sides, simultaneously. we included seven patients (three male; mean age . years) with a mean disease duration of . months (range - ) and a confirmed diagnose of sn. patients presented an asymmetrical loss of cold and warm threshold on hands or feet compared with healthy control (p< , ). regarding the tst results, we found a striking variation of sweating disturbances, ranging from small areas of anhidrosis on the trunk to complete failure of the sympathetic nervous system. two patients underwent the axon reflex test and there was an asymmetrical and mostly distal pattern of sweating loss in one of them and a distal-symmetrical on the second one. our findings indicate a great variability of sweating losses in sn, not overlapped to the sensory loss areas. currently, we are testing more patients in order to confirm our results. funding: grants # / - , # / - and # / - , são paulo research foundation (fapesp). diet, exercise, and inflammation are established modulators of peripheral nervous system function, including pain. prior work examining exercise consistently demonstrates a benefit on heightened pain from a number of acute and chronic pain models. in the present work, we investigated several parameters of peripheral nerve function relevant to pain in rats bred for high (high capacity runners, hcr) or low running capacity (low capacity runners, lcr). the longtime selective breeding of these rat substrains has created divergent intrinsic aerobic capacities and predisposition of metabolic conditions between lcr and hcr rats. examination of the role of sex in the development of chronic pain has established key differences in males and females. to understand gender specific differences, this study focused on female rats to understand the role of metabolic status and peripheral nerve function in females. our analysis identified numerous parameters of peripheral nerve function relevant to pain and neuropathy that are different among lcr and hcr female rats. lcr female rats display reduced hind paw mechanical sensitivity, increased hind paw intraepidermal nerve fiber density and trka-positive epidermal axons, increased numbers of langerhans and mast cells in the hind paw dermis, and increased overall fat mass relative to body weight compared to female hcr rats. examination of sensory and motor nerve conduction velocities, thermal sensitivity, and mrna expression of selected genes relevant to peripheral sensation found no differences between hcr and lcr females. together these results suggest that a genetic component of aerobic capacity and metabolic status can influence sensory sensitivity and specific aspects of inflammation and immune responses in peripheral tissues, which may lead modify the animal's responses to tissue damage and painful stimuli. the lcr and hcr rat model will provide a useful model in the future to assess the involvement of metabolic status in the development of pain. ivig is often considered treatment of first choice in chronic inflammatory demyelinating polyradiculoneuropathy (cidp) because of its rapid onset of action and its relatively safe long-term adverse event profile. clinical trials published so far focused on a loading dose of . g/kg ivig and/or a standard maintenance dosage of . g/kg ivig once every weeks, but have not investigated different dosing options. this study is a prospective, double-blind, randomized, parallel group, multi-center phase iii efficacy study and will be conducted in centres in canada, eu, russia, ukraine and australia. adult patients with definite or probable cidp according to the efns/pns criteria will be enrolled and randomized : : to receive either . g/kg or . g/kg or . g/kg ivig (panzyga ® ) for seven maintenance infusions at -week intervals during the dose-evaluation phase. the starting loading dose will be . g/kg ivig (panzyga ® ) for all patients. primary objective: efficacy measured as percentage of responders (decrease in adjusted incat score by at least point) in the . g/kg ivig (panzyga ® ) arm (given every weeks) at week as this should corroborate the existing and published evidence on efficacy of ivig in cidp. secondary outcome: percentage of responders at week in the . g/kg and . g/kg ivig (panzyga ® ) arms relative to baseline and compared to the . g/kg arm. the procid study aims to confirm published clinical results obtained with the . g/kg standard dose and will in addition evaluate one higher and one lower maintenance dose, with the aim to offer cidp patients a more adequately dosed and effective treatment policy. * este medicamento no se encuentra comercializado en españa. understanding the rate of disease progression in patients with charcot-marie-tooth disease (cmt), both within and between subtypes is important for clinical prognosis and is crucial for clinical trial design. due to the progressive nature of cmt, intervening at the earliest stages of the disease is a priority. measuring progression of a disease with both motor and sensory deficits requires a multi-item composite scale. the cmt pediatric scale (cmtpeds) is a well-tolerated psychometrically robust -item scale measuring fine and gross motor function, strength, sensation and balance for children and adolescents aged - years with cmt. the aim of this study was to determine the rate of disease progression of children and adolescents within and between genetic subtypes of cmt. ( female) participants aged - years enrolled in the inherited neuropathies consortium were included in this study. demographic, anthropometric and diagnostic information were collected at baseline and -year follow-up. disease progression was measured with the cmtpeds. on average cmtpeds scores progressed at a rate of . points over -years ( % change from baseline, p< . ). there was no difference in rate of disease progression between males and females. of the most common genetic subtypes, participants with cmt a/pmp duplication progressed by . points ( % change from baseline, p< . ), nine participants with cmt b/mpz mutation progressed by . points ( % change), six participants with cmt a/mfn mutation progressed by . points ( % change) and seven participants with cmt c/sh tc mutation progressed by . points ( % change). participants with cmt a progressed faster than those with cmt a (p= . ). children with cmt a progressed consistently during childhood and adolescence while children with cmt b and cmt a progressed faster during childhood than adolescence. overall, children with cmt progress at a significant rate over -years according to the cmtpeds. understanding the rate at which affected children deteriorate is essential for adequately powering clinical trials of disease-modifying interventions. queen square, london, uk; department of neurology, university of iowa carver college of medicine, iowa city, ia, usa; department of neurogenetics, the national hospital for neurology and neurosurgery, ucl institute of neurology, london, uk; department of molecular neuroscience, ucl institute of neurology, london, uk and national hospital for neurology and neurosurgery, queen square, london, uk. in recent years, targeted ngs panels have changed the diagnostic work-up in patients with inherited neuropathies. however, there is limited data on the impact of targeted ngs panels on the diagnosis of cmt patients in everyday practice. the aim of this study was to investigate the impact of targeted ngs panels on the diagnosis of cmt across two tertiary referral centres in the united kingdom (london) and united states (iowa). in london, patients with a diagnosis of cmt (previous pmp duplication and common cmt genes excluded in appropriate cases) underwent targeted ngs panel sequencing covering genes associated with cmt and additional genes associated with hsp or als. a variable number of genes, ranging from to , were analysed depending on the clinical phenotype of the patients. a definite molecular diagnosis was achieved in cases ( %) including pathogenic and likely pathogenic mutations in sh tc ( cases), gjb ( cases, including cases with mutations in the promoter and 'utr regions), gdap ( cases), fgd ( cases), aars ( cases), ighmbp ( cases), mpz ( cases), nefl ( cases). vus were further identified in patients. the diagnostic rate was higher in demyelinating cmt cases ( / , %), compared to cases with axonal cmt ( / %), dhmn ( / , %) and hsn ( / , %). in iowa, patients were investigated by ngs panels covering to genes associated with cmt. a molecular diagnosis was reached in / ( %), and in particular / ( %) demyelinating and / ( %) axonal cmt cases. the most frequent genes identified were gjb ( cases), mfn ( cases), sh tc ( cases) and ighmbp ( cases). vus were identified in patients, including cases with novel variants in aars, warranting additional testing such as segregation of the variant in the family or functional validation studies. in clinical practise, targeted ngs panels represent an effective approach for the diagnosis of cmt. the lower diagnostic rate in london is likely to be due to prior sanger sequencing and exclusion of mutations in common cmt genes in this patient population. more than genes are known to cause cmt and an even larger number are known to cause peripheral neuropathy as part of a more complex neurological disorder. despite the use of custom panels, a significant proportion of patients with inherited neuropathy have no molecular diagnosis. the aim of this study was to investigate the diagnostic yield of a disease-associated gene exome (sureselect focused exome, agilent technologies, santa clara ca, usa) in the diagnosis of cmt and in cases with complex neurological syndromes associated with neuropathy. thirty-one patients with molecularly undiagnosed inherited neuropathy were analysed with sureselect focused exome sequencing. six patients had a more complex phenotype including learning difficulties, cerebral white matter changes, ataxia and pyramidal tract involvement. a genetic diagnosis was achieved in / ( %) of cases by detecting a mutation in cmt-associated genes mpz ( cases), aars, nefl, bscl , bicd and trpv . of note, six cases had mutations in genes which are not covered by currently available diagnostic targeted ngs panels, including kif a, polg, mme ( cases), dnajb , and a novel candidate gene. the average coverage was higher compared to the usual coverage of whole-exome sequencing; % of the targets were covered at x or more, and % of the targets were covered at x or more. this study provides evidence that the sureselect focused exome is a useful tool for the diagnosis of cmt and complex neurological disorders and provides further insight into the phenotypic spectrum of genes associated with inherited neuropathy. changes in the cis-regulatory sequences of a gene's untranslated regions (utr) are increasingly recognised as a significant cause of inherited disease in humans. for example, variants in the non-coding region of gjb account for % of all mutations in our cohort of cmtx patients. one of the biggest challenges in analysing the large number of non-coding variants in a gene is identifying those that are disease-causing and those which are polymorphisms. the aim of this study was to implement a reliable method for the in-vitro functional validation of non-coding variants in the promoter and 'utr regions of gjb . in our cohort of cmtx patients we have previously identified seven mutations (c.- _ inst, c.- g>a, c.- t>g, c.- t>c, c. t>c, c.- g>c, c.- c>t) in the promoter region and one novel mutation in the 'utr (c. c>t), which were considered likely to be pathogenic based on the clinical phenotype, segregation in affected family members and absence in control databases. we have now generated a luciferase-based reporter system and optimised it in a hela cell line. mutations in the promoter region were generated by site-directed mutagenesis using a commercially available gjb promoter clone (genecopoeia). our preliminary results show a reduction of luciferase activity for the c.- t>c and c.- _ inst mutations compared to the wild-type promoter. this difference was increased when transcription factors sox or egr were co-transfected with the t>c and c.- _ inst mutations respectively. validation of other variants is currently ongoing. if successful, our study will provide a useful tool for the validation of mutations in non-coding regions of gjb . moreover, it will constitute a proof-of-principle approach to the functional validation of non-coding variants in other cmt genes known to cause disease by a loss of function. recessive and dominant mutations in leucin-rich repeat and sterile alpha-motif-containing (lrsam ) have been associated with cmt p. lrsam is a ubiquitin e ligase containing a ring domain in its c-terminal, which is crucial for correct protein folding and ubiquitination activity. to date, the majority of dominant mutations reported have resulted in a frame shift disrupting a major portion of the ring-domain, although point mutations in this domain have also been described. the aim of this study was to report the prevalence, clinical features and genetic findings of patients with cmt p in our centre. we performed targeted next-generation sequencing in genetically undiagnosed cmt patients and identified cases with heterozygous mutations in lrsam ( . %) from unrelated families. the mutations identified included frameshift insertions and deletions, a non-frameshift deletion and non-sense and missense point mutations. all of the mutations were novel and were located in or flanking the ring domain. the average age of disease onset was in the rd decade but an earlier onset was reported in two cases. four had a positive family history in keeping with autosomal dominant inheritance. symptoms at presentation were heterogeneous and encompassed distal numbness, unsteadiness, distal weakness of upper or lower limbs and foot deformities. positive sensory symptoms, including tingling and shooting pains, and cramps were also frequently reported. neurological examination showed mild to moderate distal atrophy and weakness, with early ankle plantar flexion involvement in three patients. loss of vibration and reduced joint position sense were often prominent in the lower limbs and appeared to be disproportionate to the degree of weakness and impairment of pinprick sensation. ankle jerks were absent but knee and upper limb reflexes could be normal or brisk. after an average disease duration of years, all but one patient was able to walk independently. nerve conduction studies showed a sensory and motor axonal neuropathy with normal conduction velocities. our study highlights that mutations in lrsam are a relevant cause of cmt and are associated with prominent large fibre sensory loss. in recent years, the implementation of ngs panels for the molecular diagnosis of cmt has increased the number of patients with a genetic diagnosis. nevertheless the interpretation of a particular variant as disease causing can be challenging especially when multiple variants are identified in a single patient. we report two illustrative cases of such challenges. the first index case was born of non-consanguineous healthy parents. he presented with falls in early childhood. over the years he developed foot deformities and progressive length dependent weakness. he had multiple orthopaedic operations to his feet. the past history was also notable for kyphoscoliosis, sensorineural deafness from the age of and bilateral cataracts. nerve conduction studies at the age of revealed a demyelinating neuropathy consistent with the clinical phenotype of cmt . his younger brother had a similar, although more severe phenotype. a litaf (c. c>t,p.pro ser) mutation was identified by sanger sequencing and was present in both affected brothers but also in the unaffected sister. ngs for cmt -associated genes was therefore performed and identified two compound heterozygous pathogenic mutations detected in sh tc (c. c>t, p.arg *; delg, p.arg serfs* ), which segregated with the disease in the family. the second case describes a brother and sister with early onset demyelinating cmt associated with scoliosis and cranial nerve involvement. the male proband underwent ngs and a single previously reported pathogenic intronic splice-site mutation in sh tc (c. - a>c) was found. relative read-depth analysis of ngs was performed to look for possible copy number variants in sh tc , thus identifying a deletion of exon , which was confirmed by long pcr. cruz-velásquez gj , miramar-gallart md , alarcia-alejos r , roche-bueno jc , rodríguez-valle a , capablo-liesa jl . neurology service, university hospital miguel servet, zaragoza, spain; genetics unit, clinical biochemistry service, university hospital miguel servet, zaragoza, spain. charcot-marie-tooth disease (cmtd) defines a clinical and genetically heterogeneous group of inherited peripheral neuropathies characterized by chronic motor and sensory impairment. the type cmtd- also known as cowchock syndrome, is the product of the mutation in the apoptosis inducing factor mitochondria associated gene (aifm ). it is a slowly progressive, recessive, x-linked disease characterized by axonal neuropathy, deafness, and cognitive impairment. our purpose is to describe new cases, brothers, children of non-consanguineous parents, with a characteristic phenotype and a new mutation in the aifm gene. both siblings present from childhood, progressive weakness in lower limbs with diffuse amyotrophies. needing tenotomy before the year due to equinovarus foot. likewise they develops sensory deafness and one of them requires unilateral support at and the other wheelchair at , this one need a pacemaker for an atrioventricular block at . with brain functions and normal language, sensorial deafness, proximal and distal weakness in the four limbs with intense amyotrophies, predominantly distal. tactile and painful sensitivity decreased in glove and sock pattern. an extensive metabolic and biochemical study was normal. the electroneurography demonstrates an axonal neuropathy without response in most of the nerves explored. the electromyography shown a myogenic pattern with distal predominance. brain mri was normal in both cases. through a genetic study by exoma targeting genes associated with cmt and inherited related neuropathies was identified the homicigosis mutation in the aifm gene (p.glu lys), located in the chromosomal region xq . (cmtx ). cowchock syndrome is a rare entity, with few cases described in the literature. the in silico analysis indicates in of the predictors used (provean, sift, polyphen , lrt, mutationtaster, mutationassessor and condel) , that it is a deleterious variant. we audited all patients with peripheral neuropathy caused by a paraprotein, who received treatment and attended king's college hospital peripheral nerve service between - . patients were identified retrospectively from our database of patients attending the peripheral nerve outpatient clinic. clinical information was obtained retrospectively from hospital electronic patient records. we excluded patients with poems syndrome or in whom the neuropathy was not felt to be caused by the paraprotein. we identified patients with a diagnosis of paraproteinaemic neuropathy. we excluded four who did not fulfill the diagnostic criteria and eleven who had received no treatment or were under diagnostic study. we included patients in the final audit. ( %) had igm paraprotein. the haematological diagnosis was monoclonal gammopathy of undetermined significance (mgus) in %, waldenstroḿs macroglobulinaemia %, and lymphoma or plasmacytoma %. after treatment, overall ( %) patients improved neurologically, ( %) stabilised, and ( %) worsened. in the patients who received more than one type of treatment, we analysed outcomes according to the most powerful treatment received. patients received rituximab alone of which ( %) improved, ( %) stabilised and ( %) worsened. nine patients received rituximab combined with cyclophosphamide or bendamustine, of which ( %) improved, ( %) stabilized and ( %) worsened. eight patients received intravenous immunoglobulin, of which ( %) improved, ( %) stabilized and ( %) worsened. four patients received other chemotherapy, of which improved, stabilised, worsened. two patients received corticosteroids and both worsened. there was improvement in / ( %) with mag antibodies and / ( %) without. there was improvement in / ( %) with mgus and / ( %) with haematological malignancy. there was improvement in / ( %) with kappa light chains and / ( %) with lambda. factors associated with better outcome (by univariate analysis) were negative mag antibodies, kappa light chain, and haematological malignancy. there was no significant difference between treatments in the proportions who improved. cumberbatch m , cox a . addenbrooke's hospital, cambridge, uk. treatment of patients with autoimmune neuropathies such as chronic inflammatory demyelinating polyradiculoneuropathy (cidp) and multifocal motor neuropathy (mmn) has centred on the use of intravenous immunoglobulin (ivig). however, ivig therapy is associated with systemic side-effects, treatment wear-off effects and regular hospital attendance. subcutaneous immunoglobulin (scig) is an efficacious alternative that can be flexibly dosed, self-administered at home and avoids the 'peaks' and 'troughs' observed with ivig. these factors may combine to improve patient satisfaction and alleviate hospital capacity issues. here, we report on clinical and patient experience of switching from hospital-based ivig to home-based manual push scig for the treatment of cidp and mmn. this was a clinical case series of patients ( cidp, mmn; mean age . years) who were clinically stable on ivig and wished to switch to manual push scig. starting scig dose was equivalent to the final ivig dose for each patient (mean . g/kg week). clinical efficacy (medical research council sum score, -m walk, modified inflammatory neuropathy cause and treatment score, overall neuropathy limitations scale, romberg test) and patient-reported outcomes ( -item short form health survey , life quality index [lqi] ) were assessed at baseline and at regular intervals until the final visit ( - months after switching). at baseline, patients cited 'convenience' as their primary reason for switching to scig. eight patients completed the full assessment period and successfully undertook administrations at home (via hospital-at-home service in cases). dose adjustments, based on clinical need, were required in patients. treatment efficacy and patient quality of life, measured by sf- , were maintained after switching to scig; overall patient satisfaction, measured by lqi, increased from % to %. in the lqi, 'convenience', 'travel time/cost' and 'interference-work' were significantly improved (p< . ) after switching to scig therapy. adverse events included mild erythema and localised swelling, as expected for a ml subcutaneous injection. these findings suggest that manual push scig therapy is a viable alternative to ivig for patients with cidp and mmn, as it maintains disease stability, is more convenient for patients and may help ease hospital capacity concerns. cumberbatch m , soares regua d , cox a . addenbrooke's hospital, cambridge, uk. intravenous immunoglobulin (ivig) is used to treat a number of chronic autoimmune neurological diseases. in most centres, infusions are given at slow rates as there is a perception that this reduces the risk of adverse events (aes). this results in longer in-patient admissions, or frequent day case attendances, impacting on both the patients' quality of life and hospital capacity. however, there is little evidence to suggest that slow infusion rates are required. we used the manufacturer recommendations to optimise infusion rates and reduce the time patients spend in hospital. we report a retrospective audit which describes the impact of different ivig infusion rates on patients' clinical condition. the audit comprised three -month assessment periods: january-june (cohort ; infusion rates of . ± . ml/kg/hr, n = ); january-june (cohort ; . ± . ml/kg/hr, n = ) and july-december (cohort ; . ± . ml/kg/hr, n = ). clinical data were reviewed to determine: patient demographics, duration of infusion; time spent in hospital; and incidence of aes. the three cohorts were well matched in terms of patient demographics ( patients were treated in all treatment periods). cohorts and had significantly shorter treatment episodes than cohort ( . and . vs. . hours, p< . ), spent less time on the unit over the month period ( . and . vs. . hours, p< . ) and had fewer admissions/patient ( . and . vs. . , p< . ). the overall incidence of confirmed aes (mainly headaches) was similar across the cohorts (cohort : %; cohort : %; cohort : %). these findings indicate that increases in ivig infusion rate are well tolerated and significantly reduce treatment time, which benefits patients and offers potential cost savings and reduced pressures on hospital capacity for healthcare providers. cunningham me , yao d , meehan gr , barrie ja , willison hj . university of glasgow, glasgow, uk. one mechanism of injury in the acute motor axonal neuropathy (aman) form of guillain-barré syndrome (gbs) is the attack of peripheral nerve axons by anti-ganglioside antibodies (agabs). rodent models have demonstrated that that binding of these antibodies activates the complement cascade, resulting in the insertion of the terminal component, membrane attack complex (mac) into the axonal membrane. complement activation also results in the release of anaphylatoxins, which are known to recruit phagocytic immune cells to the site of injury. our current in vivo mouse model of agab and complement-mediated injury are acute and severe, resulting in respiratory distress over several hours of such magnitude to warrant termination of experimental procedures. to observe and potentially target immune cell infiltration following agab and complement-mediated injury, a subacute model extending over days is required. here, we demonstrate the development of such a model. to compare differences in immune cell infiltration subacutely under control and injury conditions, mice with endogenous expression of egfp in monocytes and macrophages underwent a modified agab and complement-mediated injury, resulting in a less severe phenotype than previously published models. six days following injury, immune cells in the diaphragm were compared by immunofluorescence and flow cytometry. flow cytometry found overall presence of neutrophils was significantly increased in the diaphragm. macrophages were also increased in injured mice, although did not achieve statistical significance at this timepoint. these results were reflected in immunofluorescent staining of the diaphragm where egfp+ macrophages were quantified surrounding the neuromuscular junction (the primary injury target in this model). the development of an extended mouse model of agab and complement-mediated injury is important, since acute models do not take into consideration either the late-term effects of complement-mediated activation at the nerve membrane, or the recovery phase. future studies will look at the effect of inhibiting complement activation on the presence of immune cells in distal motor nerves. family- is a large australian family with an autosomal dominant form of dhmn (dhmn : omim % ) -a group of length-dependent neurodegenerative disorders affecting the lower motor neurons leading to chronic disability. we recently reported a novel . mb chromosomal insertion within the dhmn locus which we hypothesise is likely to cause disease by dysregulating the expression of one or more nearby genes. studying gene dysregulation in peripheral nerve disease is challenging as the relevant tissues (spinal cord and peripheral nerve) are not easily accessible in patients. therefore, alternative strategies are needed to elucidate the disease mechanisms and pathways involved in peripheral nerve degeneration. to address this problem, we have devised a two-tiered strategy to assess dysregulation of candidate genes using patient lymphoblast and fibroblast cell lines. these cell lines can be easily established, are minimally invasive to obtain, and will harbour the natural mutation and genetic background of patients. our strategy firstly uses lymphoblast gene expression profiles as an initial screening tool to prioritise candidate genes for assessing altered expression. differentially expressed genes will then be modelled in c.elegans where behavioural and nerve morphology can be assessed. using rt-pcr, we have screened dhmn candidate genes in patient and control lymphoblast cell lines. eighteen candidate genes were expressed in lymphoblasts. twelve of the eighteen genes were prioritized for further analysis based on expression in both lymphoblast and neural tissues. quantitative analysis using qrt-pcr taqman assays revealed that ube c, was differentially expressed between patients and controls. it is important that patterns of differential expression can be recapitulated in neural cell-specific models. as part of our second strategy, we have generated patient and control induced pluripotent stem cell derived motor neurons (ipsc-mns) from reprogrammed fibroblasts. using this model, we will perform rna-seq and qpcr experiments to examine disease-relevant alterations in gene expression in neural tissue. we predict that utilization of these two strategies will shed light on the pathogenic mechanisms underlying the dhmn insertion and provide useful insights of pathways leading to peripheral nerve degeneration. the outcome of guillain-barré syndrome remains unchanged since plasma exchange and intravenous immunoglobulin were introduced over years ago. pathogenesis studies on gbs have identified the terminal component of complement cascade, the membrane attack complex, as a key disease mediator and thus a therapeutic target. the inhibition of complement in guillain-barré syndrome (ica-gbs) trial looked at the first use of c pathway inhibition with eculizumab in humans with gbs in a randomised, double-blind, placebo-controlled trial. its primary outcome was to look at safety and tolerability of administration concomitantly with ivig and in the context of severe (gbs disability score or greater) disease. participants were recruited for a month period, with regular follow up. subjects were screened, with ( %) being randomised. the two main causes for failure to proceed were participant concerns around eculizumab side effect profile, specifically the meningitis risk, and also intercurrent infection precluding treatment. five received eculizumab for four weeks, alongside standard intravenous immunoglobulin treatment, with receiving placebo. the safety outcomes, monitored via adverse events capture at each trial visit, showed eculizumab to be well tolerated and safe when administered in conjunction with ivig. the most common adverse events were mild derangement in transaminases or infection. there were no infusion reactions. primary and secondary efficacy outcomes were captured via gbs disability scores, mrc sum scores, rasch overall disability scores and overall neuropathy limitation scores. for the primary efficacy outcome at weeks after recruitment, of placebo and of eculizumab-treated subjects had improved by or more grades on the gbs disability score. all patients had improvements in other measured parameters. this trial highlights the challenges in recruiting acutely unwell patients, due to time constraints and intercurrent infection. although the small sample size precludes a statistically meaningful analysis, these pilot data indicate further studies on complement inhibition in gbs are warranted. charcot-marie-tooth disease (cmt) affects about one in . people. currently more than genes have been identified, with the most different phenotypes. the majority of cases in western countries are autosomal dominant and classified as demyelinating and axonal according to electroneuromyography (enmg). the clinical condition is characterized by weakness and predominant sensory changes in the feet and hands. sometimes there are different phenotypes. recently, variants in heterozygotes in the hars gene (histidil-trna synthetase) have been described associated with cmt called type w.to report a case of cmt-sensitive phenotype with a probable new mutation in hars gene (p.leu arg).a male patient, adopted son, caucasian, drug addict, for three years suffered pain in lower limbs, of great intensity, refractory to drug treatment. the examination showed retrognathism, abolition of patellar and achilleas reflexes, painful and thermal anaesthesia and apalesthesia in the feet.the enmg showed reduced sensory action potentials in sural and superficial fibular nerves. laboratory investigations for painful polyneuropathy of thick and fine fibres was normal. sural nerve biopsy revealed axonal predominance neuropathy. exome sequencing revealed a mutation in the hars gene with a pathogenic variant in heterozygosity, with replacement of the amino acid leucine at position by arginine. our patient, although we did not know the antecedents, presented painful polyneuropathy, whose genetic research, although not unequivocal, indicated a variant called cmt w. few cases of this variant were described, with several mutations. our case revealed mutation hitherto unknown (p.leu arg). we conclude by the importance of a thorough genetic evaluation, in cases of sensory polyneuropathy of unknown cause. small fiber neuropathy (sfn) is a condition that affects the small a -and c-fibers, leading to severe neuropathic pain and autonomic dysfunction. several sodium channel gene mutations have been found in patients with sfn, with scn a-gene mutations being the most frequent. because current available sodium channel blockers are not selective for na v . , these treatments often result in numerous side effects. lacosamide is an anticonvulsant that targets specific sodium channels with a slow-inactivation state, while sparing those with normal activity. several mutations of the scn a-gene with an impaired slow-inactivation of na v . have been found in patients with sfn. therefore, a positive effect of lacosamide on pain reduction in these patients is expected. the primary objective of this study was to determine the effect of lacosamide versus placebo on pain in subjects with scn a-associated sfn. secondary objectives were to determine the effect of lacosamide on autonomic symptoms, sleep interference, and quality of life, and to examine the safety and tolerability. the lacosamide-efficacy-'n'-safety in sfn (lenss) study was a randomized, placebo-controlled, double-blind, crossover-design study. subjects were randomized to start with lacosamide and end with placebo or vice versa. during both of the two phases of the study, the subjects were treated for a period of eight weeks of mg bid, preceded by a titration period, and ended by a tapering period. patients filled in a pain diary twice daily and scored a set of validated questionnaires on autonomic symptoms, sleep interference, and quality of life at multiple study visits. in total patients with scn a-associated sfn were included between november and february . the subjects had a median age of years, ranging from to years. sixty percent of the patients were female. the final results of the study, including the primary and secondary outcomes, will be presented. de la oliva n , del valle j , navarro x . department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. intraneural interfaces must be in intimate contact with nerve fibres to have a proper function, but it has been shown that this is compromised due to the foreign body reaction (fbr). this fbr is the first response of the nonspecific immune system against an implanted device and is characterized by a first inflammatory phase followed by a second antiinflammatory and fibrotic phase. this process results in the formation of a tissue capsule around the interface causing function loss due to the physical separation between the active sites of the electrode and nerve axons. taking this into account, here we have tested several anti-inflammatory drugs such as dexamethasone, ibuprophen and maraviroc to reduce macrophage activation as well as clodronate liposomes to reduce monocyte/macrophage infiltration. moreover, sildenafil have been administered as an antifibrotic drug to reduce collagen deposition in a fbr model with longitudinal parylene c-based intraneural devices implanted in rat sciatic nerve. briefly, animals were systemically treated with dexamethasone, ibuprophen, sildenafil, maraviroc or clodronate liposomes for two weeks, and nerve damage, inflammatory reaction and matrix thickness around the implant were assessed. treatment with dexamethasone, ibuprophen or clodronate liposomes significantly reduced the inflammatory response in the nerve in comparison to saline group while sildenafil or maraviroc had no effect on iba positive cells infiltration in the nerve. however, only dexamethasone was able to significantly reduce the matrix deposition around the implant after two weeks of treatment. these results support the idea that inflammation triggers the foreign body response in peripheral nerves and a potent anti-inflammatory treatment with dexamethasone could have a beneficial effect on lengthening intraneural interfaces lifespan. de la oliva n , del valle j , navarro x . department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. intraneural interfaces functionality decreases over time, among other factors, due to the foreign body response (fbr), which encapsulates the implanted devices and physically separates the active sites from the nervous tissue. here we have studied the fbr to parylene c or polyimide thin devices implanted in rat sciatic nerves, assessing thickness of the tissue capsule, signs of inflammation and nerve damage. we have characterized the responsible cells of this response and several molecular mediators over months of implant to find differences between the fbr to both materials. after weeks of implant, the inflammatory response due to the surgery was already decreased, whereas in the implanted nerves it reached its highest levels to then decrease at chronic time points. besides, the amount of foreign body giant cells (fbgc), as a result of macrophage fusion, found in the tissue capsule around the implant also increases progressively to reach a maximum after weeks. on the other hand, molecular analysis of the environment revealed a peak of inflammatory cytokines during the first day of implant to return to standard levels thereafter. however, an increase on ccls molecules was found at later time-points for both materials. with regard to the capsule thickness, all the devices were surrounded by a tissue deposition which appeared soon after the implantation. however, in the case of polyimide devices, the tissue capsule showed a peak weeks after the implant and signs of remodeling thereafter, while the parylene c devices showed a second increase from to weeks in comparison to polyimide devices. immunohistochemical and electron microscopy analysis revealed two different cell types implicated in the fbr in nerve to both materials: macrophages, in close contact with the interface, and fibroblasts which appear after weeks surrounding the tissue capsule. although further analyses are needed to elucidate the differences in the fbr to parylene c and polyimide polymers, these results can help to determine therapeutic targets in order to reduce this response and to improve the intraneural interfaces lifespan. delmont e , antoine jc , paul s , boucraut j , attarian s . referral centre for als and neuromuscular diseases, marseille, france; referral centre for neuromuscular diseases, saint etienne, france; immunology laboratory, saint etienne, france; immunology laboratory, marseille, france. peripheral neuropathies with antibodies against myelin associated glycoprotein (mag) are chronic sensory neuropathies characterized by the presence of an igm monoclonal gammopathy and high levels of anti-mag antibodies. these antibodies recognize a specific epitope called human natural killer (hnk ) shared by nk lymphocytes and several components of the peripheral nerve (mag, p , pmp , sgpg, phosphocan). recently an elisa test has been developed to detect antibodies against hnk epitope. our objectives were to determine the sensitivity and the specificity of anti-hnk antibodies in the diagnosis of anti-mag neuropathy and to know if these antibodies were correlated with the severity of the disease. anti-hnk antibodies were assessed in anti-mag neuropathies and in negative controls: chronic inflammatory demyelinating polyradiculoneuropathies (cidp), miller fisher syndromes, sensory neuronopathies, length-dependant axonal sensory polyneuropathies, healthy controls. in anti-mag neuropathies, were recorded age, disease duration, incat sensory sum score (iss), overall neuropathy limitation scale (onls), rasch-built overall disability scale (rods), mrc sum score, anti-mag antibodies titer, peak dosage of the igm monoclonal gammopathy. anti-hnk antibodies were measured with gangliocombi™ mag elisa test and anti-mag antibodies with anti-mag autoantibodies elisa test both from buhlmann company. anti-hnk antibodies were positive in / anti-mag neuropathies, and in / controls (sensitivity %, specificity %). in anti-mag neuropathies, anti-hnk titer was correlated with sensory deficiency evaluated with the iss score (r= . , p= . ) and with disability evaluated with the rods (r= − . , p= . ) and onls scales (r= . , p= . ). anti-hnk titers were not related to age, disease duration, mrc sum score, anti-mag antibodies titer, peak dosage of the paraproteinemia. anti-mag antibodies titers were associated with none of the characteristics of the patients with anti-mag neuropathy. anti-hnk antibodies have good sensitivity and specificity in the diagnosis of anti-mag neuropathy. compared to anti-mag antibodies, their value is that their titers are related to the disease severity. these results need to be confirmed in a larger prospective cohort. chronic inflammatory demyelinating polyradiculoneuropathy (cidp)is a heterogeneous and treatable immune-mediated disorder that critically lacks biomarkers to support diagnosis. recent evidences indicate that paranodal proteins (contactin- , contactin-associated protein- , and neurofascin- ) are the targets of autoantibodies in a subset of patients with cidp showing distinct clinical presentations. particularly, these biomarkers appear to have clinical relevance and help to orientate therapeutic choice. here, we examined five patients presenting an igg reactivity against the nodes of ranvier and the axon initial segment. using a proteomic approach, cell-based assays and elisa, we identified neurofascin- (nfasc ) and neurofascin- (nfasc ) as the main targets of autoantibodies at the nodes of ranvier. four patients displayed predominantly antibodies of the igg subclass, whereas one patient presented igg antibodies that activated the complement pathway in vitro. these antibodies recognized different epitopes than the previously described anti-neurofascin- igg suggesting different pathogenic functions. accordingly, patients with anti-nfasc / igg showed a distinctive clinical presentation. most patients had a severe phenotype associated with conduction block or decreased distal motor amplitude. tremors or neuropathic pain were not observed. four patients presented with a subacute-onset and sensory ataxia. of interest, the neuropathy occurred concomitantly with nephrotic syndromes in two patients and with an igg -related retroperitoneal fibrosis in one patient. this suggested that autoantibodies could be responsible for the occurrence of both disorders. intravenous immunoglobulin and corticosteroids were effective in three patients, and one patient improved following cyclophosphamide and rituximab treatment. clinical remission was found to correlate with the depletion of anti-nfasc / antibodies and the loss of igg reactivity toward the nodes of ranvier. in addition, recovery of conduction block and of distal motor amplitude were observed following remission and suggested a nodo-paranodopathy. our data demonstrate that nodal antigens are the target of autoantibodies in a subgroup of patients with cidp. this emphasizes that the pathogenic mechanisms involved in chronic immune-mediated demyelinating neuropathies are broad and may include dysfunctions of the nodes of ranvier. mutations in the neurofilament heavy (nefh) gene have been recently identified as a rare cause of autosomal dominant, axonal charcot-marie-tooth disease (cmt ). the clinical spectrum of this condition remains to be delineated. we report two french families with an axonal, predominantly motor, dominantly inherited form of cmt caused by two previously unreported mutations in the nefh gene. twelve patients belonging to two different families were included in the study. they displayed an axonal motor and sensory neuropathy, with no mutations in known axonal cmt genes. a remarkable feature in all patients was the early involvement of proximal muscles of the lower limbs, occurring approximately to years after the onset of motor deficit. proximal weakness affected predominantly the iliopsoas muscle, whereas quadriceps and hamstring muscles were relatively preserved. muscle weakness and muscle wasting progressed rapidly, with most of the patients requiring walking assistance after years of disease evolution. three patients in family had brisk reflexes. nerve-conduction velocity studies displayed evidence of a motor and sensory axonal neuropathy predominantly affecting the lower limbs. original deletions of nucleotides near the end of the coding sequence of nefh were identified: in family , c. _ del (p.lys argfs* ), and in family c. _ del (p.lys glyfs* ) causing a frameshift. interestingly, this frameshift leads to the loss of the terminating codon and to the translation of additional amino acids encoding a cryptic amyloidogenic element, suggesting that this type of mutations could induce protein aggregation. consistently, we showed that overexpression of the mutated forms of nefh in a human neuroblastoma cells induced the formation of protein aggregates. we also observed that it triggered caspase activation and apoptosis. using electroporation of chick embryo spinal cord, we confirmed in vivo that mutated nefh formed aggregates and triggered apoptosis of spinal cord neurons. altogether, this suggests that these mutations in nefh cause protein aggregation and neurotoxicity in neurons expressing nefh. progressive loss of such neurons would explain the early motor involvement and the pyramidal signs observed in some patients. our results provide a physiological explanation to the presence of cmt and als clinical features in affected patients. del valle j , , delgado-martínez i , righi m , santos d , , cutrone a , bossi s , d'amico s , micera s , , navarro x , . neuroprosthetic devices that are aimed to restore sensorimotor limb function of amputee patients require highly selective electrodes designed to establish a tight relationship with the nerve, allowing the bidirectional transduction of signals between nerve fibres and the interface and enabling close-loop control from the user. differently from extra-or intraneural interfaces, regenerative nerve electrodes are designed to enable electrical interface with regrowing axonal bundles of injured nerves, aiming to achieve high selectivity for recording and stimulation. however, most of the developed designs pose an obstacle to the regrowth mechanisms due to low transparency and cause an impairment of the nerve regeneration. in this work, we present a novel double-aisle planar regenerative electrode, a new type of highly transparent, non-obstructive regenerative electrode, which allows the selective stimulation and recording of separated nerve fascicles. the design consists of a thin and flexible double-sided electrode longitudinally inserted across a conduit thus creating two separated aisles in which regenerating fascicles can independently regrow after nerve transection. electrodes implanted in acutely transected nerves of rats showed the capability of selectively stimulating and recording different fascicles inserted in the aisles. moreover, chronic implantation of the electrode in a nerve gap of mm after sciatic nerve section allowed for fascicle regeneration and reinnervation of distal muscles as confirmed by the high number of myelinated axons inside each aisle, good biocompatibility, and adequate nerve conduction. in addition, three and six months after implantation, independent stimulation and recording of each separately regenerated fascicle were possible. our results demonstrate the potential contribution of the doubled-aisle regenerative electrode to selectively interface different fascicles of an injured nerve with no deleterious effects on nerve regeneration. therefore, this multi-aisle regenerative electrode may be suitable for neuroprosthetic applications, such as prostheses for the restoration of hand function after amputation or severe nerve injuries. demichelis c , garnero m , franciotta d , cortese a , callegari i , mancardi gl , schenone a , leonardi a , benedetti l . department of neuroscience, rehabilitation, ophthalmology, genetics, maternal and child health, university of genoa and irccs aou san martino-ist, genoa, italy; laboratory of neuroimmunology, irccs, "c. mondino" national neurological institute, university of pavia, pavia, italy; u.o. neurology, asl imperiese, imperia, italy. querol et al. showed that neurofascin (nf ) antibodies identify a chronic inflammatory demyelinating polyradiculoneuropathy (cidp) phenotype characterized by severe polyradiculoneuropathy, poor response to intravenous immunoglobulins (ivig), and disabling tremor. neurological improvement after therapy with rituximab has been previously reported in three patients with cidp with igg anti-nf antibodies. herein we describe the acute-onset of a case of cidp positive for nf igg antibodies resistant to conventional therapies and responsive to rituximab. the patient is a year-old woman who presented acute onset ataxia and gait disturbances; her symptoms progressed over two weeks and distal weakness, numbness and paresthesias appeared too. the nerve conduction study was suggestive for a motor-sensory polyradiculoneuropathy mainly demyelinating. the cerebrospinal fluid analysis showed elevated protein level and normal cellular count. the patient was initially diagnosed with guillain-barré syndrome (gbs) and treated with plasma exchange without improvement. an ivig cycle was started with a partial relief but at the time of admission to the rehabilitation center the patient still had a marked weakness in all four limbs. after six months she presented a further clinical deterioration and she was restricted to wheelchair. there was no response to additional treatment with ivig, while pulse corticosteroid treatment determined a significant clinical improvement. during the next months, despite the maintenance of steroid therapy, the patient presented a progressive deterioration and she was again restricted to wheelchair. postural and intention tremor appeared at upper limbs and became progressively more disabling. anti-nf ab dosage resulted positive. rituximab was administered at a dosage of mg/m /weekly for weeks. after three months the tremor improved, allowing her to eat independently and the patient was able to walk with bilateral support. antibodies anti-nf were negative. after six months she walked without support and she was able to stitch crochet. as previously reported, in this case a cidp positive for igg nf developed severe polyradiculoneuropathy with predominant distal weakness, ataxia, disabling tremor and resistance to conventional therapies. interestingly the onset was gbs-like. the correct identification of these cidp subtypes has diagnostic, prognostic and therapeutic implications. rituximab con be useful in these patients. demir Ö , yazıcı t . department of neurosurgery, university of gaziosmanpaşa school of medicine, tokat, turkey; department of neurosurgery, kent hospital, giresun, turkey. it is still challenging problem to maintain motor and sensory functions of peripheral nerve after nerve transection. after the nerve injury, calcium concentration in the damaged area increases. then the calcium ions act like cytotoxic agents in the damaged area. nifedipine is calcium channel blocker. we aimed to investigate the effects of nifedipine on nerve regeneration by modulating calcium in the damaged area. twenty-four swiss albino male rats were divided into two groups. left sciatic nerve transection surgery was performed to the all rats in both groups. then the all transected nerves were sutured primarily with epineural interfascicular method. in the experimental group, the anastomosis sites were wrapped with a piece of gel foam soaked into diluted nifedipine solution. in the control group, the anastomosis sites were wrapped with a piece of gel foam soaked into saline solution. we evaluated the effect of nifedipine by using functional, electro-physiological and histopathological studies after the surgeries. in the postoperative second week, walking test was performed and sciatic function index was calculated. in the postoperative third week electroneuronography (enog) was performed. there are significant differences between two groups. nifedipine improved nerve recovery functionally (p< . ) and electro-physiologically (p< . ). in the postoperative fourth week, we performed histopathological examination. in the experimental group with nifedipine there were more organized axons that reached the aim. we conclude from these results that nifedipine is an effective nerve protective agent when used locally at the anastomosis site after the transection of the nerve. the lack of effective, disease-modifying therapies for cmt highlights the need for novel preclinical models suitable for drug discovery. studies in rodent models of cmt tend to be time-consuming, and findings so far have translated poorly into clinical trials. primary and induced pluripotent stem cell (ipsc)-derived neuronal cultures are an established model of neurological diseases. however, due to the random distribution of neuronal bodies and neurites that happen when plating these cells, this system is not ideal to investigate axonal, length-dependent processes like peripheral neuropathies and particularly cmt. to optimize this well-established model system, we developed a robust human platform to study axonal morphology and physiology based on motor neuron neurospheres. we differentiated motor neurons from human induced pluripotent stem cells, purified them by magnetic sorting and cultured them in suspension until they formed neurospheres. floating neurospheres can be maintained in agitation for months as a reliable source of motor neurons. after neurospheres are platted, axons rapidly grow out of them in a radial fashion, resembling dorsal root ganglia cultures. this configuration allows for a better visualization of axons in imaging studies and for continued axonal growth over at least a -day period. axons grew at an average rate of micrometers/day and reached up to cm in length. neurospheres can be fixed and stained allowing for morphological analysis and investigation of protein distribution in axons. this system is also ideal for time-lapse imaging to study axonal transport of organelles and neurofilament kinetics. lastly, our motor neuron neurosphere system lends itself well for high content screening platform. neurospheres can be plated in -well plates where multiple compounds can be tested and the axons easily imaged by a high content screening microscope. in summary, we developed a new platform to investigate motor axons in vitro, which are particularly useful to study length-dependent processes such as inherited peripheral neuropathies and may facilitate the identification of new therapeutic compounds using high content screening systems. mutations in the neurofilament light chain (nfl) gene cause autosomal dominant axonal charcot-marie-tooth neuropathy (cmt e). nfl is a major component of the neuronal cytoskeleton, and is believed to function in conjunction with nfm and nfh to provide structural support for the axon and regulate axon diameter. despite the significant advances in understanding its biological basis, there is still no effective, disease-modifying therapy for cmt e, in part due to the paucity of preclinical models suitable for drug discovery. the development of novel preclinical platforms that can faithfully mimic mechanisms of axonal degeneration in vitro would be an essential and valuable resource to better understand the biology of cmt e and identify potential targets for therapy development. to address this, we generated control and cmt e patient-derived motor neurons and cultured them in suspension until they formed neurospheres. immunostaining of cmt e neurospheres with nfl and tubb antibodies revealed numerous areas of nfl accumulation in n s cmt e axons, resembling the accumulations of mutant nfl protein seen in the processes of catecholaminergic neuronal cell line cad overexpressing several nfl mutants. further analysis demonstrated that areas of nfl accumulation were also immunopositive for nfh, pnfh and nfm and that at least nfl and nfm co-localized in the same areas of deposits. taken together, these results demonstrate that abnormal axonal neurofilament distribution is a feature of cmt e ipsc-derived motor neurons and involve all three neurofilament subunits. we also developed an image analysis routine to allow for automatized quantification of neurofilament distribution. preliminary quantification of nfl signal intensity revealed that axons from patients have a weaker nfl signal compared to control axons, but present several signal peaks above the range observed in controls, which related to the areas of nfl accumulation. these results suggest that nfl accumulates in certain regions of cmt e axons but is reduced in the areas with no accumulation. these findings can be readily adapted into a high content screening platform and will be used to identify compounds able to reverse this axonal phenotype. in summary, we identified a strong axonal phenotype in human cmt e motor neurons with potential as a screening platform for drug discovery. nodal and paranodal proteins have been identified as antigens in peripheral inflammatory neuropathies, however the frequency and clinical relevance of antibody responses against these targets remain poorly investigated in gbs. patients with acute onset inflammatory neuropathies were identified by exploration of the local databases of the departments of neurology and the institute of neurology of the medical universities in innsbruck and vienna. patient data, electrophysiological classification and presence of anti-gangliosid antibodies were retrospectively retrieved by review of patient records. only patients with typical clinical presentation and electrophysiological results consistent with one of the subtypes of gbs were included in the study. among forty-nine patients, thirty-five were classified as aidp, six aman, three amsan, three mfs, and two pharyngo-cervico-brachial gbs. of the included patients had anti-ganglioside-antibodies. ten patients with the initial suspicion of aidp had a disease duration of more than months and were reclassified as cidp. all patient and twenty sera of control patients with non inflammatory polyneuropathy were screened by an optimized tissue based assay using rat brains for immune responses against surface antigens, and by cell-based assays with transfected hek cells for antibodies against contactin (cntn ), contactin (cntn ), contactin-associated-protein (caspr ) and neurofascin- (nf ). in the tissue based assay some of the patients showed a light neuropil staining. none of gbs patient's sera had antibody reaction to cntn , cntn , caspr or nf in cell-based assays. among the cidp patients, two patients demonstrated reactivity against cntn with similar clinical presentation as previously described. none of the control patients had any antibody reaction to the performed tests. our results suggest that antibody responses to cntn , cntn , caspr or nf are absent in austrian gbs patients, although more patients will be screened to substantiate these preliminary results. furthermore, it remains to be established whether antibodies against cntn may predict a chronic course in acute onset inflammatory neuropathies. a few variants of chronic inflammatory demyelinating polyradiculoneuropathy (cidp) have been described with a frequency of - %. their relation and possible evolution into typical-cidp remain unclear, as is their treatment response possibly because of differences in diagnostic criteria. we used the data from a web-based database on italian patients with cidp to determine the frequency and characteristic of these variants, the possible evolution into typical-cidp, and their treatment response. all the patients were assessed at study entry and the disease course before inclusion was analyzed. by february- , we included patients ( men, women), aged - years (median years) with a mean disease duration of . years (range . - years) and complete data available from . based on the clinical data and our revised diagnostic criteria, patients ( %) were classified to have atypical cidp at onset and for the following two years including with dads ( %), with motor cidp ( %), with sensory cidp and cisp ( . %), with lewis-sumner syndrome ( . %), and with recurrent cranial neuropathy. at study entry, patients ( %) had progressed into typical cidp after - years (median years) while ( %, % of total) still had atypical cidp after . - years (median years) with a similar proportion of progression ( - %) within each group. the diagnosis of atypical cidp at entry fulfilled efns/pns criteria in ( %). csf studies were diagnostic in / ( %) patients, nerve biopsy in / ( %), and nerve imaging in / ( %) tested patients. similarly to typical cidp, % of treated patients with atypical cidp improved after treatment with a proportion of response varying from % to % in the different forms. most patients with sensory or motor cidp had however an unsatisfactory response to steroids. this study shows that he proportion of patients with atypical cidp varies during the course of the disease with almost % of the patients evolving into typical cidp within years from onset. in addition, response to treatment is frequent in atypical cidp even if not all the forms respond to the same therapies. only few studies investigated the frequency of antecedent events and comorbidities in patients with chronic inflammatory demyelinating polyradiculoneuropathy (cidp), and little is known on the role of possible predisposing factors, dietary, and lifestyle habits, on the onset and progression of the disease. we used the data from a web-based database on italian patients with cidp to determine the frequency of antecedent events and comorbidities and the possible role of predisposing factors including lifestyle and dietary habits and exposure to toxic agents, using a structured questionnaire. partners of patients served as controls. impairment was evaluated using the mrc sumscore and disability with incat and r-ods scales. logistic regression was used to calculate odds ratio (or) with % confidence interval (ci) for the risk of cidp. sex and disease-duration were included as covariates. by february- , patients were enrolled, with complete data on patients for antecedent events and comorbidities and patients and controls for lifestyle habits. ninety-two patients ( %) reported an antecedent event, mostly infection or vaccination ( %). one or more comorbidity were present in % of the patients including hypertension ( . %), thyroid disorders ( %) and diabetes ( . %) and in % influenced the choice of initial therapy. exposure to toxic environmental agents (odds ratio [or] = . ; % ci, . - . ), cigarette smoke (or = . ; % ci, . - . ), and dietary supplements (or = . ; % ci, . - . ) were associated with a higher risk of cidp while rice consumption was associated with a reduced risk (or = . ; % ci, . - . ). concerning disease severity, more severely affected patients more frequently consumed raw-meat (or = . ; % ci, . - . ) and white meat (or = . ; % ci, . - . ), while rice (or = . ; % ci, . - . ) and soft drink consumption (or = . ; % ci, . - . ) and physical activity were associated with lower disability (or = . ; % ci, . - . ). this study confirms that comorbidities are frequent in patients with cidp and often influence the choice of initial therapy. in addition preliminary data show that toxic exposure and some lifestyle and dietary habits may influence the onset and progression of cidp. doppler k , schuster y , weishaupt a , sommer c . department of neurology, university hospital würzburg, würzburg, germany. autoantibodies against the paranodal protein contactin- have recently been described in patients with cidp. in most patients, autoantibodies of the igg subclass are predominant and are supposed to be pathogenic. the role of igg anti-contactin- is so far unclear. in the present study, igg of three different patients, one with igg anti-contactin- , one with a low titer of igg anti-contactin- and one with a high titer of igg anti-contactin- , and of controls were injected into the sciatic nerves of lewis rats. nerve conduction studies of the injected nerve and motor and sensory testing were performed before and after injection. conduction blocks and motor deficits were detectable in the two patients with high titers of igg and igg , not in the patient with low titers. the percentage of conduction blocks was . % in rats injected with igg of the igg patient and % in those injected with igg . motor deficits were detectable in both patients with conduction blocks but were most apparent in the patient injected with igg of the igg patient. no differences in sensory testing were observed. conduction blocks and motor deficits improved after five days and were normal after seven to eight days. our data give the first evidence of pathogenicity of igg anti-contactin- autoantibodies, not only igg . igg of the igg patient induced a more severe clinical and electrophysiological phenotype compared to the igg patient. remarkably, this reflected the clinical phenotype of the patients, as the igg patients showed an acute-onset of sensorimotor symptoms at the time of blood withdrawal whereas the igg patient presented with a more chronic course of disease. doppler k , frank f , koschker a-c , reiners k , sommer c . department of neurology, university hospital würzburg, würzburg, germany; endocrinology and diabetes unit, department of medicine i, university hospital würzburg, würzburg, germany. axoglial dysjunction and paranodal demyelination have been discussed as potential mechanisms of nerve fiber damage in diabetic neuropathy. studies on human tissue are limited, as nerve biopsies are invasive and only rarely performed in patients with confirmed diabetic neuropathy. skin biopsy has recently been suggested as a good tool to analyze paranodal and nodal changes of myelinated fibers. in the present study, we analyzed the paranodal and nodal region in myelinated fibers of skin biopsies of patients with diabetic neuropathy, patients with diabetes mellitus without neuropathy, and normal controls. immunofluorescence of skin sections with antibodies against caspr, neurofascin, sodium channels and myelin basic protein was performed to assess paranodal/nodal architecture, segmental demyelination and myelinated nerve fibers. staining with antibodies against protein gene product . was used to quantify unmyelinated nerve fibers. we found an increase of elongated ranvier nodes and a dispersion of neurofascin at the distal leg in patients with diabetes mellitus with and without neuropathy and at the finger in patients with diabetic neuropathy. an increased dispersion of caspr was only found in biopsies of the finger in patients with diabetic neuropathy. our data show that skin biopsy is an appropriate tool to analyze nodes of ranvier in patients with diabetes mellitus. structural nodal changes are detectable in diabetic neuropathy, and even in diabetic patients without neuropathy. dourado me , fernandes u , vital al , ramos e , urbano jc , sena a , queiroz jw , jeronimo smb . federal university of rio grande do norte, natal, brazil. the erasmus gbs outcome score (egos) is a validated prognostic model that uses acute phase and easy-to-obtain clinical characteristics to determine outcome at months in patients with gbs. this study aims to assess the validity of egos in rio grande do norte, brazil, and to compare with another european study. data collected prospectively from a cohort of patients with gbs of rio grande do norte, brazil, between june and august , was assessed. ninety patients were excluded for missing data or diagnoses of miller fisher syndrome and atypical forms of gbs. to calculate the egos, the gbs disability score was assessed in the second week of disease and at months. to compare this study with the european one in independent group proportions, we used the student's t-test, being considered statistically significant p< , . the patients included were divided in four groups based on egos. thus, patients had egos between and ; had egos between . and . ; had egos and had egos between . and . in the first, second, third and fourth group, ( %), ( . %), ( . %) and ( . %) of the patients were unable to walk independently after six months of the disease, respectively. overall, of the patients analyzed, ( . %) had poor outcomes in this study. in the european paper, based on the same group division, of ( . %), of ( %), of ( %) and of ( %) were unable to walk independently. comparing both studies, the patients of this study were younger, more seriously ill in the first weeks and with more sensitive deficits. there were no difference relative to sex, cranial nerves deficits and presence of anti-gangliosides antibodies. using the student's t-test for ability to walk after months according to egos stratification, we achieve in the first group p= . ; in the second p= . ; in the third p= . ; and in the fourth p< . .the egos did not have a good capacity to predict the ability to walk after months of gbs in rio grande do norte, brazil. historically, guillain-barré syndrome (gbs) epidemics are rarely seen. between and , we treated and followed cases of gbs in the state of rio grande do norte with a yearly incidence of . / , . no seasonality was observed. the mean age of the patients was years (range, - ), with % of the cases younger than years. demyelinating variant was the most frequent subtype of gbs. in march , the first report of autochthonous transmission of zika virus (zikv) was determined in natal, brazil. later that month, we documented an increase in incidence of gbs in natal, brazil. the incidence in was of . / . . of the cases of gbs diagnosed in , were diagnosed from march through may, which coincided with the outbreak of zikv in natal, brazil. eighteen patients ( % of the cases) had a history of rash and fever prior to onset of gbs symptoms, with the median age of years (range, - ) . the electroneuromyography studies of these patients indicated that ( . %) had acute inflammatory demyelinating polyneuropathy, ( . %) had acute motor axonal neuropathy, and ( . %) was inconclusive. the mean time from onset of zikv infection symptoms to onset of the gbs were days (range, - ). the mean time of nadir was days (ranged, - ) . cranial neuropathies were present in patients ( . %). nine patients were bedridden ( %) and ( . %) required mechanical ventilation. the mean protein content of the central spinal fluid was . g/l, with the white blood cell count below /mm in all patients. they were all treated with intravenous igev. they all improved quickly. anti-gm was negative in all patients. rt-pcr was negative for dengue, chikungunya and zika. serum mac-elisa igm for zika and dengue was made in patients and it had % of positivity. prnt for zika and dengue had % positivity. in summary, we report a geographically and temporally defined cluster of gbs associated with an outbreak of acute rash in the state of rio grande do norte, brazil. as the prevalence of diabetes mellitus continues to increase worldwide, diabetic complications represent a growing burden to patients and society. distal symmetrical polyneuropathy (dsp) is a common complication that affects up to % of diabetic patients. dsp reduces patient quality of life due to chronic pain, ulcerations, and may lead to lower extremity amputations. despite its high prevalence, the mechanisms underlying diabetic dsp are poorly understood and several mechanisms are believed to play a role. we hypothesize that diabetic dsp arises from microvascular complications characteristic to diabetes. specifically, capillary dysfunction -disturbances in capillary flow patterns -is a likely candidate to explain development of dsp, as it can limit oxygen and nutrient delivery to nervous tissue, causing nerve dysfunction and damage, and thus development of dsp. we will study this hypothesis utilizing the state-of-the-art blood flow imaging techniques to visualize and quantify endoneureal blood flow and then link these findings with measures of dsp (e.g. nerve conduction velocity; intra-epidermal nerve fibre density) in animal models. we will include several animal models of diabetic dsp caused by either type or type diabetes. two photon microscopy and optical coherence tomography allow visualisation and quantification of capillary transit times and blood flow within peripheral nerves at high resolution. we hypothesise that changes in blood flow patterns and subsequent impairment of nutrient and oxygen delivery to nervous tissue precede the onset of diabetic dsp. if our experiments support this prediction, we will attempt to develop interventions that improve capillary blood flow to prevent or delay the development of dsp. duman o , saracoglu m , haspolat s , bozkurt o . department of child neurology, akdeniz university hospital, antalya, turkey. axonal polyneuropathies are very heterogeneous group of diseases which are very rarely seen during infantile age. some of them may be accompanied by developmental retardation, severe muscle weakness and progressive course. we aimed to present two cases of axonal sensorymotor neuropathy with infantile onset and atypical course. our -year-old boy patient was admitted to our clinic for progressive gait loss since one month. he was the first offspring of consanguineous parents with normal prenatal and natal history. electromyography revealed axonal sensorymotor polyneuropathy. metabolic and cerebrospinal fluid (csf) examinations for etiology were all normal. brain and spinal magnetic resonance imaging (mri) were normal. he had partial benefit from oral steroid treatment. in the course of disease along with four neuropathy attacks he had significant benefit from serial intravenous immunoglobulin treatments in two years clinical course. a month-old girl who is the first offspring of nonconsanguineous parents was admitted to the clinic for acute tetraparesis. axial sensorymotor polyneuropathy was detected in the electromyography. metabolic and cerebrospinal fluid (csf) examinations were normal. she had three more acute polyneuropathy attacks during ivig cessation period. both patients revealed with serial immunoglobulin treatments but unresponsive to riboflavine treatment. we aimed to discuss our rarely seen and the pathogenesis is not completely understood cases' course. serial ivig treatment may be helpfull for such patients' treatment. transthyretin-related familial amyloid polyneuropathy (ttr-fap) is an autosomal dominantly inherited disorder caused by mutations of the transthyretin (ttr) gene. the mutant amyloidogenic transthyretin protein causes the systemic accumulation of amyloid fibrils that result in organ dysfunction. ttr-associated fap is a progressive and fatal disease, if left untreated, and should be considered in the differential diagnosis of any person presenting with a progressive polyneuropathy, particularly with accompanying autonomic involvement. the clinical, electrophysiological, histopathological, and genetic characteristics of patients from turkey ( female, male) from eleven families with polyneuropathy and mutations in ttr were evaluated. two patients had no family history of ttr-fap and were considered as sporadic cases, and the remainders were familial cases displaying an autosomal dominant inheritance pattern. sequence analysis of the ttr gene revealed five mutations (p.val met, p.glu gln, p.gly glu, p.glu gly and p.gly glu). most common mutation was p.val met (in unrelated families). mean age at disease onset was . ± . years (range - years). the most commonly reported initial complaint was paresthesia in the feet (asymmetric in three patients). four patients ( male) with the p.glu gln mutation presented with carpal tunnel syndrome. two patients with the p.gly glu mutation showed episodes of dysarthria and hemiparesis, consistent with this genotype. seven patients died during the follow-up period as a result of systemic involvement. this study suggests that our cohort of ttr-fap patients from turkey exhibits clinical and genetic heterogeneity. ebenezer gj , liu y , judge dp , cunningham k , truelove s , carter nd , sebastian b , byrnes k , polydefkis m . department of neurology, johns hopkins university, baltimore, md, usa; division of cardiology, johns hopkins university, baltimore, md, usa; department of epidemiology, johns hopkins bloomberg school of public health, baltimore, md, usa. effect of amyloid deposition on cutaneous nerves was assessed in subjects with pathogenic ttr variants and control subjects. three groups of subjects each including ttr-fap patients, age/gender-matched healthy subjects and disease controls as well as ttr mutation carriers without neuropathy (ttr-nopn) and with al-amyloid underwent neurological examination and mm skin biopsies. micron sections were stained with anti-pgp . , anti-ttr and congo red. amyloid burden with imagej, intraepidermal (ienfd) sweat gland (sgnfd) and pilomotor densities (pmnfd) measured and correlations between amyloid burden, fiber subtype, neuropathy impairment score-ll (nis-ll) and nis sensory subscore were evaluated. ienfd, sgnfd, and pmnfd were all significantly reduced in ttr-fap patients vs. healthy controls while mutation carriers had intermediate reductions. lower nerve fiber densities were associated with nis-ll (p< . ). congo red staining revealed brilliant red amyloid deposits with apple-green birefringence within dermal collagen, sweat glands, and arrector pili muscles. amyloid infiltration was observed in the endoneurium and perineurium of small fiber sensory and autonomic nerves that innervate sweat glands and arrector pili muscles. cutaneous amyloid deposition was detected in % of ttr-fap and not in healthy or disease controls subjects. both al and / ttr-nopn subjects were congo red positive. amyloid burden was inversely correlated with ienf (p< . , r=− . ) sgnf (p< . , r=− . ), pmnf (p= . , r=− . ) distal leg densities, and correlated with nis-ll (p= . , r= . ) and nis sensory subscore (p= . , r= . ). wild-type ttr staining was less prominent in pathogenic ttr carriers. the diagnostic sensitivity and specificity to detect amyloid in skin were % and % in ttr-fap. the repeat measurement of the amyloid burden from the same section with imagej was r = . , p< . and different sections from the same biopsy was r = . and p< . . we conclude that endoneurial amyloid contributes to sensory and autonomic nerve injury. amyloid burden correlated strongly with sensory/autonomic axon densities and nis-ll. skin punches offer a convenient alternative to establishing a tissue diagnosis. amyloid burden is an attractive biomarker marker for ttr-fap and treatment effect. the study was supported through a grant from pfizer. ebenezer gj , truelove s , polydefkis m . department of neurology, johns hopkins university, baltimore, md, usa; department of epidemiology, johns hopkins bloomberg school of public health, baltimore, md, usa. we investigated differences of unmyelinated sensory nerve fibers in the distal limb among healthy-weight subjects (bmi < kg/m ) and overweight/obese subjects (bmi ≥ kg/m ), aged - years. subjects underwent neurological examination and mm skin punches from distal leg (dl), thigh (dt), and proximal thigh (pt) sites, from which micron sections were stained with anti-pgp . antibody; intraepidermal nerve fiber density (ienfd; fibers/mm) and epidermal thickness were assessed. a second dl biopsy was processed for electron microscopic examination and both thick and thin sections were examined for ultrastructural changes. multivariable linear regression models were used to assess the effect of age, gender, height and weight. after controlling for height, age, and obesity, females were found to have lower distal leg ienfd (− . ; p= . ). increasing age and height were significantly associated with decreasing dl ienfd, with decreases of − . fibers/mm per years (p<. ) and − . fibers/mm per cm (p< . ), respectively. even after controlling for height, being overweight/obese was associated with reduced dl ienfd, with . fibers/mm lower dl enfd than healthy-weight individuals (p=. ). these findings remained consistent across distal thigh and proximal thigh enfd, though not all associations remained significant. the epidermis was thicker in obese subjects across the lower limb, most pronounced at the distal leg (μm, mean± sd, healthy-dl: ± . , dt: . ± . , pt: . ± . , obese-dl: . ± . , dt: ± . , pt: . ± . ). under em very few intact dermal nerve bundles were identified at the proximal thigh sites. the atrophic and degenerating axons were seen with perineurial infiltration by dense collagen in obsess/overweight subjects but not age/gender matched controls. obesity further accelerates attenuation of epidermal nerve fibers across the lower limb even after controlling for other associated factors. echaniz-laguna a , geuens t , petiot p , péréon y , adriaenssens e , haidar m , capponi s , maisonobe t , fournier e , dubourg o , degos b , salachas f , lenglet t , eymard b , delmont e , pouget j , juntas morales r , goizet c , latour p , timmerman v , stojkovic t . strasbourg university hospital, strasbourg, france; peripheral neuropathy group, vib department of molecular genetics and institute born bunge, university of antwerp, antwerpen, belgium; lyon university hospital, lyon, france; nantes university hospital, nantes, france; hôpital de la pitié-salpétrière, paris, france; nice university hospital, nice, france; marseille university hospital, marseille, france; montpellier university hospital, montpellier, france; bordeaux university hospital, bordeaux, france. in this study, we describe the phenotypic spectrum of distal hereditary motor neuropathy caused by mutations in the small heat shock proteins hspb and hspb and investigate the functional consequences of newly discovered variants. among unrelated patients with distal motor neuropathy, we identified mutations in hspb ( index patients/ ; . %) and hspb ( index patients/ ; . %) genes. patients have slowly progressive distal ( %) and proximal ( %) weakness in lower limbs, mild lower limbs sensory involvement ( %), foot deformities ( %), progressive distal upper limb weakness ( %), mildly raised serum creatine kinase levels ( %) and central nervous system involvement ( %). we found a broad range of disease onset with some patients presenting with foot drop at the age of years, and others presenting symptoms only after years. disease progression was slow in all patients, and even with a disease duration of more than years patients were still able to walk. none of our patients were wheelchair dependent. muscle pathology, nerve pathology and electrophysiology showed in all cases a slowly progressive, mostly symmetrical and predominantly distal motor axonal neuropathy. mild sensory involvement was observed upon nerve conduction studies, mostly the lower limbs, in % of cases. we identified hspb and hspb mutations, including respectively and not previously reported. transmission was either dominant ( %), recessive ( %) or de novo ( %). three missense mutations in hspb (pro ser, gly asp, gln arg) cause hyperphosphorylation of neurofilaments, while the c-terminal mutant ser leu triggers protein aggregation. two frameshift mutations (leu fs, ala fs) create a premature stop codon leading to proteasomal degradation. two mutations in hspb (lys met/asn) exhibited increased binding to bag . we demonstrate that hspb and hspb mutations are a major cause of inherited motor axonal neuropathy. mutations lead to diverse functional outcomes further demonstrating the pleotropic character of small heat shock proteins. eftimov f , querol l , rajabally ya and on behalf of all participants of the st enmc workshop. academic medical center, amsterdam, the netherlands; hospital de la santa creu i sant pau, universitat autònoma de barcelona, spain; aston university, birmingham, uk. although chronic inflammatory demyelinating polyneuropathy (cidp) is a treatable neuropathy further research is urgently needed to define the diagnostic clinical and electrophysiological boundaries of cidp and its subtypes, and to define the role of biomarkers in supporting the diagnosis, monitoring disease activity and predicting response to treatment and outcome. in recent years, several national registries and biobanks have been developed to enable systematic data collection in cidp. an international registry with large number of patients is needed to allow answering many important questions and develop validated prognostic models to predict outcome in individual patients with cidp. at the inflammatory neuropathy consortium (inc) meeting in , the inc members agreed that a european neuromuscular center (enmc) workshop would be the ideal setting to reach a consensus on the infrastructure of database and biobanks. the st enmc workshop will take place on may - , with participants representing different countries. primary objective of the workshop is to reach a consensus on inclusion and exclusion criteria, core sets and recommended sets of clinical data, diagnostic data and follow-up points and a manual of operations for collection of biomaterials. a secondary objective is to construct an infrastructure to allow sharing data between different databases and biomaterials. conclusions of the consensus meeting and outline of further perspectives will be presented at the peripheral nerve society meeting in . eichinger kj , burns j , cornett k , bacon c , shepherd m , mountain j , sowden j , shy r , shy me , herrmann dn . clinical outcome assessments that measure functional ability are important endpoints for clinical trials. dr. burns has led the development/validation of a functional outcome assessment (cmtpeds) for individuals with charcot marie tooth disease (cmt) ages - years in the inc rdcrn. the cmtpeds is reliable and sensitive to change. however a validated functional outcome measure (fom) for adults with cmt is needed. our data in - year-olds indicated that the cmtpeds could be modified for adult use. however, some items of the cmtpeds (e.g. balance beam and jumping) have floor effects in adults with cmt. we have developed an adult cmt-fom modeled on the cmtpeds, and refined based on literature review, patient interviews, a large-scale cmt patient survey and expert opinion. the cmt-fom is a performance-based scale comprising items that are combined to form a composite score to quantify functional ability of adults with cmt. the cmt-fom shares items with the cmtpeds. four items were added to measure functional abilities relevant to adults (sit to stand, meter walk/run, stair climb, and timed up and go test). the cmt-fom scoring mirrors the cmtpeds. to generate a score ranging from - , raw item scores are converted to z scores, based on age-and sex-matched normative reference values form the norms project and categorized to a - likert along a continuum of impairment levels. we have conducted a pilot study of the cmt-fom in adults with cmt a ( male, female, age . ± . yrs) of differing severity (cmt exam score (cmtes) range - ). the cmt-fom is feasible, individuals were able to complete all items, and takes minutes to perform. the mean cmt-fom score was . ± . (range - ). concurrent validity of the cmt-fom is supported by an association with the cmtes (r = . ). the overall score did not demonstrate floor or ceiling effects. in summary the adult cmt-fom is well-tolerated and captures upper and lower limb strength, dexterity, balance, speed, ambulation and endurance. the cmt-fom requires validation in a large longitudinal cohort, prior to application in clinical trials. estilow t , glanzman am , burns j , cornett kmd , menezes mp , shy r , moroni i , foscan m , pagliano e , pareyson d , laurà m , bhandari t , muntoni f , reilly mm , finkel rs , sowden j , eichinger k , herrmann dn , shy me , yum sw , ramchandren s and on behalf of the inherited neuropathies consortium. cmt is associated with progressive impairment of the hands. reducing this impairment by treating children who are in the early stages of the disease is crucial. studies assessing measures of cmt hand function in children and their associations with patient-reported outcomes are lacking. we analyzed the upper extremity items from the cmt pediatric scale (cmtpeds) and pediatric cmt quality of life scale (pcmt-qol) in children ages - years enrolled in the inherited neuropathies consortium, to explore the relationships between measures of hand function (impairment, activity, and activities of daily living), and patient-reported outcomes. weak grasp ( %), hand pain ( %), and tremor ( %) were prevalent impairments. performance on activity level tasks, the nine hole peg test ( hpt) and functional dexterity test (fdt), were impaired in % and % of the cases, respectively. patients reported difficulty "sometimes" to "always" in opening a jar/lid ( %), zipping/buttoning ( %), writing ( %), carrying a plate without spilling food ( %) and putting on shoes ( %). patients reporting tremor showed significant differences on the hpt (p=. ). grip strength was shown to have a moderately significant correlation with performance on the fdt (r=. ; p<. ). stepwise multiple linear regression showed that grip strength (beta=−. ; p<. ), hand pain (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to open a jar (adjusted r =. ; p<. . grip strength (beta=−. ; p<. ), and fdt (beta . ; p<. ) were predictive of ability to carry plate without spillage (adjusted r =. ; p<. ). grip strength (beta=−. ; p<. ), hpt (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to put on shoes (adjusted r =. ; p<. ). hand pain (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to zip/button (adjusted r =. ; p<. ). hand pain (beta=. ; p<. ) and hpt (beta . ; p<. ) were predictive of ability to use a pen/pencil (adjusted r =. ; p<. ). children with cmt present with frequent limitations in adl performance impacting qol. the upper limb measures of the cmtpeds are associated with hand performance and interventions to improve grip strength and reduce pain should be investigated further with respect to their impact on improving function, and ultimately qol. evans me , morrow jm , wastling s , sinclair cdj , fischmann a , shah s , emira ak , hanna mg , yousry ta , thornton js , reilly mm . mrc centre for neuromuscular diseases, ucl institute of neurology, london, uk; neuroradiological academic unit, ucl institute of neurology, london, uk; university of basel hospital, basel, switzerland. responsive outcome measures are needed in charcot-marie-tooth disease (cmt) to allow adequately powered clinical trials to test novel therapeutics. we have shown high responsiveness of mri quantified intramuscular fat accumulation in calf muscles of cmt a patients over months. the aim of the present study was to assess the responsiveness and longitudinal validity of quantitative mri over a year follow-up period. we undertook two further sets of quantitative mri, myometric and clinical assessments in the original mrc centre cmt a quantitative mri cohort. mri sequences included fat quantification using the point dixon fat-water separation method, t quantification and magnetisation transfer imaging. of the patients with genetically confirmed cmt a were assessed at baseline ( male, mean age . ± . years), underwent repeat assessments a median on months (data already published), underwent repeat assessments at a median of months, and underwent a final assessment at a median of months. the primary outcome measure currently being analysed is mean calf muscle fat fraction at a single axial slice a fixed distance distal to the knee joint. results of this analysis and correlation with clinical measures will be presented at the peripheral nerve society meeting. fainmesser y , dori a , , drory ve , . department of neurology and neuromuscular service, tel-aviv medical center, tel-aviv, israel; department of neurology, sheba medical center, ramat gan, israel; sackler faculty of medicine, tel aviv university, israel. the use of anabolic drugs by those who wish to increase lean body mass is widespread and not well supervised medically. a years old man was referred for evaluation of a slowly progressive sensory and motor disturbance in the distribution of the left ulnar nerve. his symptoms began months after repeated self-injections of anabolic steroids and vitamin e into the biceps and triceps brachii muscles bilaterally. his examination showed increased muscle mass of the injected muscles with a hard-rubbery consistency, atrophy and weakness of left interossei, mild weakness of the bilateral biceps brachii and sensory loss in an ulnar nerve distribution. nerve conduction studies showed a left ulnar neuropathy with reduced motor and sensory response amplitudes and denervation in the left first dorsal interosseus, as well as mild myopathic changes with significantly reduced insertional activity in both biceps muscles. mri of the soft tissues of the arms showed massive fibrosis and infiltration of fat in the arm muscles compressing the ulnar and median nerves on the left. neurolysis of the left ulnar nerve was performed, and the ulnar nerve was found enclosed in a fibrotic mass throughout the entire length of the upper arm. the brachial artery and the median nerve were similarly enclosed in fibrotic tissue and were released. a biopsy of the affected muscles showed muscle necrosis and fibrosis. the patient was treated with physiotherapy and losartan with only mild improvement in the consistency of the muscles, but without clinical improvement in ulnar nerve function, and worsening of nerve conduction. this case illustrates severe peripheral nerve damage due to entrapment in massive muscle fibrosis following improper intramuscular injection of anabolic steroids for cosmetic purposes. fabrizi gm , testi s , høyer h , braathen gj , squintani g , bertolasi l , ferrarini m , taioli f , cabrini i , pancheri e , cavallaro t , tonin p . department of neuroscience, biomedicine and movement, university of verona and department of neuroscience, aoui verona, italy; section of medical genetics, department of laboratory medicine, telemark hospital, skien, norway. inherited diseases of nerve and muscle may overlap phenotypically or coexists as facets of the same disorder. two families whose probands were initially diagnosed with a lower motor-neuron (lmn) syndrome and a hereditary distal motor neuropathy (dhmn) turned out to represent a vcp (valosin-containing protein)-related syndrome and a gne (udp-n-acetylglucosamine -epimerase/n-acetylmannosamine kinase)-related "distal myopathy" . both conditions shared the presence of rimmed vacuoles (rv) in muscle biopsies. in family , the year-old male proband had a lmn syndrome manifesting at age years (wasting/weakness, cramps, fasciculations of thigh muscles, later involving the distal upper limbs). his year-old brother had lower limb weakness and diffuse pain in bones/joints since age . the father was diagnosed with a "muscular dystrophy" thirteen years before dying a years; by age years he had developed a behavioural frontotemporal dementia (ftd). electrodiagnosis (edx) disclosed myopathic changes with ongoing denervation together with a mild sensory-motor axonal polyneuropathy in the proband, and a chronic sensory-motor axonal polyneuropathy in the brother. mri showed fatty replacement of weakened muscles in both siblings and diffuse changes of bones consistent with paget disease (pd) in the elder sibling. in family , a year-old african woman was affected by weakness and wasting starting at the distal lower-limb muscles at age years and soon progressed proximally; parents were not consanguineous and two sisters out of six siblings, deceased in their thirties for post-partum complications, had a similar disease. edx mainly disclosed a neurogenic process with ongoing denervation. muscle biopsies from the three family- patients and from the family- proband showed a myopathy with rv. next-generation sequencing demonstrated a heterozygous c. c>t change of vcp leading to a known pathogenic p.arg cys substitution in all family- patients, and two compound heterozygous c. g>a and c. g>a changes of gne in the family- proband leading to known p.ala thr and p.ala thr substitutions. vcp is known to cause autosomal dominant amyotrophic lateral sclerosis- , charcot-marie-tooth disease type y or inclusion body myopathy (ibm) with pd and ftd (ibmpfd); gne causes the autosomal recessive nonaka myopathy (alias ibm ). both cases emphasize the clinical and neurophysiological heterogeneity of those disorders. fargeot g , vandendries c , labeyrie c , viala k , theaudin m , adams d . neurologie, crmr nnerf, aphp, filnemus, chu bicêtre, le kremlin-bicêtre, france; neuroradiologie, crmr nnerf, aphp, filnemus, chu bicêtre, le kremlin-bicêtre, france; neurologie, groupe hospitalier universitaire pitié salpêtrière, paris, france. the diagnosis of cidp is often challenging, especially when electrophysiological signs of demyelination are lacking. plexus mri has documented nerve abnormalities in small series of typical cidp but its contribution in patients with no electrophysiological signs of demyelination remains to be proved. we report the results of plexus mri in a serie of patients suspect of having cidp without efns pns definite electrophysiological criteria. we did a retrospective study of patients consulting in kremlin bicetre and pitié salpétrière hospital. lumbar or brachial plexus mri (or both) were performed and we assessed nerve trophicity, t -stir signal intensity and gadolinium enhancement as well as the topography of abnormalities. a consensus diagnosis was made by a group of experts (based on clinical data and other supportive criteria) and allowed us to classified patients in "cidp" or "other diagnosis". the practical contribution of plexus mri to the diagnostic algorithm has been studied. diagnosis of cidp was made in patients. mri was abnormal in % of cidp patients and showed nerve roots hypersignal/hypertrophy/enhancement in respectively . / . / . % of patients. the pattern of abnormalities was often asymetrical ( . %), diffuse ( . %) or multifocal ( . %). after unblinding, the mri confirmed the diagnosis of experts in . % of patients and changed the diagnosis in % of patients. plexus mri has shown to be useful in our serie to confirm the diagnosis of experts or to modify it in patients ( %) . the "classical" pattern described in definite cidp (diffuse nerve root hypertrophy and hypersignal) was documented in % of our cidp patients whereas less typical pattern (focal or multifocal abnormalities, hypersignal without hypertrophy) was found in %. plexus mri seems usefull when facing patients suspected of having cidp when electrophysiological criteria are not met: both symetrical diffuse or asymetrical multifocal patterns can be found and should be always correlated to the clinical examination and other supportive criteria. the specificity of such abnormalities remains to be studied. feely sme , rebelo a , abreu l , tao f , bacon c , zuchner s , shy me . university of iowa, iowa city, ia, usa; dr. john t. macdonald department of human genetics and hussman institute for human genetics, miller school of medicine, university of miami, miami, fl, usa. mutations in bcle-associated athanogene (bag ) have been shown to cause a distal myofibrillar myopathy and cardiomyopathy that can severely affect children or only affect adults depending upon the particular mutation. children with severe cardiomyopathy and myopathy have also developed axonal peripheral neuropathy, consistent with the known localization of bag in neurons as well as in muscle. we have identified two large autosomal dominant families with adult onset charcot-marie-tooth disease (cmt ) with the identical novel missense mutation pro ser, a codon previously shown to cause severe or mild myopathy depending on the amino acid substitution. these families expand the phenotypes caused by mutations in bag to include cmt and provide an additional example of adult onset cmt that may previously have been diagnosed as chronic idiopathic axonal neuropathy (ciap). feely sme , saade d , shy me . carver college of medicine, university of iowa, iowa city, ia, usa. myelin protein zero (mpz), expressed only by myelinating schwann cells, has sequence alterations that have previously been reported to cause demyelinating, intermediate, and axonal forms of charcot marie tooth (cmt) disease. we describe a rare duplication in mpz which is causing an early onset, demyelinating form of cmt in our patient. patient was a product of a normal pregnancy and delivery. early milestones were delayed. she began walking at months of age. she was not able to keep up with her peers and was the slowest runner. she started wearing smos at years of age and afos by years of age. she was diagnosed with scoliosis at years of age and started wearing a brace at years. her examination at years of age showed that her fdi, apb, and adm were all / bilaterally. weakness in her lower extremities included / foot eversion and / great toe dorsi flexion. she had tight heel cords bilaterally. pinprick sensation was reduced throughout in her upper and lower extremities and absent at her toes bilaterally. vibration sensation with a rydel tuning fork was absent at her toes and ankles and reduced at her knees and fingers. nerve conduction studies were performed and revealed no responses in all sensory nerves tested with the exception of the radial nerve which had normal latency and mildly slowed conduction velocity ( m/s). prolonged latencies, demyelinating range slowing (between - m/s), and low cmap amplitudes in almost all segments of the median and ulnar motor nerves were also observed. these findings were consistent with a hereditary sensorimotor demyelinating polyneuropathy. she was diffusely areflexic and her total cmt pediatric score (cmtpeds) was / which is in the severe range. her cmt neuropathy score (cmtns) was / in the moderate range. the duplication of mpz has previously been identified as a rare cause of cmt b. inherited neuropathy consortium is part of the nih rare diseases clinical research network (grant# u ns - ). feely sme , saade d , shy me . carver college of medicine, university of iowa, iowa city, ia, usa. mutations in egr cause a severe, demyelinating form of cmt, cmt d. we describe a novel mutation in egr which led to extreme variability in severity in a family. proband was a year old girl who was the product of a normal pregnancy and delivery. early milestones were on time. problems with walking started at years of age. at . she was unable to use stairs, run, or jump. at she was wearing bilateral afos. by she was using a wheelchair and started breathing assist at night. she lost arm lift but could still hold a pen. by she could not ambulate independently and breathing assist was required day/night. she lost the ability to write and developed a head drop. by years she could not sit up. on exam her head and neck muscles were / . upper limbs, deltoids, biceps, wrist ext/flex, finger ext, fdi, and adm were / bilaterally. triceps were / ; finger flexors and apb were / bilaterally. lower limbs were / and she had contractures on the right. sensory examination was normal. she was diffusely areflexic. ncvs were absent. both the cmtns and cmt pediatric score were severe at / and / . exome sequencing revealed a r l variant in egr which was likely pathogenic. the probands' mother also had this mutation. she had no reported symptoms at the age of with strength at / throughout with the exception of left foot eversion which was +/ and great toe dorsi flexion which was -/ bilaterally. sensory exam showed a decrease of vibration and pinprick sensation at left toe. she had a normal gait, tandem gait and could toe walk, but not heel walk. she was diffusely areflexic. ncvs showed absent sensory responses. motor ncvs showed her latencies were prolonged and velocities reduced ( - m/s). cmtns was mild ( / ). additional family members who had the r l mutation were evaluated including maternal grandmother who was moderately impaired ( / ) and maternal aunt who was severe ( / ). no other mutations that could cause another known neuropathy or myopathy were identified and mitochondrial sequencing was normal. inherited neuropathy consortium is part of the nih rare diseases clinical research network (grant# u ns - ). fisgun a , luan x , hoke a . johns hopkins university, baltimore, md, usa. molecular mechanisms that underlie slow distal axonal degeneration seen in chemotherapy induced peripheral neuropathy (cipn) are unclear. however, several identified molecular targets suggest shared mechanisms with wallerian degeneration. since spontaneous mutation in wlds mice and genetic deletion of sarm gene lead to slow wallerian degeneration, we asked if wlds or sarm knockout (ko) mice are resistant to distal axonal degeneration induced by several chemotherapy agents. we chose chemotherapeutic drugs from different classes of agents, paclitaxel (taxane), cisplatinum (platin-based drugs) and bertozomib (proteasome inhibitor), to model cipn in mice. primary outcome measure was evaluation of epidermal nerve fibers in the hind paw plantar footpads. secondary outcome measures included thermal sensation and nerve conduction studies. sarm ko mice were almost % protected against development of sensory neuropathy but the protection in wlds mice was partial. this study confirms the pivotal role sarm plays in mediating axonal degeneration and identifies inhibition of sarm activity as a potential therapeutic target for prevention of cipn. florio f , scapin c , ferri c , feltri ml , wrabetz l , d'antonio m . myelin biology unit, san raffaele scientific institute, milan, italy; hjkri-university of buffalo, ny, usa. myelin protein zero is the most abundant structural protein in myelin of the pns. in humans, more than mutations in p are associated with hereditary neuropathies. deletion of serine causes charcot-marie-tooth (cmt) b disease in humans and a similar demyelinating neuropathy in mice (wrabetz et al., ) . p s del protein is misfolded and is retained in the er where it gives rise to a dose-dependent unfolded protein response (upr) (pennuto et al., ) . the upr results in the activation of transcriptional and translation control programs that reduce protein synthesis and increase the folding and degradative capacity of the cell. usually, when this first response is not sufficient the cells may activate apoptosis resulting in cell death. however, in p s del schwann cells there is no cell death suggesting that these cells may respond differently to chronic stress. transcriptomic analysis performed on p s del nerves showed increased expression of transcription factors normally present only in the early phases of differentiation such as c-jun, sox and id (d' antonio et al. ) . in order to understand the role of the expression of these factors in the peripheral nerve myelination we used ex vivo and in vivo approaches and we showed that sox and id act as negative regulators of myelination. these results suggest that the expression of sox and id may contribute to the hypomyelination observed in p s del mice. as such, we reasoned that their ablation could ameliorate the phenotype. surprisingly, the ablation of these factors in the p s del mouse severely worsens the neuropathy bursting schwann cell differentiation and increasing the expression of both p wild type and mutant allele with concomitant exacerbation of the upr. this suggests that the overexpression of early differentiation factors in schwann cell under chronic er-stress is an adaptive mechanism that limits differentiation and reduces the expression of toxic proteins. intravenous immunoglobulin (ivig) is the treatment of choice for the guillain-barré syndrome (gbs). the working mechanism of ivig in gbs is undefined, but most likely all potential effects are dose dependent. the pharmacokinetics (pk) and pharmacodynamics (pd) of ivig in gbs are highly variable between patients and a rapid consumption or clearance of ivig is associated with poor recovery. in the current study we developed a model to predict the pk of a standard dosage of ivig ( . g/kg for consecutive days) in individual patients with gbs. non-linear mixed-effects modelling (nonmem) was used to construct a model based on a cohort of gbs patients, with a total of sequential serum igg levels. the final model accurately describes the day to day increment in igg levels during the -day course and the initial rapid fall and graduate decline to steady-state levels thereafter. we explored several potential covariates that improved the predictive capabilities and decreased the between-subject variation in the model. the model including these covariates were evaluated successfully (bootstrap analysis) and through numerous simulation studies each based on (simulated) gbs patients. in conclusion, a first accurate and robust nonmem model for the pk/pd of standard ivig treatment in gbs was developed. the model can be used to predict the pk in individual patients applying a few simple baseline characteristics. in addition, the effect of different treatment regimens of ivig in gbs on a population pk/pd level can be simulated. this modeling technique is a new tool to optimize the pk in individual patients and the study design for new trials with ivig in gbs. forese mg , pellegatta m , rivellini c , podini p , quattrini a , previtali sc , taveggia c . axonal neuregulin (nrg ) type iii is an essential instructive signal for peripheral nervous system (pns) myelination, as its expression determines whether axons are myelinated as well as the thickness of the myelin sheath. we recently demonstrated that gamma-secretase cleavage of nrg type iii generates an axonal intracellular fragment, which translocates in the nucleus to upregulate the expression of the prostaglandin d synthase (l-pgds) gene in neurons. l-pgds catalyzes the conversion of prostaglandin h into prostaglandin d (pgd ). we also showed that specific inhibition of l-pgds activity impairs in vitro myelination. accordingly, myelin in l-pgds null mice is noticeably thinner, thus indicating that l-pgds is a new modulator of developmental pns myelination. previous studies have shown that prostaglandins are involved in the process of wallerian degeneration (wd) and axonal regeneration after injury. thus, to determine whether l-pgds and pgd could be important in pns regeneration and remyelination, we performed sciatic nerve crush injury in months old l-pgds null and wild type control mice and we analysed nerves by morphologic, biochemical, histological and molecular approaches at different time points (t) after crush. we focused on three phases: degeneration (t -t ), axon regeneration (t -t ) and remyelination (t ). our results indicate that in l-pgds null mice the amount of myelin proteins synthesized after crush as well as the number of remyelinated fibers do not change, suggesting that l-pgds might be dispensable for remyelination. however, we observed an increased number of macrophages in null nerves during regeneration (t ), possibly as a consequence of an increase in the blood-nerve barrier (bnb) permeability, indicating potential alteration in the regeneration process in l-pgds null mice. these results suggest that l-pgds could have a different role in developmental pns myelination and after injury. whether other prostaglandins and synthases might compensate for l-pgds activity is currently under investigation. fornasari be , , raimondo s , , ronchi g , , crosio a , budau ca , el soury m , muratori l , , tos p , battiston b , geuna s , , gambarotta g . department of clinical and biological sciences, university of turin, italy; neuroscience institute cavalieri ottolenghi, turin, italy; microsurgery unit, health and science city, cto, turin, italy; hand microsurgery and surgery, gaetano pini hospital, milan, italy. to repair nerve gaps following severe peripheral nerve injuries, chitosan tubes were proved to give good results, comparable with those obtained with nerve autografts, the gold standard technique. to further improve peripheral nerve regeneration using chitosan tubes, a conduit enrichment strategy was developed using longitudinal skeletal muscle fibres, which have been previously shown to be good fillers in the "muscle in vein" experimental paradigm, where they played a trophic and a structural role. to this aim, rat median nerve gaps were repaired using two different conduits: mm chitosan tubes filled with a longitudinal piece of pectoralis major muscle ("muscle in tube") and hollow chitosan tubes. samples were harvested at early time points ( , , , days) for biomolecular and morphological analysis, and later ( months) for stereological analysis. autologous nerve grafts were used as gold standard positive control in the early time points. biomolecular analysis carried out on in vitro degenerating muscle and on "muscle in tube" at early time points show that the muscle produces high levels of soluble isoforms of neuregulin , a key factor for schwann cell survival and activity, usually released by schwann cells after nerve injury. functional assay and stereological analysis carried out on the distal part of regenerated nerve months after nerve repair, show no significant differences in the regeneration outcome between hollow chitosan tube and "muscle in tube" groups. therefore, we conclude that for short gaps (≤ mm), both hollow chitosan tube and "muscle in tube" are good techniques to repair nerve defects and we suggest that the "muscle in tube", which spontaneously releases neureg-ulin , might be a promising strategy to promote regeneration when the gap is longer or the repair is delayed in time. foucquier j , bertrand v , jouve e , truillet r , mandel j , laffaire j , blin o , magy l , lehert p , , hajj r , guedj m , cohen d , attarian s . pharnext, issy-les-moulineaux, france; aix marseille université, aphm, marseille, france; hôpital dupuytren, limoges, france; university of melbourne, melbourne, vic , australia; faculty of economics, louvain, belgium. charcot-marie-tooth disease type a (cmt a) is a rare disease belonging to a group of inherited, progressive, chronic motor and sensory peripheral neuropathies. the charcot-marie-tooth neuropathy score (cmtns) (shy et al., ) and the overall neuropathy limitations scale (onls) (graham & hughes, ) are considered as the main clinical scales for evaluating progression of disability associated with cmt. as cmt a is a slowly progressive neurodegenerative disease, the choice of endpoints and their ability to monitor small changes over time remain a major concern for clinical drug development. with this in mind, we studied a cohort of french cmt a patients with a follow-up ranging from months to . years resulting from the merge of two multicentre clinical trials (micallef et al., ; attarian et al., ) and a non-interventional study (unpublished) . the sensitivity to change of both cmtns and onls were assessed using a mixed effect model estimating annual progression with time in years, cmtns or onls baseline value as covariates, study centre as a fixed factor and patients as a random effect to account for the repeated measures. disease progression was estimated to be + . points per year on the cmtns (p = . ) and + . points per year on the onls (p = . x − ), both corresponding to a deterioration of impairment and disability. while both endpoints have similar and favourable properties, our set of observations led us to conclude that the onls could be more promising to monitor disease progression in cmt a. frasquet m , , lupo v , mas f , , vílchez r , chumillas mj , , espinós c , sevilla t , , . hospital universitari i politècnic la fe, valencia, spain; instituto de investigación sanitaria la fe, valencia, spain; centro de investigación príncipe felipe, valencia, spain; eresa, valencia, spain; centro de investigación biomédica en enfermedades raras (ciberer), valencia, spain; departamento medicina, universitat de valencia, valencia, spain. mutations in the bicd gene are a cause of dominant spinal muscular atrophy, lower extremity predominant (smaled). we report six patients belonging to three spanish families who carry three different novel mutations in the bicd gene. we describe clinical, electrophysiological and magnetic resonance imaging (mri) data. we provide results of muscle biopsy of one patient and skin biopsy for the study of epidermal nerve fiber density (enfd) of other two patients. genetic diagnosed was reached using a gene panel for genetic testing of cmt and dhmn. three novel mutations in the bicd gene that segregated with the disease were detected: p.val gly; p.tyr his and p.s l. the most frequent clinical phenotype consisted of mild weakness in proximal muscles of lower limbs combined with foot deformities. one patient had prominent sensory symptoms and abnormalities on sensory examination. other two patients had minor sensory abnormalities on examination. in one patient sensory and motor nerve action potentials were reduced, in the rest of patients electrophysiological studies showed normal motor and sensory nerve responses, with chronic denervation predominantly in muscles of lower limbs. mri studies at the level of tight and calf were performed in all patients. the most affected muscles were rectus femoris, vastus lateralis and medial gastrocnemius. mri studies at the level of feet were obtained from five patients and showed that there was not fatty infiltration in intrinsic foot muscles. mri at the level of pelvis muscles performed in four patients showed marked fatty infiltration of gluteus medius muscle in two of them. muscle biopsy performed in one patient showed myopathic features. skin biopsy was performed in two patients of the same family. in the older patient, who had minor sensory abnormalities on examination, there was a marked reduction of enfd that followed a length-dependent pattern. in conclusion, we report three new pathogenic mutations in the bicd gene. in our study we include mri findings at the level of pelvis and feet, which allow us to better define the pattern of muscle involvement related with this gene. our results also raise the subject of a possible sensory involvement in the disease. hereditary neuropathy with liability to pressure palsies (hnpp) is an autosomal dominant disorder that usually results from deletions in the pmp- gene. the neuropathy is unique in that it manifests with recurrent mono-neuropathies at common sites of compression. while spontaneous recovery from episodes of nerve injury usually occurs, it is often incomplete, and over time patients may develop a length dependent polyneuropathy. given the relapsing/remitting nature of hnpp symptoms, standard clinical scores, such as the cmt neuropathy score, are not effective at capturing the severity or progression of the disease. a specific tool is therefore needed for measuring clinical severity of hnpp in preparation for emerging clinical trials. in the current study, we evaluate a new pilot measure, the hnpp severity score (hnpps). the score is composed of patient reported questions addressing current and prior sensory and motor symptoms, and the impact of symptoms on quality of life, followed by items based on a motor examination. total scores vary from - , with higher scores indicating increased disease severity. in this study, the hnpps was administered to patients with genetically confirmed hnpp at the ucl institute of neurology. subjects included males and females with a mean age of years (+/− , range - ). the mean hnpps was . points (+/− . , range - ) and the data did not demonstrate major skew. the cronbach alpha for the hnpps was . , and items based on the physical examination showed the least variability. a modest correlation was observed between the hnpps and the cmt examination scores (pearson correlation . , ci . - . ). we conclude that the hnpps may be a useful measure of clinical severity in hnpp, and should be refined in larger patient cohorts. fridman v , sillau s and on behalf of the inherited neuropathies consortium (inc) . university of colorado hospital, aurora, co, usa; university of iowa hospitals and clinics, iowa city, ia, usa. the most common of the hereditary neuropathies (hn) is cmt a, an autosomal dominant demyelinating neuropathy that results from duplications in the pmp- gene. recent advances in defining the pathomechanisms of the disease have led to an increasing number of potential therapies; however, the absence of reliable natural history data and the paucity of sensitive clinical outcome measures have been barriers to effective clinical trials. the charcot marie tooth neuropathy score (cmtns) was developed to quantify impairment and measure progression in hn. it was observed that while the score discriminates well between mildly and severely impaired patients, it tends to cluster together patients in the middle range of severity. to improve the score's sensitivity, rasch analysis-based weighted category responses were developed. we report a longitudinal study of weighted cmtns and cmt examination scores (cmtes) over a three-year time frame in patients with cmt a. baseline, one year, two year and three year wcmtnsv /wcmtesv scores were available for / , / , / and / patients respectively. mean wcmtns (sd)/wcmtes (sd) scores were as follows: . ( . )/ . ( . ) at baseline, . ( . )/ . ( . ) at one year, . ( . )/ . ( . ) at two years and . ( . )/ . ( . ) at three years. a mixed regression model showed significant change in wcmtns and wcmtes at years (mean change from baseline at years was . points (p=. ) for wcmtns and . points (p=. ) for wcmtes. significant change as compared to baseline was also seen at years (mean change from baseline . points (p= . ) for wcmtns and . points (p= . ) for wcmtes. we conclude that weighted cmtnsv scores show change over the first three years of the inherited neuropathies consortium natural history study and are a helpful measure of progression in cmt a. fridman v , novak p , david w , macklin ea , mckenna-yasek d , walsh k, oaklander al , brown r , hornemann t , eichler f . massachusetts general hospital, boston, ma, usa; university of massachusetts medical school, worcester, usa; university hospital zurich, zurich, switzerland. hsan is an autosomal dominant, severe sensory motor polyneuropathy caused by mutations to serine palmitoyl-coa transferase. mutations shift the substrate preference from serine to alanine leading to formation of neurotoxic -deoxysphingolipids ( -deoxysl). treatment with high-dose l-serine has been shown to reduce -deoxysl accumulation and improve neuropathy in transgenic hsan mice. we report a two-year, delayed-start, placebo-controlled clinical trial evaluating the safety and efficacy of oral l-serine in hsan . eighteen hsan subjects were equally randomized to l-serine ( mg/kg/d) or placebo for year. at -weeks, the placebo group crossed-over and all participants took open-label l-serine for one additional year. sixteen subjects completed their -week visit, and no serious adverse events related to l-serine were reported. participants randomized to l-serine experienced a decline in charcot marie tooth neuropathy scores (cmtns) over year relative to placebo (− . units, % ci − . to − . , p = . ). both groups improved in the second year of the study, with a diminished difference in cmtns at weeks (− . units, % ci − . to . , p= . ). skin biopsies from the distal leg site were largely devoid of intra-epidermal nerve fibers (ienf), but at year, a greater increase in ienf density was seen in participants on l-serine versus those on placebo (median change of vs. fibers/μm , p= . ). -deoxysl levels declined among participants on l-serine versus those on placebo after one year of treatment ( % decrease in deoxysphinganine vs. % increase on placebo, p < . ), and placebo participants experienced similar declines in -deoxysl levels after crossing over to l-serine. we conclude that l-serine is a safe and potentially efficacious treatment for hsan . fu liong h , yusuf r . regional neuromuscular clinic, queen elizabeth hospital, university hospitals of birmingham, birmingham, uk; school of life and health sciences, aston brain centre, aston university, birmingham, uk. the relationship between guillain-barré syndrome (gbs) and malignancy is uncertain. under the diagnostic criteria of paraneoplastic neurological syndrome (pns) by euronetwork, , neuropathy with no definite onconeural antibodies identified due to gbs has been classified as "non-classical" paraneoplastic disorder. we retrospectively analyzed data of consecutive patients admitted with gbs from birmingham, u.k. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) to calculate the relative cancer risk using different definitions and determined characteristics of malignancy-associated gbs. patients were classified according to definitions: ) all cases of malignancies excluding preceding diagnoses > year before gbs and with no evidence of malignant disease activity at the time of gbs diagnosis, and ) malignancies diagnosed > years post-gbs onset as per pns criteria. total number of gbs patients with malignancy was ( . %). a total of patients ( . %) fulfilled requirements for inclusion as malignancy-associated gbs as per existing criteria for pns. associated malignancy consisted of angioimmunoblastic t-cell lymphoma ( ), poorly differentiated squamous cell carcinoma of nasal septum ( ), gastric adenocarcinoma ( ), hepatocellular carcinoma due to hepatitis b ( ), rectal carcinoma with liver metastasis ( ) and myelodysplastic syndrome ( ). malignancy was globally commoner in our gbs cohort compared to the general population (odds ratio: . ; ci: . - . ; p = . ). however, this was unconfirmed if paraneoplastic criteria were applied. gbs patients with cancer were significantly more likely to be older (p = . ), hyponatremic (p = . ) and demonstrate more electrophysiological axonal loss (p < . ). cerebrospinal fluid (csf) protein levels were lower in the malignancy group (p = . ) and neurological improvement was less likely (p = . ). in-patient mortality was significantly higher in patients with malignancy (p < . ). none of the patients in the malignancy group had positive anti-ganglioside antibodies or anti-neuronal antibodies (anti-hu, yo, ri, crmp ). we conclude global cancer risk is higher in gbs than in the general population, although definition-dependent. application of the strict published criteria for paraneoplastic syndrome reduced the number of cases and suggested absence of a link. malignancy requires consideration in elderly, hyponatremic subjects with normal csf protein, severe electrophysiological axonal loss who fail to improve post-treatment. fu liong h , aude-marie g , anne-catherine an , emilien d , julien c , shahram a , yusuf r . anti-myelin associated glycoprotein antibody (anti-mag) neuropathy is a slowly progressive neuropathy resulting in disability through distal sensory more than motor deficits and tremor. there are currently no evidence-based treatments for anti-mag neuropathy and the effect of the disease on quality of life (qol) in this patient population is unknown. our objective was to assess determinants of qol in patients with anti-mag neuropathy. the sf- questionnaire was assessed in patients, from marseille, angers (france) and birmingham (united kingdom). routine clinical evaluations included mrc sum score, incat sensory score, inflammatory rasch built overall disability score (i-rods), ataxia score, jamar grip dynamometry, timed meter-walk, neuropathic pain symptom inventory (npsi) score, and fatigue severity score (fss). there were males and females. mean age was . years (sd: . years). mean disease duration was . years (s.d.: . years). there were no significant differences between the french and u.k. cohorts in terms of gender distribution, age, disease duration, anti-mag antibody titre. all physical assessments, including mrc sum score (p= . ), jamar grip dynamometry (p= . ), onls (p= . ), irods (p= . ), incat sensory score (p= . ), -meter timed walk (p= . ), ataxia score (p= . ), tremor score (p= . ), were comparable. prevalence and/or severity of pain (p= . ), fatigue (p= . ), restless legs syndrome (p= ) and cramps (p= . ) were also similar. physical component summary (pcs) and mental component summary (mcs) of the sf questionnaire were significantly lower than in reported normal subjects of both countries (p< . ). all sf- qol domains correlated with i-rods, except mcs for which significance was however approached (p= . ). pcs correlated with mrc sum score, ataxia score, timed m-walk, tremor, jamar grip dynamometry, npsi pain score, fss and level of social support. mcs correlated exclusively with fss and level of social support. in multivariate regression, pcs was associated independently with i-rods (p< . ) and npsi pain score (p= . ), whereas mcs was associated independently with fss (p= . ). qol is accurately predicted in anti-mag neuropathy by the i-rods and fss, lending support to their use in clinical and research settings. effective measures to improve qol should include tremor and neuropathic pain treatment, fatigue management and improved social support. fu liong h , yusuf r . regional neuromuscular clinic, queen elizabeth hospital, university hospitals of birmingham, birmingham, uk; school of life and health sciences, aston brain centre, aston university, birmingham, uk. the electrophysiological diagnosis of guillain-barré syndrome (gbs) is dependent on a number of abnormalities affecting different parameters in peripheral nerves on nerve conduction studies (ncs). diagnostic sensitivity varies with different electrodiagnostic criteria, practices as well as extensiveness of nerve study. however, the most efficient method of performing electrophysiology for guillain-barré syndrome (gbs) is unknown. we retrospectively analyzed electrophysiological data of consecutive gbs patients from birmingham, uk ( - ), studied ≤ weeks post-onset. we first identify abnormal nerves from various regions which produced gbs electrodiagnosis using the recently described criteria by rajabally et al. we subsequently used pre-established hypothetical nerve conduction study protocols to determine the potential optimal method of achieving electrodiagnosis. we found the sensitivity of electrophysiology for each gbs subtype was dependent on the upper and lower limb nerves tested. in acute inflammatory demyelinating polyneuropathy (aidp), abnormalities were predominant in the arms, whereas leg abnormalities predominated in axonal gbs. in aidp, the most common abnormal parameters were distal motor latency ( . %) and conduction block ( . %), and the most frequently affected nerve was the median ( . %). prolonged f-waves were present in % and f-waves were absent in %. mcv was the least frequently abnormal ( . %) with demyelinating range slowing, significantly lower than cb or dml prolongation (p< . in both cases). in axonal gbs, reduced motor amplitudes ( . %) and conduction block ( . %) were the most common parameters, and the most frequently abnormal nerve was the tibial ( . %). f-waves were absent in . %. . % of all motor nerves were unexcitable, significantly more common in lower limb nerves compared to upper limb nerves (p= . ). on comparison of different hypothetical ncs protocols ( unilateral protocols with nerves, with nerves as well as the protocols with -nerves and with exclusive bilateral upper limb and lower limb testing), unilateral -nerve (median, ulnar, common peroneal and tibial) testing produced the highest diagnostic sensitivity for both aidp ( . %) and axonal ( . %) gbs. electrodiagnostic sensitivity in gbs is thus dependent on nerves tested and parameters considered. each subtype preferentially involves specific nerves and parameters. these findings may help per-procedure interpretation, improve electrodiagnostic sensitivity, and reduce patient discomfort. funakoshi k , nagashima t , kokubun n , hirata k , yuki n . department of neurology, dokkyo medical university, tochigi, japan; department of neurology, mishima hospital, nagaoka, japan. recently, igg anti-ganglioside complex (gscs) antibodies have been reported in patients with guillain-barré and miller fisher syndrome. some researchers (nobile-orazio et al., ) reported igm anti-gscs antibodies in multifocal motor neuropathy (mmn) and other chronic immune-mediated neuropathies. in ten of eleven mmn patients with anti-gm antibody, there was a decreased reactivity to gm /gd a compared to single antigen gm . similarly, in one of two chronic inflammatory degenerative polyradiculoneuropathy (cidp) patient with anti-gm antibody, there was a decreased reactivity to gm /gm , gm /gd a, gm /gd b, gm /gt b compared to gm . these relationships were defined negative interaction. in one cidp with anti-gd b antibody, anti-gm /gt b and -gm /gt b reactivity increased although gm and gm were negative (positive interaction). in japan, on the other hand, this correlation remains unclear. sera were investigated from one mmn patient ( y/o male) with anti-gm , and anti-gd a antibody, one cidp patient ( y/o female) with anti-gm , and one subacute sensory motor polyradiculoneuropathy ( y/o male) with anti-gd b, and anti-gq b. igm antibodies to gscs gm /gd a were tested with a mixture of gm and gd a (each pmol/well) as antigen. anti-gm /gd a antibodies were judged to have positive interactions when the optical density was . greater than the sum of the antibodies against individual gm and gd a, and negative interactions when the optical density was % or less compared to the antibodies against individual gm or gd a. antibodies to at least one combination of two of the six gangliosides (gm , gm , gd a, gd b, gt b, and gq b) were similarly tested and judged for positive or negative interactions. in one mmn, anti-gm /gd a had negative interactions. in one cidp, anti-gm /gd a, and anti-gm /gq b had negative interactions. in one subacute sensory motor polyradiculoneuropathy, various anti-gscs antibodies including gd b or gq b had negative interactions. in the cidp patient mentioned above, anti-gm /gt b or anti-gm /gt b had no positive interactions. the relationship between this attenuated reactivity presumably driven by adjacent gangliosides and the mechanism of chronic immune-mediated neuropathies needs to be clarified. galino j , cervellini i , zhu n , stöberl n , fricker fr , lee g , hütte m , lalli g , bennett dl . the nuffield department of clinical neurosciences, john radcliffe hospital, university of oxford, oxford, uk; wolfson card, king's college london, guy's campus, london, uk. schwann cells (scs) are the myelinating cells in peripheral nerve system and are important for normal peripheral nerve development and repair. rala and ralb are small gtpases that have been implicated in neural tube closure, neurite branching and have been described as upstream effectors of proteins involved in cell migration and membrane dynamics. due to their potential role in sc function here we investigated, by genetic ablation in transgenic animals of one or both gtpases, their importance in sc function in adulthood and in nerve repair. we showed that ral gtpases are dispensable for sc function in the naïve state. however ral signalling (provided by rala or ralb) is required for effective remyelination of axons following nerve injury. moreover, absent ral signalling produced defects in axon reinnervation of distal organs and a delay in motor function recovery after nerve injury. we also studied the ral dependent cellular mechanisms that may be responsible for impaired sc remyelination and noted abnormal sc lamellipodia formation that prevent normal axial and radial axon remyelination. this work demonstrates for the first time a novel mechanism for ralgtpases that controls sc lamellipodia formation and their importance in normal sc function during peripheral nerve repair. garcía-lareu b , , , ariza l , cobianchi s , , chillón m , , , , navarro x , , , bosch a , , . tumor necrosis factor (tnf) alpha has been implicated in the pathogenesis of diabetic peripheral neuropathy (dpn), among other inflammatory demyelinating diseases and neuropathic pain. tnf alpha is a pro-inflammatory cytokine that can act at several stages in the demyelination process. it is produced by schwann cells (scs) in the peripheral nervous system (pns) after nerve injury and released into the local environment to attract and activate macrophages at the site of injury, contributing to wallerian degeneration. in vivo studies demonstrated a local inflammation in the sciatic nerve of rats after injection of tnf alpha, followed by demyelination and axonal degeneration. furthermore, the application of tnf alpha resulted in acute mechanical hiperalgesia, a main characteristic of neuropathic pain and therefore tnf alpha is postulated as a biomarker for painful changes after nerve injury. with the aim to characterize tnf alpha effects in chronic neuropathic pain and in diabetic neuropathy, a transgenic mouse model overexpressing tnf alpha cdna under the peripheral myelin protein p promoter was generated. here we characterized the overexpression of tnf alpha in myelinated scs at different stages of myelination (postnatal days , and ) showing that high levels of tnf alpha in sciatic nerve leads to the downregulation of the major pns myelin proteins (p , mbp, pmp , mag) compared to wild type mice, correlating with the loss of structured myelin and an increase in p ntr, a marker for immature and non-myelinated scs in the sciatic nerve. iba staining showed high levels of macrophage infiltration in both sciatic nerve and spinal cord tissues, compared with wild type animals. stress conditions were induced by sciatic nerve crush surgery after which recovery and subsequent remyelination were delayed in the transgenic mice, as evaluated by the sciatic functional index (sfi) and electrophysiological tests. on the other hand, mechanical and thermal nociception seemed to be unaltered, with or without lesion. this model could be helpful in the characterization of the role tnf alpha in pain development, injury and dpn as well as in developing efficient therapeutic strategies to modulate such pathological conditions. garcía-sobrino t , , blanco-arias patricia , , vidal-lijó mp , quintáns bea , , sobrido mj , , pardo j , . department of neurology, hospital clínico, santiago de compostela, spain; neurogenetics research group, instituto de investigaciones sanitarias (idis), santiago de compostela, spain; genomic medicine group (u ), centre for biomedical network research on rare diseases (ciberer), spain; department of neurophysiology, hospital clínico, santiago de compostela, spain. charcot-marie-tooth (cmt) disease is a genetically heterogeneous group of hereditary motor and sensory neuropathies. more than genes were involved in the disease pathogenesis. the objective of this study was to assess the genetic distribution of cmt disease in galicia (northwestern spain). patients were diagnosed as cmt if they had a consistent neurological and/or neurophysiological examination or if they had sensory motor neuropathy with a positive family history. a total of cmt adult patients ( % females) were evaluated with a median age of [ - ] years. the molecular diagnosis was achieved in patients ( %) with a higher success in cmt ( %) than cmt ( %). globally, pmp duplication was the most frequent finding ( %), followed by mutations in mpz ( %), mfn ( %), gjb ( %) and gdap ( %). in cmt , with expception of the pmp duplication, pathogenic variants in egr , nefl and mpz gene were most common. pathogenic variants in mfn and gdap accounted for % of cmt patients. several patients referred to our institution as cmt were diagnosed as hereditary motor neuropathy (hmn) and pathogenic variants in the bscl gene were the most frequent. pathogenic variants not previously related to cmt were identified in mpz, mfn , gjb , egr , nefl and sh tc genes. sporadic or autosomal recessive (ar) cmt accounted for the % of all diagnoses. the genetic epidemiology of cmt in galicia follows a similar pattern to other populations, although some remarkable features are axonal gdap and demyalinating egr pathogenic variants as well a seemingly elevated proportion of ar cases. mutations in mpz gene are associated to a wide range of phenotypes. the r c, in particular, is associated to a severe and early onset disease. some cmt patients respond to immunosuppressive or immunomodulatory treatment, some of them harbouring mpz mutations. we present the case of a -year-old brazilian female with a severe cmt b (r c) that presented an acute episode of worsening after a febrile exanthematic disease. she complained of tingling, stopped walking without support, and could not raise her arms. her emg showed an asymmetrical reduction of motor cv, reduced amplitudes, severe temporal dispersion and possibly conduction block. csf protein was mg%, with normal cell count. diagnosed as gbs, she received ivig. curiously, she markedly improved, regaining the functional status of youth, recovering functions that she had long lost. on emg, motor amplitudes improved significantly, but cv remained the same. reviewing carefully her past history, we identified some motor fluctuations along her adult life. at age , while pregnant, she developed positive sensory symptoms, became unable to get up from the chair and could not walk without assistance. after delivery, she improved, but did not return to baseline. one year later, she presented a new transitory functional worsening after an influenza illness. her parents were healthy but two out her children have a severe cmt. her first daughter never walked and died at the age of six. six months after treatment with ivig, she faced a new relapse. we pulsed with corticosteroids, with great response. she remains stable to date, four months after treatment. the episodic fluctuations and the evident response to treatment clearly suggest an associated immunomediated process. the fast improvement of the amplitude with maintenance of cv suggests that another mechanism in addition to demyelination and axonal degeneration is involved. we propose the existence of an associated channelopathy. funded by cnpq, faepa, pronas (ministry of healthy) gbs is an acute, immune-mediated neuropathy that comprises three major subtypes: aidp, aman, and amsan. on clinical grounds, this distinction is based on nerve conduction studies (ncs): aidp is a demyelinating neuropathy, aman is an axonal motor neuropathy, and amsan, an axonal sensory and motor neuropathy. recently, it has been demonstrated that axonal gbs, in addition to axonal degeneration, may present conduction block (cb) that can progress to axonal regeneration or revert, characterising a reversible conduction failure (rcf), a finding usually associated to good prognosis. patients presenting axonal cb are frequently diagnosed as having aidp, a mistake that can be avoided with two sets of ncs, one as early as possible and the second after three weeks of disease. following these recommendations, we retrospectively classified a brazilian group with gbs followed in our institution that had been submitted to two ncs sets. from september to january , patients fulfilled clinical criteria for gbs at clinical hospital of ribeirão preto and had more than one ncs accomplished. at least four motor nerves (median, ulnar, peroneal, and posterior tibial) and five sensory nerves (medial, ulnar, radial, sural, and peroneal superficial) were evaluated in each examination, according to routine procedures. first ncs revealed patients with aidp ( %), cases of axonal gbs ( aman, amsan, %), and patients with equivocal results ( %). at follow-up study, patients ( %) had their classification changed. the main shifts were from aidp to axonal group and from equivocal results to aidp. aidp increased to % (n = ), axonal gbs increased to % (n = , aman, amsan), and there were no more equivocal patients. although the majority of studies have shown that aidp is more frequent in western countries while axonal gbs predominates in eastern countries, we found a different pattern of distribution in our population, with predominance of the axonal subtypes. the considerable increase of axonal gbs at follow-up studies reinforces that serial emg are mandatory for accurate diagnosis of gbs subtypes. prospective studies are now being carried out in order to confirm these results. in amyloid light chain (al) amyloidosis, misfolded light chain (lc) accumulates and causes progressive peripheral neuropathy (pn) and progressive failure of critical organs such as the heart and kidneys. progressive ascending sensorimotor neuropathy is often a related clinical finding. the deposition of toxic lc amyloid in peripheral nerves is associated with paresthesias, pain, muscle weaknesss, orthostatic hypotension, and diarrhea or constipation in approximately % of patients with al amyloidosis. there are no approved treatments for al amyloidosis. current therapeutic approaches for al amyloidosis are intended to limit lc production but do not directly target misfolded lc deposited as amyloid in organs. we report phase / trial results of neod , an investigational monoclonal antibody that targets misfolded lc and may neutralize circulating lc aggregates and clear insoluble deposits. trial inclusion criteria were one or more plasma cell-directed treatment completed before enrollment, partial or greater hematologic response to any previous therapy, and persistent organ dysfunction. neod was administered intravenously every days during a dose-escalation phase ( patients; . , , , , , , or mg/kg in a + study design) and an expansion phase ( patients; mg/kg). we assessed safety, tolerability, pharmacokinetics, immunogenicity, organ responses based on consensus criteria, and pn responses using the neuropathy impairment score-lower limb (nis-ll). neod treatment was not associated with dose-limiting toxicities or serious adverse events, drug-related discontinuations, or antidrug antibody development in any patient (n = ). of patients with measurable pn at baseline (n = ), % achieved pn response based on the nis-ll score after months of treatment, which resulted in a median % nis-ll score reduction (mean baseline nis-ll, . ). in a best response analysis, % of cardiac-evaluable patients (n = ) and % of renal-evaluable patients (n = ) met respective criteria for organ response. improvements in neuropathy have not been previously shown in al amyloidosis. these results demonstrated that monthly neod infusions were safe, well tolerated, and associated with responses across three different organ systems. gervais cbl , ross ma , goodman bp , khoury ja , muzyka i , smith be . mayo clinic in arizona, scottsdale, az, usa. this work describes clinical, examination and electrophysiologic findings in a cohort of patients with length dependent sensorimotor peripheral neuropathy (ldsmpn) with ventral abdominal sensory loss. ldsmpn affects the longest nerve fibers, namely those innervating structures in the feet and hands. what is less well appreciated is that length dependent involvement of sensory nerve fibers in ldsmpn from the thoracic segments gives rise to ventral abdominal sensory loss. consecutive patients seen for ldsmpn (n= ) were evaluated for the presence or absence of ventral abdominal sensory loss. demographic variables, symptoms and quantitative neurologic findings (neuropathy impairment score [nis]) were examined using descriptive statistics. final diagnoses were noted. ventral abdominal sensory loss to pinprick (which was asymptomatic in all patients tested) was documented in / ldsmpn patients ( . %), mean age was . years (range - ), m:f gender ratio was . ( : ), mean nis was . (range - ), ncs/emg abnormalities were found in / patients (the remaining showing objective evidence of small fiber sensory involvement). ldsmpn patients without ventral abdominal sensory loss (n= ) had a mean age of . (range - ), m:f of . ( : ), and mean nis of . (range - ). no patient ( / ) had dorsal torso sensory loss between the shoulder and buttock levels on either side. diagnoses of the ldsmpn patients with vs. without ventral abdominal sensory loss included charcot marie tooth or other hereditary neuropathy (n= vs. ), abnormal carbohydrate metabolism (n= vs. ), idiopathic (n= vs. ), hypothyroidism (n= vs. ), inflammatory (n= vs. ) and other (n= vs. ). ventral abdominal sensory loss appears to be common in patients diagnosed with ldsmpn of a variety of causes including inherited neuropathies; in addition to those innervating distal limb territories, distal sensory fibers from the thoracic region represent another category of length dependent involvement in ldsmpn; ) the clinical examination of ldsmpn should include the ventral abdomen. gibbons c , garcia j , casasola m , freeman r . beth israel deaconess medical center, harvard medical school, boston, usa. objective: to determine the relationship between measures of autonomic function, electrochemical sweat conductance (esc) and intra-epidermal (ienfd) and sudomotor nerve fiber density (sgnfd). background: structural and functional measures of small fiber neuropathy have been studied in patients with diabetes, but little information comparing these techniques exists. design/methods: we studied patients with diabetes (ages ± yrs, gender f) and healthy control subjects (age ± yrs, gender f). subjects underwent examination scores (nis-ll), quantitative sensory testing, autonomic testing (heart rate variability, valsalva maneuver, tilt test), esc, and millimeter punch skin biopsies at the distal leg and distal thigh for ienfd & sgnfd. results: there were strong correlations between exam scores (nis-ll) and biopsy ienfd (r=− . , p< . distal leg; r=− . , p< . distal thigh), and sgnfd (r=− . , p< . distal leg; r=− . , p< . distal thigh). moderate correlations were noted between exam scores and qst (r values . - . , p< . ), ienfd and qst (r= . - . ). modest correlations were noted between esc and parasympathetic function (r= . - . , p< . ). modest correlations were noted between esc and ienfd (r= . , p< . , but only at the distal leg) and esc and nis-ll (− . , p< . ). no correlations between exam scores and autonomic function were noted. no correlations were detected between esc and sgnfd (r=− . - . ) or esc and sympathetic adrenergic function (r= . ). conclusions: differences between tests are expected based on our understanding of the pathophysiology and natural history of diabetic neuropathy. exam scores, qst results and biopsy results do correlate, and are consistent with prior studies. in contrast, there was little relationship between tests of autonomic function and exam scores, similar to prior studies and our understanding of autonomic function and differences in p. esc had little correlation with either exam scores or biopsy ienfd or sgnfd. there was a modest correlation between esc and parasympathetic function. the exact relationship between esc and diabetic peripheral neuropathy is not clear, and further research studies are needed to determine the role this technique has in clinical practice. objective: to determine the optimal tissue thickness of skin biopsy sections for studies of cutaneous nerve fibers. background: although analysis of intra-epidermal nerve fiber density (ienfd) is routinely reported using μm thick tissue sections, many recent studies of peripheral alpha-synuclein deposition use or μm frozen sections, or μm paraffin embedded tissue sections. design/methods: we compared the results of biopsies from patients with parkinson's disease (pd), using tissue sections each of , and μm thickness. tissues were stained with pgp . and stained for phosphorylated alpha-synuclein (p-syn). the total number of dermal structures (hair follicles, sweat glands, pilomotor muscles) were quantified, nerve densities analyzed, and the frequency of p-syn positive results. we also studied μm paraffin embedded tissue samples from patients with pd. results: in the biopsies of patients with pd there were no differences in the number of sweat glands, hair follicles or pilomotor muscles in , or μm sections. there were significantly fewer blood vessels noted in and μm sections compared to μm sections (p< . ). ienfd and sgnfd declined with tissue thickness (p. . , all) and there was increased variability in results in thinner tissue sections. there was a highly significant reduction in p-syn positive sections in thinner tissue sections (p< . , all tissues compared to μm sections). paraffin embedded tissue sections had significantly lower nerve densities and positive p-syn results (p< . ) compared to all frozen tissue sections of , and μm thickness. thinner tissue sections carried a greater risk of false positive result or indeterminate results due to difficulty interpreting overlap with pgp . . conclusions: tissue sample thickness plays a critical role in interpretation of skin biopsy results. thinner tissue sections, or paraffin embedded tissue sections, do not provide equivalent data and significantly underestimate nerve densities and positive alpha-synuclein results with increase false positive results despite similar numbers of dermal tissue structures. ace- is an investigational protein therapeutic that acts as a localized ligand trap for myostatin and other negative regulators of muscle growth. local injection of ace- into the gastrocnemius muscle of wild-type, mdx, and sod mice produced dose-dependent increases in muscle mass and force without systemic effects. in a phase single-center, double-blind, placebo-controlled dose escalation study in post-menopausal women, unilateral injections of ace- into the rectus femoris (rf) or tibialis anterior (ta) muscle were generally safe and well tolerated. mean percent changes from baseline in muscle volume of the injected muscle were + . % in the rf and + . % in the ta at the highest dose administered with minimal changes observed in the contralateral side and placebo-treated subjects. frequent related aes (≥ %) included injection site pain, pain in extremity, injection site discomfort, and muscle twitching, with similar incidence in ace- and placebo-treated groups. all aes were grade - and reversible. together, these preclinical and clinical results support further studies of ace- in myogenic and/or neurogenic diseases with focal loss of muscle strength and function, including cmt. a phase study is ongoing in facioscapulohumeral muscular dystrophy (fshd). study a - is an ongoing multicenter, two-part, phase study to evaluate the safety, tolerability, pharmacodynamics, efficacy, and pharmacokinetics of ace- in patients with cmt and cmtx. part is open-label and will enroll up to dose-escalating cohorts ( patients per cohort); part is randomized, double-blind, and placebo-controlled, and will enroll an additional patients. ace- will be administered bilaterally to the ta muscle once every three weeks for up to five doses. a safety review team will meet periodically throughout the study to review safety data and make dosing recommendations, including the recommended dose level for part . eligible patients must have genetically confirmed cmt or cmtx with mild-moderate weakness in ankle dorsiflexion. safety and tolerability will serve as the primary outcome for part , muscle volume evaluated by mri for part . additional outcome measures of interest include strength by quantitative muscle testing, function by motor tests, and quality of life by the cmt-health index questionnaire. our study was aimed to evaluate the functional and morphological consequences of cellular and humoral responses in chronic inflammatory demyelinating neuropathy (cidp), using extensive standardized high-resolution sonography (hrus) and nerve conduction study (ncs) protocols in incident treatment-naive patients. we enrolled consecutive, newly diagnosed, treatment naive patients with cidp. in addition to all relevant clinical examinations, all patients underwent a standardized ncs and extensive hrus protocol, of median, ulnar, tibial, fibular and sural nerves. we assessed standard nerve and fascicle size, and echogenicity. we found focal sonographic enlargements in multiple nerves and nerve segments with and without ncs abnormalities. the degree of nerve hypertrophy was not associated with presence of ncs features of demyelination, i.e. / ( %) of median nerve segments showed enlargement without strong decrease in motor conduction velocity and / ( %) hypertrophic median nerve segments revealed no conduction block. a lower distal cmap of median nerve was related with lower mrc sums-scores (p < . ). we found no correlation between age, disease duration or mrc sum-score and nerve size. cellular and humoral responses in cidp may lead to nerve enlargement along the length of nerves, that can be detected by hrus, whereas ncs allows identification of its' specific focal disruption in nerve function. the most common complication of diabetes is peripheral neuropathy, which has prevalence as high as % and is characterized by damage to neurons, schwann cells and blood vessels within the nerve. the concept of schwannopathy as an integral factor in the pathogenesis of diabetic neuropathy is re-emerging, and it is now known that schwann cells cultured in hyperglycemic environments underproduce neurotrophins and exhibit loss of axonal associations, further indicating a non-optimal glial cell activation and function. furthermore, the increased expression of p ntr in myelin sheaths around fibers that are susceptible to axonal degeneration in diabetic neuropathy suggest an important role for this molecule in disease progression. with this project, it is our main goal to evaluate how disruption of p ntr signaling in the schwann cells affects the pathophysiology of diabetic neuropathy. by using a fluorescent live/death cell viability assay, our preliminary data indicate that wild-type schwann cell cultures present increased cell death rate h after stimulation with high levels of glucose. the p ntr has a highly recognized role in the activation of death signals and when absent, we observed that schwann cells are significantly more resistant to apoptosis in hyperglycemic conditions. the role of p ntr receptor signaling in neuron-schwann cell communication and myelination under in vitro diabetic conditions was investigated with primary schwann cell-sensory neuron co-cultures. after eight days of ascorbic acid stimulation, both under euglycemic and hyperglycemic conditions, myelination was assessed by confocal microscopy using specific markers for neurons and myelin. results highlight a compromised ability of wild-type schwann cells to myelinate axons when exposed to a hyperglycemic environment, which was even intensified in co-cultures with schwann cells lacking the p ntr . to complement the in vitro studies, we are modeling type diabetes in a p ntr schwann cell conditional ko mouse model and plan to investigate nerve mrna expression profile to disclose genetic regulation depending on this receptor signaling and its modulatory role in endoneurial hypoxia and neuroinflammation. the results from this project will provide an integrated vision of how impaired schwann cell activity guides neuropathy progression. gondim faa , barreira aa , cruz mw , cunha fmb , de freitas m , frança mc jr , marques w jr , nascimento ojm , oliveira asb , pereira rc , pupe c , rotta ft , schestatsky p . panelists on behalf of the scientific department of peripheral neuropathy, brazilian academy of neurology, brazil. neuropathy is one of the most common neurological manifestations of several diseases and sfn has been progressively receiving more attention in the medical literature. the aim of this study is to generate a set of recommendations to define and diagnose sfn in brazil. a group of neurologists, members of the scientific department of peripheral neuropathy from the brazilian academy of neurology reviewed a preliminary draft prepared by the first author that was distributed by email. the panelists got together on . . at the city of fortaleza, brazil, to discuss and finish the text for the first submission of the manuscript. sfn can be defined as a subtype of neuropathy characterized by selective involvement of unmyelinated or thinly myelinated sensory (sometimes also autonomic) fibers. it is usually characterized by sensory (pain/dysesthesias/pruritus) or combined sensory and autonomic complaints, associated with an almost entirely normal neurological examination (except for sensory changes). electromyography is normal. a growing list of medical conditions has been linked to sfn, although there is no evidence-based literature to support the use of any specific set of screening tests to diagnose the etiology of sfn (the panelists will suggest a basic screening panel). sfn may also serve as a fallacious but useful terminology to uncover discrepancies in the normal values from different neurophysiology laboratories. in brazil, skin biopsy is not usually performed and initial forms of leprosy may have predominant small fiber involvement. there are several tests to demonstrate involvement of small sensory and autonomic fibers. skin wrinkling test, sympathetic skin responses & heart rate variability (conducted on emg machines) and thermoregulatory sweat test may be low-cost screening alternatives. after the final meeting on . . , we finished the first draft for submission to arquivos de neuropsiquiatria (together with a translation to portuguese as supplementary material), the official journal of the brazilian academy of neurology to serve as a source for the definition and diagnosis of sfn in brazil. the final draft will be submitted after presentation at the pns meeting in barcelona on . . the lower cranial nerves (lcn) include the paired glossopharyngeal, the vagal, the accessory and the hypoglossal nerves. these are involved in the execution of swallowing, speech, phonation, tasting, as well as sensory and autonomic functions. lcn can be affected in central and peripheral nervous system diseases. this study investigates the use of ultrasound (us) to detect lesions of the peripheral course of lcn. in addition mri can be used in unilateral lcn local lesions to demonstrate indirect signs of nerve lesions such as muscle atrophy. the patients were examined in supine position with the neck in maximum extension with either a regular or a portable us system (logiq e and logiq e ge healthcare, milwaukee, wisconsin) and high frequency transducers ( to mhz). according to the detected pathology longitudinal or transversal images were recorded. a series of exemplary observations are demonstrated. in one case a paraganglioma in the glossopharyngeal nerve at the carotid sinus was found. in another patient an us of the thyroid gland revealed a nerve tumor, which was later identified to be a schwannoma. in another case multiple neurofibroma were identified in the vagal nerve during a routine neurofibromatosis screening. us has become also important in investigations of lesions of the accessory nerve, following damage by surgery. nerve continuity and scar formation can be distinguished. lesions of the hypoglossal nerve can be caused by tumor infiltration. in one patient an infiltration of the nerve by a squamous cell carcinoma, and in another case by a low grade sarcoma were detected. in addition mri demonstrated an atrophy of the tongue. identification and assessment of the lcn from the point of exit of the skull to their endpoint can be pursued by us techniques, which have been described for the individual nerves. us is easily applicable and -in addition to the "static" image of the mri -allows assessing nerve positions during different movements, as well as detecting muscle movements as fasciculations. mri can also be used to detect not only more proximal lesions, but also changes of the skeletal muscle by denervation. grümme l , hattenhauer st , wolffram k , kleinschnitz c , mausberg ak , stettner m . university hospital essen, essen, germany; heinrich-heine-university, düsseldorf, germany. in chronic inflammatory demyelinating polyneuropathy (cidp), t cells are suspected to play a crucial role in myelin destruction. cd and cd t cells contribute to the inflammatory process, while the exact mechanism of myelin damage is still under debate. to elucidate the molecular interaction of t cells and myelin sheets, we compared neuritogenic and control cells in a live imaging in vitro model. the myelinated fibres of rat dorsal root ganglia (drg) served as model for the peripheral nervous system. drgs of embryonic (e ) lewis rats were cultured; myelination was initiated after one week in vitro and continued for two additional weeks. lewis rats immunized with p and neuritogenic t cells of the lymph nodes were obtained ten days after immunization. control t cells were prepared from healthy rats. for vital tracking, neuritogenic t cells were stained with orange cell tracker, while the control cells were labelled with cfse. myelin detection in vital cultures was assessed by incorporation of c fatty acids conjugated with a fluorophore into the myelin layer. experiments were performed in a conditioned microscope chamber, the two t cell populations were added simultaneously to the drg culture and migration was tracked using live cell imaging. we observed differing migration patterns for neuritogenic and control t cells. the velocity as well as the directionality was altered. after initial contact, the non-neuriotgenic t cells in close proximity to myelin subsequently decreased over time, while the numbers of neuritogenic t cells close to myelin remained stable. long term incubation with neuritogenic t cells affected the myelin integrity in regard to the intermodal length as well as the myelin ratio. further experiments will elucidate the specific effects of neuritogenic t cells to decipher the role of t cells during inflammation in the pns, which could be useful in developing targeted therapies. gundapaneni b , sultan mb , keohane dj , schwartz j . inventiv health inc., burlington, ma, usa; pfizer inc., new york, ny, usa. transthyretin familial amyloid polyneuropathy (ttr-fap) is a rare, life-threatening disorder caused by protein destabilizing ttr gene mutations, broadly classified as val met (the most common mutation worldwide) and non-val met genotypes. tafamidis, a highly-specific ttr-stabilizer, is the only medicine approved to delay neurologic progression in ttr-fap. the objective of this post-hoc analysis was to compare the effects of tafamidis on neuropathy progression in patients with val met and non-val met genotypes. val met patients were participants in a randomized, double-blind, placebo-controlled clinical trial of tafamidis, while non-val met patients were participants in an open-label tafamidis study. patients were grouped into three cohorts: val met tafamidis (n= ); val met placebo (n= ); and non-val met tafamidis (n= ). baseline disease severity and change in disease severity from baseline to month was assessed using the neuropathy impairment score-lower limbs (nis-ll). the effect of tafamidis in the val met and non-val met cohorts versus the val met placebo cohort was determined using a mixed-effects model for repeated measures (mmrm). at baseline, patients in the non-val met cohort were older, had longer symptom duration, and more advanced neurologic impairment than the val met cohorts. at month , the baseline-adjusted mean ± standard error change in nis-ll was comparable between the val met tafamidis and non-val met tafamidis cohorts ( . ± . and . ± . , respectively). these changes were smaller than that observed in the val met placebo cohort ( . ± . ; p= . vs val met and p= . vs non-val met) indicating less disease progression. based on predicted values from the mmrm analysis, the size of the change in nis-ll across the full range of baseline nis-ll scores was remarkably similar in the val met tafamidis and non-val met tafamidis cohorts and was consistently smaller than that observed in the val met placebo cohort. moreover, in all three cohorts, as baseline nis-ll increased, the predicted level of disease progression also increased. in conclusion, while controlling for baseline disease severity, tafamidis delayed disease progression to a comparable extent in val met and non-val met patients. the similar trajectories of disease progression across val met and non-val met patients suggest that these two genotype groups may be more similar than previously considered. clin-icaltrials.gov identifiers: nct ; nct . gutmann l , shy m . university of iowa, iowa city, ia, usa. post tetanic potentiation (ptp) is a physiological phenonmenon seen with disorders of the neuromuscular junction (nmj). it is caused by the influx of ca++ into the terminal axon during the tetanus resulting in an increased number of acetylcholine (ach) vescicles released by each axonal action potential. in myasthenic syndromes it results in improved nmj function by increasing the probability of achieving a large enough end plate potential to generate a muscle action potential. ptp has different appearances depending on whether the nmj defect is pre-synaptic or post-synaptic. in pre-synaptic defects (the most common being lambert-eaton syndrome) there is an increased amplitude of the muscle action potential after a tetanus that persists up to minutes. prolonged ptp has now been reported in disorders, one on a genetically determined neuropathy/myasthenic basis and the other on an acquired toxic origin. prolonged ptp up to minutes was reported in families with a motor neuropathy (having leg weakness and foot deformites) and a congenital myasthenic syndrome caused by a heterozygous mutation of the synaptotamin ii (syt ) gene (c t>g p.pro [p.asp ala] and c g>a [p.pro leu]). electrophysiological testing showed features of a presynaptic defect with prolonged ptp persisting up to minutes. the same phenomenon has been noted in the acquired pre-synaptic defect caused by botululinum toxin. the ptp continued up to minutes. syt is the synaptic vescicle calcium sensor in the terminal axon, allowing for fusion of ach containing vescicles with the presynaptic membrane and the synchronous release of ach. the fusion requires a complex assembly process involving snare proteins. both the syt gene mutation and botulinum toxin affect normal syt function, the mutation by altering amino acids in the calcium-binding domain and the toxin by binding to syt as well as gangliosides gd a and gt b on the neural membrane. the mechanism for the ptp prolongation remains unknown. prolonged ptp appears to be a unique physiological abnormality resulting from altered syt . this phenomenon has been decribed to occur in syt mutations causing congenital motor neuropathy/myasthenic syndrome and botulism. the abnormality may represent a physiological marker for a presynaptic nmj defect involving altered syt . hachisuka a , , senger j , curran m , chan km , . division of physical medicine and rehabilitation, university of alberta, edmonton, canada; department of rehabilitation and medicine, university of occupational and environmental health, kitakyushu, japan; division of plastic surgery, university of alberta, edmonton, canada. background: motor unit number estimation (mune) techniques are valuable tools in neuromuscular disease. among them, the multiple point stimulation (mps) is one of the most common used. contamination by distant single motor unit potentials (smups) generated by neighboring muscles is a potential confounding factor. this is particularly problematic in ulnar neuropathy, one of the most common neuropathies in humans. reason being that the ulnar nerve innervates the majority of hand muscles. the goals of this study are to test the hypotheses that ) distant smups all have an initial positive deflection and ) elimination of smups generated by distal muscles will significantly lower the mune results in the hypothenar muscles. methods: to address the first hypothesis, we tested subjects by stimulating their median nerve while recording smups simultaneously over the hypothenar and thenar muscles. for the second hypothesis, we carried out mps mune of the hypothenar muscles using multi-channel recordings placed over ulnar innervated intrinsic muscles across the hand. when a smup with an initial positive deflection was detected at the hypothenar electrodes, its original was systemically tracked through all the recording channels. results: in the first series of experiments, in accordance with the dipole theory, all smups recorded at the hypothenar recording electrodes had an initial positive polarity. in the second series of experiments, of the studies carried out in subjects, distant smups generated by muscles other than those in the hypothenar eminence represented ± . % (mean±sd) of the overall sample. mune calculated using only smups generated by the hypothenar muscles was ± , compared to ± if all smups were included (p < . ). the extent of increase in mune was highly correlated with the proportion of distant smups found in each study (r = . , p < . ). conclusion: in contrary to some studies suggesting that smups from distant muscles could have an initial negative deflection, we found all smups from distant muscles had a positive deflection. exclusion of those smups from the sample had a significant impact on the mune results. hagiwara w , konno s , kihara inoue m , fujioka t . toho university, tokyo, japan. matrix metalloproteinase (mmp) plays crucial roles in developing immune-mediated neuritis as guillain-barré syndrome (gbs) and its animal model experimental autoimmune neuritis (ean). to investigate the intraneural expression of mmps during ean and the effect of a phosphodiesterase- inhibitor cilostazol (clz) on it, ean rats were treated with either mg/kg/day of clz or vehicle from one day post immunization (dpi). to induce ean female lewis rats were immunized with synthetic peptide from bovine p protein. cauda equina (ce) were removed in several time points, total rna was extracted and reverse-transcribed to obtain cdna that was subjected to real-time pcr analysis for expression of mmp- , mmp- and tissue inhibitor of matrix metalloproteinase- (timp- ) messages. mmp- and mmp- messages peaked at dpi, that is presymptomatic phase of ean. all rats developed motor paralysis at dpi, mmp- and mmp- messages subsided at this moment. however, timp- message reciprocally increased at dpi, persisted through dpi. treatment of clz suppressed motor paralysis of ean significantly. mmp- message peaked at dpi and mmp- message peaked at dpi. on the other hand, both messages at dpi were suppressed compared to untreated ean rats. timp- message in clz treated rats peaked at dpi coincided with motor paralysis peak. both mmp- and mmp- messages might result in subsequent upregulation of timp- that finally downregulates mmps activity and inflammatory process. clz treatment suppressed and delayed expression of mmps and facilitate timp- expression, resulting suppression of ean. the precise mechanism of expression of mmps and timp- remain unclear, however, that mmp- and mmp- messages peaked at dpi suggests involvement of pro-inflammatory cytokines such as tumor necrosis factor-alpha or interleukin- . clz might suppress these cytokines resulted in mmps down regulation. mmp- less affected by clz and thus stimulated timp- expression at dpi. clz might rational treatment for immune-mediated neuropathy via mmps modulation although further investigation especially in-vivo study is needed. haidar m , de winter v , asselbergh b , bouhy d , timmerman v . peripheral neuropathy research group, vib, university of antwerp, antwerp, belgium. the small heat shock protein hspb (hsp ) gene is ubiquitously expressed and encodes for a chaperone protein with essential cellular functions. our lab was the first to identify missense mutations in hspb responsible for axonal charcot-marie-tooth neuropathy (cmt f). since then we became interested in understanding the physiological functions of hspb and its association with cmt neuropathies. we demonstrated the involvement of hspb in microtubule stability. because of the link between autophagosome formation and its intracellular transport, and microtubules stability, we believed that the macro-autophagy process could be regulated by hspb . macro-autophagy is a cellular housekeeping process during which autophagosomes target, envelop and degrade aberrant protein aggregates and damaged organelles. there is strong evidence for an essential role for autophagy in the maintenance of neuronal homeostasis; hence its impairment can lead to a neuropathic condition. our data indicate that macro-autophagy is disrupted by hspb cmt-causing mutations. combining novel microscopy and interactomics techniques we unravelled the way different cmt-causing mutations in hspb impair the autophagic pathway. our data present the impairment of autophagy as a possible pathomechanism for cmt-causing hspb mutations. hajjar h , gautier b , berthelot j , gonzalez e , gess b , young p , tricaud n . institute of neurosciences of montpelllier, inserm, university of montpellier, montpellier, france; universitätsklinikum münster, klinik für schlafmedizin und neuromuskuläre erkrankungen, münster, germany. cmt a, the most common of charcot-marie-tooth diseases, results from the duplication of peripheral myelin protein (pmp ) gene. this gene encodes for a small protein of kda, pmp , mainly produced by schwann cells and the excess of pmp leads to demyelination. there is no cure for this disease but one approach for a treatment is gene therapy. a transgenic rat model exists for cmt a, which possesses copies of the mouse pmp gene. our goal is to provide a proof of principle for gene therapy in peripheral nerves using this rat model of cmt a. our strategy is to reduce the overexpression of mouse pmp protein in rats schwann cells using short hairpin rnas (shrnas). shrnas are small non-coding rnas that specifically bind to targeted mrnas resulting in their degradation. we tested for the efficiency of several shrnas targeting mouse pmp in vitro to find two shrnas that reduce pmp levels. the shrnas have been cloned in an adeno-associated serotype (aav ) viral vector together with green fluorescent protein in order to detect infected cells. aav was selected for its high transduction rate of myelinating schwann cells, for its good diffusion and low immunogenicity. we plan bilateral injections in the sciatic nerve of control and diseased rats. the efficiency of this gene therapy will be checked by assessing muscle strength (grip test), way of walking (catwalk), mobility (rotarod) and nerve conduction velocity of treated cmt a rats versus non-treated. the process of myelination and myelin maintenance in schwann cells will be analyzed by biochemistry and electron microscopy. biochemical tests include western blot for pmp protein expression in sciatic nerve, immunohistochemistry for pmp protein expression in myelinating schwann cells and pcr for mrna pmp expression. if the therapy is successful in rats, it could possibly be later on used in clinical trials. ultrasound is a widely-used tool in diagnosing carpal tunnel syndrome (cts). several different methods for sonographical evaluation of median nerve damage exist, such as calculating the ratio of cross sectional areas (csa) of median nerves in various sites. this enables detection of actual nerve swelling proximal to the carpal tunnel as an expression of median nerve damage inside the carpal tunnel (as seen in cts). in comparison to other diagnostic methods no data exist about the prevalence of cts like changes in an unselected population. a series of non-selected fresh cadavers were examined. arms of fresh, non-embalmed whole body cadavers were examined. the medical record did not allow to obtain conclusive information on the peripheral nerves. using a regular ultrasound system with a mhz transducer, median nerves were identified and tracked along their course in the forearms. csa measurements of the median nerves were performed at two sites in each arm: .) halfway between the elbow joint and wrist, .) directly proximal to the carpal tunnel. csa ratio was calculated with the following formula: csa ratio = csa wrist (cm ) csa forearm (cm ) csa ratio < . was found in ( . %) arms, csa ratio ≥ . in ( . %) arms and csa ratio ≥ in ( %) arms. csa ratio ≥ . was detected in . % of women and % of men. the overall mean (± sd) age was . ± . years. men ( . ± . ) were significantly younger than women ( . ± . ; p = . ). a weak but significant correlation between age and csa ratio was found in women (spearman -− . ; p= . ), but not in men (p= . ). the mean bmi for csa ratio ≥ . was . ± sd . . based on a csa ratio ≥ . as a criterion for cts, the present ultrasound results are consistent with the average cts prevalence reported in previous studies, which were obtained with electrophysiological methods. this study on a large unselected series of cadavers confirms the comparability of both methods. instrumental activities of daily living (badl and iadl), falls and gait patterns in the large, prospective, population based rotterdam study. in total, participants of this study (mean age years, % women) underwent a polyneuropathy screening involving a symptom questionnaire, neurological examination and nerve conduction studies. screening yielded four groups: no, possible, probable and definite polyneuropathy. participants were interviewed about badl (stanford health assessment questionnaire), iadl (instrumental activities of daily living scale) and frequency of falling in the previous year. in a random subset of participants, gait was assessed with an electronic walkway (gaitrite). associations of polyneuropathy with badl and iadl were analyzed continuously with linear regression, and dichotomously with logistic regression. history of falling was evaluated with logistic regression and gait changes were evaluated with linear regression. we found that participants with definite polyneuropathy had more difficulty in performing badl and iadl than participants without polyneuropathy. polyneuropathy related to worse scores of all badl (especially walking) and three iadl components (housekeeping, traveling, and shopping). participants with definite polyneuropathy were two times more likely to fall, and these falls more often resulted in injury. participants with polyneuropathy had worse gait parameters on the walkway, including lower walking speed and cadence, and more errors in tandem walking. in summary, chronic polyneuropathy is strongly associated with significant impairment in daily life. recognition of polyneuropathy and related disability is very important in order to inform, support and possibly treat patients, and to prevent future falls and dependence in daily functioning. patients with polyneuropathy often suffer from tingling sensations, numbness, weakness and pain. these symptoms are used in several screening questionnaires, most of which were developed for high-risk patient groups, such as individuals with diabetes mellitus. in most tools equal weights are applied to all symptoms, while some might be more informative than others. we evaluated the diagnostic value and frequency of occurrence of individual symptoms of chronic polyneuropathy and constructed and validated a simple screening questionnaire that can reliably help to diagnose polyneuropathy in low-risk patient groups. in a multi-step procedure, we initially compiled a twelve-item questionnaire concerning symptoms of polyneuropathy. the questionnaire was completed by polyneuropathy patients and controls (headache, transient ischemic attack, multiple sclerosis). we calculated the sensitivity, specificity and likelihood ratios of each individual symptom. next, stepwise multivariable logistic regression was used to create a compact model, able to discriminate cases from controls using only the most informative symptoms. a simple scoring system was subsequently developed based on the regression coefficients of this reduced model. external validation was subsequently conducted in a population of cases with chronic idiopathic axonal polyneuropathy and controls without polyneuropathy. performance was assessed with discrimination (area under the curve, auc), and calibration. numbness and tingling feet were most frequently reported by polyneuropathy patients and had the highest sensitivity. feeling as if walking on cotton wool and allodynia of the feet had the highest specificity. multivariable logistic regression yielded a model that contained these four symptoms, complemented with balance problems and tingling hands. based on this regression analysis, the erasmus polyneuropathy symptom score (e-pss) was created, a score ranging from to . this polyneuropathy symptom score had a good performance (auc . ) in de derivation set and proved to be valid in the external population (auc . ). in this study, we created a simple, validated polyneuropathy symptom score (e-pss) that takes both the individual value of only six different symptoms and its frequency into account. this tool can be helpful as screening instrument in clinical practice and for future studies on polyneuropathy. multiple sclerosis (ms) is traditionally viewed as a central nervous system disease. to date, there is no unequivocal evidence implicating involvement of the peripheral nervous system (pns). this study aims to prove whether the pns is additionally affected and if so, to detect, localize and quantify these peripheral nerve lesions in patients with multiple sclerosis (ms) by applying high-resolution mr-neurography (mrn) with large anatomical coverage in combination with standard electrophysiological and neurological tests. we prospectively enrolled patients with confirmed ms (> years), two patients with clinically isolated syndrome (cis), and age-/sex-matched healthy volunteers. any other potential causes for a concomitant polyneuropathy were excluded. all ms patients underwent detailed neurological and electrophysiological testing. tesla mrn with large anatomical coverage from lumbar plexus and spinal nerves down to ankle level was performed in all participants by using fat-saturated, t -weighted turbo-spin-echo (tse) sequences (tr/te / ms) and a dual echo tse sequence for t -relaxometry (tr ms; te /te / ms). a d t -weighted, fat-saturated space sequence (tr ms; effective te ms) was used for imaging of the lumbar plexus. manual segmentation of spinal/sciatic/tibial/peroneal nerves was performed on a total of , axial slices. besides evaluation of nerve t w-signal, detailed quantification of nerve lesions by analyzing morphometric (nerve caliber) and microstructural markers (proton-spin-density and t -relaxation-time) was conducted. mean lesion load at thigh level was higher in ms ( . ± . ) vs. controls ( . ± . ;p< . ). nerve proton-spin-density was also higher in ms (tibial/peroneal: . ± . / . ± . ) vs. controls (tibial/peroneal: . ± . / . ± . ;p< . ). in contrast, t -relaxation time was significantly higher in controls (tibial/peroneal: . ± . / . ± . ) vs. ms (tibial/peroneal: . ± . / . ± . ;p< . ). proximal tibial and fibular nerve caliber was also significantly higher in ms (tibial: p< . ; fibular: p= . ). for the first time, pns lesions in ms patients could be visualized and quantified in vivo by high-resolution mrn. lesions are indicated by an increase of proton-spin-density and a decrease of t -relaxation-time. nerve caliber as a morphometric criterion also significantly increased. this proof-of-concept study may offer new insights into the pathomechanism of ms and might have future implications on therapeutic approaches. immunoglobulin g (igg) fc-gammars confer diverse effector functions by linking the cellular and humoral arms of the immune system that has been involved in the pathogenesis of guillain-barré syndrome (gbs). in the post-polio era, the polymorphisms of fc-gammar and their relevant knowledge have become one of the main targets for new therapeutic strategies for the treatment of gbs patients. differences in severity and frequency of gbs subtypes found between south-asian and western populations can be attributed to their genetic susceptibility. therefore, we aimed to determine fc-gammar polymorphic alleles (fc-gammariia: h /r ; fc-gammariiia: v /f ; fc-gammariiib: na /na ) and their possible link with gbs on the currently available large gbs cohort in bangladesh. fc-gamma r polymorphisms of gbs patients and healthy controls were genotyped using sequence-specific pcr. for validation, we carried out the sequencing of some samples for fc-gammariia and fc-gammariiia alleles. no significant differences were found regarding the distribution of fc-gammar genotypes and allele frequencies in gbs patients and controls. fc-gammar-h/h- genotype was significantly predominant in patients with severe disease compared to patients with mild disease (p= . , or, . ; % ci, . - . ). no other significant associations were found in gbs patients for candidate alleles and disease severity. fc-gammariiia-f/f- was found to be significantly predominant in anti-gm antibody positive gbs patients compared to anti-gm antibody negative patients (p= . , or, . , % ci, . - . ). fc-gammariiia-v alleles were significantly higher in patients with poor prognosis when compared to patients with good outcome (p= . , or, . , % ci, . - . ). no significant association of fc-gammariiib genotypes and alleles were found with gbs patients, disease severity and disease outcome. extensive subgroup analysis revealed no significant association in genotype and allele frequencies between aman and aidp subtype. in conclusion, igg fc-gammar polymorphisms do not constitute significant risk markers for susceptibility to gbs, however homozygous fc-gammariia-h might be involved in the severe form of gbs. in addition, fc-gammariiia-f/f- might play an important role in the molecular mimicry against nerve gangliosides in gbs. further studies that enroll a large number of patients (e,g. igos) are required to confirm the present findings from different geographical areas. hayes jm , o'brien pd , backus c , feldman el . department of neurology, university of michigan, ann arbor, mi, usa. peripheral neuropathy (pn) is a common complication observed in patients with impaired glucose tolerance and type diabetes. male mice fed a high fat diet (hfd) develop metabolic impairments and pn serving as an appropriate animal model to study pn development and progression. it is well documented that female mice fed a hfd display a degree of protection against hfd-induced metabolic changes with mice retaining relatively normal insulin sensitivity. this protection is attributed to differences in fat accumulation and to the anti-diabetic effects of estrogen. based on these sex-dimorphisms we hypothesized that hfd-fed female mice would also exhibit resistance to developing pn. in the present study male and female c bl /j mice were fed either a standard diet ( % kcal fat; sd) or a high fat diet ( % kcal fat; hfd) from wk. at wk, wk and wk, neuropathy phenotyping was performed on all groups complemented with longitudinal metabolic assessments including insulin tolerance testing (itt). neuropathy phenotyping consisted of hindpaw latency to heat stimulus, motor and sensory nerve conduction velocities (ncvs), and terminal intraepidermal nerve fiber (ienf) counts. assessment of insulin resistance through itt demonstrated that during early hfd feeding, female hfd-fed mice exhibited relatively normal insulin responsiveness, while male hfd mice exhibited insulin resistance. despite this finding, wk female hfd mice displayed a similar pattern of pn to that of their male counterparts, with similar fold-changes in hindpaw latency and sensory and motor ncvs. therefore, although female hfd-fed mice exhibit resistance to hfd-induced metabolic changes, they display a pn comparable to male hfd-fed mice suggesting that systemic insulin resistance does not mediate pn. further studies are underway investigating the role of insulin signaling in the peripheral nerves of female hfd-fed mice. anti-mag (myelin-associated glycoprotein) neuropathy is a disabling autoimmune peripheral neuropathy caused by monoclonal immunoglobulin m (igm) autoantibodies that recognize the carbohydrate epitope hnk- (human natural killer- ). this glycoepitope is highly expressed on adhesion molecules, such as mag, present in myelinated nerve fibers. since the pathogenicity and demyelinating properties of anti-mag autoantibodies are well established, current treatments aim at a reduction of autoantibody levels. however, the therapies applied so far are primarily immunosuppressive and lack selectivity and efficacy. we therefore hypothesized that a significant improvement of the disease condition could be achieved by selectively neutralizing the pathogenic anti-mag antibodies with carbohydrate-based ligands mimicking the natural hnk- glycoepitope. in an inhibition assay, a mimetic (mimhnk- ) of the natural hnk- epitope inhibited mag-binding by pathogenic igm antibodies from patient sera, however only with micromolar affinity. therefore, considering the multivalent nature of the mag-igm interaction, polylysine polymers of different sizes were substituted with the mimetic. with the most promising polylysine glycopolymer pl (mimhnk- ) the inhibitory effect on patient sera was improved by a factor of up to , per epitope, consequently leading to a low nanomolar inhibitory potency. since clinical studies indicate a correlation between the reduction of anti-mag igm levels and clinical improvement, an immunological surrogate mouse model for anti-mag neuropathy, producing high levels of anti-mag igm, was developed. the observed efficient removal of these antibodies with the glycopolymer pl (mimhnk- ) represents a first step towards an antigen-specific therapy for anti-mag neuropathy. hinder lm , mendelson f , backus c , feldman el . university of michigan, ann arbor, mi, usa. in the united states % of children and young adults are obese and at risk of developing prediabetes. prediabetic patients largely develop the same macro-and microvascular complications as patients with type diabetes, including peripheral neuropathy (pn). moreover, recent clinical data suggest normoglycemic obese patients develop pn. central obesity, characterized by excess fat storage in visceral white adipose tissue, leads to systemic metabolic dysfunction largely due to an imbalance between pro-inflammatory/anti-inflammatory adipokine production. subcutaneous adipose tissue is considered 'benign', but adopts a visceral-like phenotype in response to metabolic stress, with reduced thermogenicity, reduced brown adipose identity, and increased pro-inflammatory gene expression. the popliteal adipose tissue (pat) depot, corresponding to subcutaneous adipose, is adjacent to the peripheral nerve affected in pn, contains the lymph node for lymphatic drainage of the hind limb, and expands following local, sterile hind paw inflammation. the aim of the current study was to characterize pat changes in the high fat diet (hfd) mouse model of obesity and pn, and consider its contribution to peripheral nerve dysfunction. we previously reported c bl /j mice fed % hfd from - wk develop obesity, 'prediabetes' and pn; and switching mice back to a standard diet from - wk improves metabolic and pn phenotypes. at and wk pat was bilaterally dissected and the lymph node removed. the left pat was processed for histomorphometry, and the right pat for rt-qpcr. at wk hfd was associated with a significant shift in adipocyte size-frequency distribution, with a greater number of larger adipocytes. switching the hfd mice back to standard chow from - wk restored the size-frequency distribution towards age-matched controls. rt-qpcr was performed to assess changes in thermogenicity (ucp ), brown adipose identify (cidea) and sterile inflammation (saa ). at and wk hfd pat had reduced thermogenicity and brown adipose identify, and increased sterile inflammation. this switch towards a visceral-like phenotype was reversed in the hfd mice switched back to standard chow. in summary, hfd-induced changes in pat histomorphometry and adipose identity closely associate with pn phenotype. these preliminary data suggest a potential role for pat-nerve signaling in pn. hinder lm , backus c , hayes jm , feldman el . university of michigan, ann arbor, mi, usa. peripheral neuropathy (pn) is a common and debilitating complication of obesity and diabetes that triggers pain and loss of sensation. substantial nerve damage occurs in many patients prior to noticeable symptoms and no treatments are currently available; therefore, there is a critical need to identify treatment strategies that impact the underlying disease pathogenesis. our in vivo fluxomics data in the bks-db/db mouse model of type diabetes (t dm) and pn suggest that 'metabolic reprogramming' occurs in the t dm nerve to downregulate mitochondrial oxidative phosphorylation of substrates derived from glycolysis and fatty acid beta-oxidation. therefore, we hypothesize that distinct systemic metabolic alterations occur in obesity and diabetes which induce tissue-specific metabolic reprogramming within the peripheral nerve, altering fuel utilization and ultimately leading to tissue dysfunction. we contend that identifying conserved bioenergetic profiles across mouse models of pn will provide insight into key pn mechanisms. the current study utilized two mouse models of pn: the % high fat diet (hfd) mouse model of obesity and prediabetes at wk of age ( wk hfd), and the leptin receptor-deficient bks-db/db model of t dm at wk of age. mitochondrial function was determined in primary dorsal root ganglia (drg) neurons and sural nerve tissue from both models using the seahorse xf analyzer. resting mitochondrial oxidative metabolism was upregulated in drg neurons from mice with pn, with increased resting atp production and maintained mitochondrial coupling. in contrast, resting atp generation was decreased in sural nerve from mice with pn, with decreased coupling efficiency. relative spare respiratory capacity was attenuated in both drg neurons and sural nerve from mice with pn, indicating that mitochondria were less able to increase respiration in response to an energetic challenge. moreover, mitochondrial copy number was unchanged in drg neurons, but decreased in sural nerve tissue of mice with pn compared with respective controls. these data suggest a change in absolute number and function of sural nerve mitochondria, and a conserved cross-model proximal-distal bioenergetic profile in pn. we are currently exploring the relationship between these changes and pn pathogenesis. hoang ttn , umapathi t . hospital, ho chi minh city, vietnam; national neuroscience institute, singapore. ho chi minh city (hcmc) is the biggest metropolitan city in southern vietnam. its population is more than ten million. adult patients with neurological disorders are seen at six city public hospitals, including , hospital. there has been no systematic study of guillain barré syndrome (gbs) at hcmc or in vietnam in general. we are in the process of starting a prospective gbs database that we hope to expand to the other public hospitals at hcmc. here we describe our experience from . we saw gbs patients at , hospital. most of the cases were admitted in the rainy season, from late april to november, when mosquito-borne flavivirus infections are more common. patients were seen in the first week of illness and reported antecedent fever. two patients had diarrhea. diagnoses were made largely on clinical features, cerebrospinal fluid analysis and nerve conduction study. clinical findings include limb weakness, numbness and vii cranial nerve palsy. extraocular eye movements were affected in one patient. none had respiratory involvement severe enough to require artificial ventilation or intensive care. there were no pure miller-fisher syndrome cases; we suspect this might be related to the mild deficits that did not prompt hospitalization. nerve conduction studies showed typical features such as loss of f waves and abnormal blink reflex. the electrophysiology of patients' was dominated by demyelinating changes, one case was largely axonal and the remaining patient had normal electrodiagnostic study. repeat nerve conduction studies were not feasible because of limited resources. neurologists use corticosteroids as the main treatment. intravenous immunoglobulin and plasma exchange are costly and not reimbursed by medical insurance. we are currently preparing to systematically study gbs in southern vietnam, specifically with regards to possible role of antecedent flavivirus infections. we are also exploring the possibility of using low volume plasma exchange as a feasible cost-effective therapeutic modality. about % of patients with guillain-barré syndrome (gbs) treated with intravenous immunoglobulin (ivig) or plasma exchange deteriorate after initial improvement or stabilization -a phenomenon that is termed treatment-related fluctuation (trf). it is important to distinguish acute onset cidp (a-cidp) from gbs-trf during early course of the disease, because their therapeutic strategies and prognoses are different. herein, we describe a patient with gbs-trf, but with an extended progression phase that exceeds weeks. a -year-old woman was admitted due to acute onset progressive leg weakness and diplopia that had developed weeks prior (onset, d ). on neurological examination, right facial palsy was also observed. lower extremity weakness was moderate in proximal and distal muscles (mrc grade iv) with absent knee and ankle jerks. sensory examination revealed no abnormality in all modalities. she denied any recent diarrhea or upper respiratory infection, and vaccination. albumino-cytologic dissociation was noted in csf analysis; white blood cell count of / l and protein level of . mg/dl. nerve conduction study revealed demyelinating sensorimotor polyneuropathy with prolonged distal latency and conduction blocks. anti-ganglioside antibodies (gm igm/g, gd b igm/g, and gq b igm/g) were all negative. following ivig treatment, she was discharged with considerable improvement (d ). days later, she was re-admitted due to deterioration of leg weakness and hand clumsiness (d ). after another ivig treatment, she was discharged with clinical improvement (d ). days later, however, she was admitted again (d ) due to another considerable deterioration with four extremity weakness being worst at this time (mrc grade ii-iv in upper extremity, grade ii in lower extremity). a-cidp was considered given the progression phase exceeding weeks, but, we decided to give another treatment with ivig instead of a switch to corticosteroids because of uncertainty regarding distinction between a-cidp and gbs-trf. she was significantly improved following ivig treatment, and finally discharged (d ). thereafter, there has been no further deterioration during long-term follow-up of year. conclusively, this is a rare case of gbs with extended progression phase and trf. we propose that this could be referred to as subacute inflammatory demyelinating polyradiculoneuropathy (sidp) with trf. hsieh s , chao c . national taiwan university hospital, taipei, taiwan. transthyretin (ttr)-related familial amyloid polyneuropathy (fap) constitutes a major etiology of adult-onset hereditary neuropathies worldwide, in particular, a mutant ttr of ala ser (ttr-a s) in taiwan, the most common cause of acquired genetic neuropathy with adult onset (> years of age) of taiwanese patients. fap is a pan-modality neuropathy involving motor, sensory, and autonomic components of the peripheral nervous system with early involvement of small fibers as a major symptom. the early symptoms of fap are sometimes minimal and difficult to ascertain, mainly related to the fact that conventional electrophysiological examinations were not sensitive enough to detect small fiber neuropathy. skin biopsy with quantification of intraepidermal nerve fibers (ienf) has become one of the standard approaches to diagnose small fiber sensory neuropathy based on pathological documentation of nociceptive nerve degeneration. to explore the issue of early biomarkers in fap, we performed skin biopsy and compared ienf density with parameters of nerve conduction studies (ncs) and quantitative sensory testing (qst) on subjects ( men, aged . ± . years) with genetic confirmation of ttr-a s: patients and carriers. the ienf densities were significantly reduced compared to the age-and gender-matched controls in carriers ( . ± . vs. . ± . fibers/mm, p = . ) and patients ( . ± . vs. . ± . fibers/mm, p = . ). the latter was consistent with our previous report (neurology, : - , ) . the abnormal rate of ienf density was significantly higher than that of ncs and qst, respectively. in conclusion, there was significant skin nerve degeneration in carriers with ttr-a s. compared with qst and ncs, ienf density assessment had the highest abnormal rate and highest sensitivity to detect neuropathic changes in the early stage of fap. charcot-marie-tooth type p (cmt p) has been associated with frame-shift mutations in the ring domain of lrsam (an e ligase). this study describes families with a novel missense mutation of lrsam gene and explores pathogenic mechanisms of cmt p. this american family with dominantly inherited axonal polyneuropathy reveals a phenotype similar to those in previously reported non-us families. the affected members in our family co-segregated with a novel missense mutation cys arg that alters a highly conserved cysteine in the ring domain. this mutation leads to axonal degeneration in the in vitro neuronal cell-line. moreover, using protein mass spectrometry, we identified a group of rna binding proteins (including fus, a protein critically involved in motor neuron degeneration) that interacted with lrsam . the interactions were disrupted by the cys arg mutation, which resulted in reduction of intranuclear rna-binding proteins. a knockin mouse of cys arg has been created for further explorations of cmt p mechanisms and therapeutic development. together, our findings suggest that the mutant lrsam may aberrantly affect the formation of transcription machinery. given a similar mechanism has been reported in motor neuron degeneration of amyotrophic lateral sclerosis, abnormalities of rna/rna-binding protein complex may play a role in the neuronal degeneration of cmt p. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr ). this double-blind, multicenter, parallel-group trial randomized ( : ) adult participants (n= ) with chronic inflammatory demyelinating polyradiculoneuropathy being treated with intravenous immunoglobulin (ivig) or corticosteroids to . mg fingolimod (n= ) or placebo (n= ) once-daily. previous treatment was discontinued (ivig) or tapered (corticosteroids). in the total trial population, there was no significant difference between the groups in the primary outcome, time-to-first confirmed worsening (≥ -point increase on the adjusted inflammatory neuropathy cause and treatment [incat] scale vs. baseline), or time-to-first any worsening (confirmed on incat assessment or unconfirmed). no significant difference between the two treatment groups was shown on secondary outcomes; change from baseline in grip strength and rasch-built overall disability scale (r-ods) at six months or trial end. analyses of pre-specified subgroups were performed for primary and secondary outcomes. this trial evaluated the efficacy and safety of fingolimod in chronic inflammatory demyelinating polyradiculoneuropathy (cidp). corticosteroids, intravenous immunoglobulin (ivig) and plasma exchange are recognized treatment options but no other immunomodulators demonstrated efficacy in a controlled trial. fingolimod has been shown to be efficacious and is approved for the treatment of relapsing multiple sclerosis. results from experimental autoimmune neuritis in rats suggested that it might show an effect in cidp. in this double-blind, multicenter, parallel-group trial, cidp participants receiving ivig or corticosteroids were randomized to once-daily fingolimod . mg or placebo ( : ). participants were stratified by inflammatory neuropathy cause and treatment disability (incat) scores and prior treatment. previous ivig treatment was discontinued after one final course before randomization. previous corticosteroid treatment was tapered over weeks. the primary outcome was time-to-first confirmed worsening (≥ -point increase on the adjusted incat score versus baseline). secondary outcomes included change in grip strength and rasch-built overall disability scale (r-ods) score from baseline to month and at trial end. the trial was stopped for futility by an independent data monitoring committee after a pre-planned interim analysis based on pre-specified criteria. in all, participants received fingolimod (ivig: , corticosteroids: ; age: . ± . years [mean±standard deviation]; male: . %); received placebo (ivig: , corticosteroids: ; age: . ± . years; male: . %). the percentage ( % confidence interval) of participants free from confirmed worsening at the trial end was not significantly different between fingolimod ( . % [ . %- . %]) and placebo ( . % ( . %- . %); p= . ). in the first days, approximately % participants experienced worsening. at trial end, approximately % participants had no worsening. there was no significant difference after six months or at the trial end (whichever occurred earlier) in the secondary endpoints. adverse events were reported in / and / participants in the fingolimod and placebo group, respectively. there were no deaths. nine participants in the fingolimod group and in the placebo group had serious adverse events. adverse events leading to trial drug discontinuation occurred in ( %) participants on fingolimod and none on placebo. no new safety signals emerged in this trial. acknowledgment: the authors consulted for or were employed by the study sponsor novartis pharma ag, basel, switzerland. myelin sheath enwraps non-nociceptive mechanoselective abeta-afferents transmitting touch/vibration sense. a prominent reduction in the mechanical stimulus required to evoke a withdrawal response in rodents, a phenomena interpreted as mechanical allodynia, arises due to peripheral nerve/myelin damage. evidence has emerged that nerve injury-induced mechanical allodynia depends on the adaptive immune/t cell activity in female but not male rodents. having previously demonstrated both the release of the cryptic - peptide regions of myelin basic protein (mbp - ) following sciatic nerve chronic constriction injury (cci) and the direct, robust and t cell-dependent ability of the pure mbp - peptides to induce mechanical allodynia after injection into the intact sciatic nerve, we hypothesized that mbp - contributes to sexual dimorphism in mechanical allodynia. the pure mbp - wild-type (wt), its histidine (his) mutant or scramble peptides were administered into an intact sciatic nerve fascicle in male and female rats or mice, followed by von frey testing. intra-sciatic mbp - -wt peptide induced robust and lasting allodynia in females. in contrast, males responded with a brief and mild decline in mechanical sensitivity for one day post-injection of both wildtype and control peptides. the algesic ability of mbp - -wt was diminished in the his mutant. we here present the molecular changes in the sciatic nerve, drg and the spinal cord after the intra-sciatic mbp - injection in male and female animals. in addition, using the biotin-labeled mbp - peptide and the hrp-labeled goat anti-rat igg/igm antibodies, we developed an elisa to quantitatively assess seropositivity for the specific anti-mbp - peptide igm/igg autoantibodies in female and male rats post-cci. human serum from female patients with multiple sclerosis was used for control. our work corroborates the findings of sexual dimorphism of mechanical hyperpathia and suggests its potentially autoimmune nature in females. iijima m , nishi r , ikeda s , kawagashira y , koike h , sobue g , katsuno m . nagoya university, nagoya, japan. non-obesity diabetic (nod) b - knockout (ko) mice are characterized by chronic and progressive neuritis and expected as models of immune-mediated neuropathies, especially cidp. hindlimb-predominant weakness due to inflammatory demyelination followed by axonal degeneration begins from around twenty week-age in all female mice until thirty week-age. to clarify the efficacy of immunoglobulins as immune-regulating therapeutics and the similarity of pathogenesis of human cidp, we injected intraperitoneally human-derived immunoglobulins (ipig, mg/mg bw/week) and saline as a control to totally forty female mice. clinical and pathological estimations in sciatic nerves were performed in time series. as a result, the ipig-treated group was protected from weight loss which could be related to axon loss followed by muscle atrophy as well as inflammatory demyelination between twenty-five week-age and thirty week-age compared to the control. in addition, the pathological findings in sciatic nerves showed that ipig apparently suppressed inflammatory infiltrates. about the subsets of inflammatory infiltrates, while macrophages (cd +) and lymphocytes (cd +) highly existed and suggested to play a main role in the neuritis until thirty week-age, only macrophages naturally disappeared after thirty week-age without any therapeutic induction. immunoglobulins effectively suppressed only macrophages although that did not suppress cd + lymphocytes. in conclusion, nod b - ko mice respond to immunoglobulins in a similar manner to human cidp and this efficacy is due to the suppression of macrophage-dominant pathogenesis. therefore, macrophage-derived pathogenesis is for the main target of immunoglobulin therapy and we should focus on the lymphocyte-derived pathogenesis which might plays an important role in non-responders to immunoglobulins. ikeda s , nishi r , kawagashira y , iijima m , koike h , katsuno m , sobue g , . department of neurology, nagoya university graduate school of medicine, nagoya, japan; research division of dementia and neurodegenerative disease, nagoya university graduate school of medicine, nagoya, japan. chronic inflammatory demyelinating polyneuropathy (cidp) is an acquired immune-mediated polyradiculoneuropathy that is characterized by heterogeneous clinical manifestations. typical cidp is defined as neuropathy manifesting in a progressive manner, stepwise manner, or with recurrent symmetrical proximal and distal weakness and sensory impairment in all four limbs. although they occur at a lower proportion than the so-called typical cidp, atypical forms, such as multifocal acquired demyelinating sensory and motor (madsam), distal acquired demyelinating symmetric (dads), pure sensory, pure motor, and focal, are considered to cidp subtypes. thus far, pathological features characterizing each clinical subtype have not been fully elucidated. we analyzed clinical and pathological correlations in consecutive cidp patients who underwent sural nerve biopsy and fulfilled the definite or probable efns/pns criteria. there were male and female patients. the age at biopsy was . ± . (mean ± sd) years, and the duration from the onset of neuropathy to biopsy was ± months. fifty-five percent (n = ) of the patients were classified as having typical cidp. regarding atypical cidp, madsam (n = , %), dads (n = , %), and pure sensory (n = , %) subtypes were the major subtypes, while pure motor (n= , %) and focal (n= , %) subtypes were rare. no significant difference was found among these subtypes in terms of sex, age at biopsy, and disease duration. sural nerve biopsy specimens revealed that the densities of large myelinated fibers significantly decreased in the madsam subtype than in the other subtypes (p = . ). in addition, the variation in nerve fibers among fascicles was more conspicuous in the madsam subtype than in typical cidp (p= . ). patients with the dads subtype tended to show the formation of onion-bulbs. in conclusion, pathological findings of sural nerve biopsy specimens were different among the cidp subtypes. further studies are needed to clarify mechanisms leading to different pathological features. small volume plasma exchange (svpe) can be an affordable and potentially effective alternative form of plasma exchange. svpe is the repeated removal of small volumes of supernatant plasma over several days via sedimentation of patient whole blood. the aim of this study is to assess the clinical feasibility and safety of svpe in patients with gbs in low-income countries. twenty adult patients with gbs diagnosed as per the criteria for gbs of the national institute of neurological and communicative disorders and stroke (ninds) were enrolled for svpe at a centre in bangladesh. serious adverse events (sae) were defined as the number of patients developing severe sepsis associated with the central venous catheter (cvc) or deep venous thrombosis in the limb where the cvc is placed for svpe. the svpe procedure was considered safe if less than of svpe-treated gbs patients have a sae, and feasible if eight litres of plasma could be removed in at least of svpe-treated gbs patients. among the cases who received svpe, ( %) patients were male and the age range between to yrs. all the patients were quadriplegic and bedbound at enrolment for svpe with a median mrc score of (iqr, - ). cranial nerve involvement, autonomic dysfunction and requirement for assisted ventilation were observed in ( %), ( %) and ( %) patients respectively. electro physiologically ( %) patients were motor axonal and ( %) patients were sensory-motor demyelinating type. during the svpe none of our patients experienced sae and one patient experienced central line associated blood stream infection. common adverse effects were transient intravenous fluid responsive hypotension during the svpe sessions in ( %), cv catheter insertion site hemorrhage in ( %) and hypersensitivity reaction to fresh frozen plasma in ( %) patients. there was no hypo-albuminemia, anemia or electrolyte imbalance observed in most patients ( %) treated with svpe. improvement in one or more grade of the gbs disability score at four weeks after the onset of svpe was observed in ( %) patients. in conclusion svpe can be a safe, feasible and cost effective alternative to standard pe in the developing countries. guillain-barré syndrome (gbs) is a descriptive disease entity defined by a set of clinical, electrophysiological and laboratory criteria. various clinical phenotypes exist that may be triggered by different antecedent infectious events. although the disease appears to affect primarily the elderly in developed countries, but, scenario is different in developing countries. bangladesh has made an impressive progress towards the eradication of poliomyelitis, and no new cases have been reported since . gbs, an acute polyradiculoneuropathy, is the most frequent cause of acute flaccid paralyis. the crude incidence rate of gbs in < years of age reported here appears to be . × to × higher than that reported in the literature. we conducted a hospital based observational study including patients fulfilling the national institute of neurological disorders and stroke (ninds) criteria for gbs patients between and in dhaka medical college hospital, dhaka, bangladesh. detailed clinical, electrophysiological, serologic and microbiological data were obtained. gbs affected predominantly in young adults males (m/f= : ) living in rural areas. antecedent events were recorded in > % of patients; frequent events being gastroenteritis (> %) and upper respiratory tract infection ( %). more than % of the patients were bed-bound (gbs disability score ) at entry and about % patients required mechanical ventilator. about % patients did not receive specific treatment either intravenous immunoglobulin (ivig) or plasmapheresis due to high expensive treatment cost. % patients had died during hospitalization. % of patients had an axonal variant of gbs and evidence for a recent c. jejuni infection ( %). c. jejuni infection was significantly associated with serum antibodies to the gangliosides gm and gd a, axonal neuropathy, and greater disability. in conclusion, the majority of the patients do not receive standard treatment with ivig in view of its high price. therefore, we developed low-cost treatment strategies and conducted a safety and feasibility trials for small volume plasma exchange (svpe) on gbs patients in bangladesh. in future, it is essential to conduct a phase ii clinical trial to assess the efficacy of svpe for low-in-come countries. prognosis of guillain-barré syndrome (gbs) has not improved in last two decades. current therapies (intravenous immunoglobulin, ivig and plasma exchange, pe) had been proved to be effective on two third of patients in developed world. unpredictable and poorly understood clinical course of gbs hamper treatment development. in bangladesh, most patients affected by gbs cannot afford specific treatments with ivig or pe instead most of them receive only supportive care. therefore, we aimed to compare the outcome of ivig treated patients with supportive care patients in improvement of gbs disability score and mrc sum score by using world's largest gbs cohort in bangladesh. we conducted a prospective observational study enrolling gbs patients between and from dhaka medical college hospital and national institute of neuroscience and hospital, dhaka, bangladesh. only gbs patients ( %) received standard ivig treatment. in current analysis, ivig treated patients and age, sex and severity matched controls (supportive care only) were considered. outcome of both groups were compared using fisher's exact or chi square test and survival analysis were performed by kaplan meier method using log rank test. among patients (cases and controls), male/female ( / ), median age years, % patients were bed-bound, one-fourth patients required mechanical ventilation and % were axonal. we did not found any significant differences of treatment outcome in both cases and control groups in gbs disability score (week : p= . , months: p= . ) and mrc sum score (week : p= . , months: p= . ). survival analysis revealed, the differences of time required for independent locomotion, improvement of one gbs disability score and improvement of mrc score were not statistically significant between treatments (ivig) and supportive care patients. in conclusion, our analysis showed that standard dose of ivig use has no considerable advantage to improve specific outcome measures among gbs patients in bangladesh. as the phenotype of gbs in bangladesh is different from developed world; therefore, an efficacy trial for ivig is needed for developing countries like bangladesh or new targeted therapeutic strategies can append beneficial effects for gbs patients. isose s , , watanabe k , omori s , sekiguchi y , beppu m , shibuya k , amino h , suichi t , misawa s , kuwabara s . graduate school of medicine, chiba university, chiba, japan; national hospital organization chiba east national hospital, chiba, japan. diabetic neuropathy is a frequent cause of neuropathic pain, suggesting the small-fiber involvement. additionally, persistent peripheral pain-related inputs could cause neuronal hyperexcitability and complex interactions of the nociceptive pathways, i.e., central sensitization. to investigate the pathophysiology of neuropathic pain in diabetic neuropathy, we studied pain-related evoked potentials (preps) after selective intraepidermal electrical stimulation (ies) to a-deltaand c-fibers in diabetes patients with neuropathic pain (n= ) and without neuropathic pain (n= ). we also conducted a longitudinal study to assess changes in preps and pain profiles in patients with neuropathic pain months after the start of treatment with duloxetine. this study is registered with the umin clinical trials registry, umin . ies was applied in the hand and foot, and preps were recorded from the cz electrode referenced to the linked earlobes. we evaluated prep latencies, amplitudes, and amplitude ratios of preps after c/a-delta -fiber stimulation. in the conventional nerve conduction studies, patients with neuropathic pain significantly showed conduction slowing and decreased snap amplitudes in the median and sural nerves compared with those in patients without neuropathic pain. in pain-related sep studies, there were no significant differences in prep amplitudes and latencies after a-delta -or cfiber stimulation between the patients with neuropathic pain and without it. prep amplitude ratios after c/a-delta -fiber stimulation tended to increase in patients with neuropathic pain compared to patients without pain. after the treatment with duloxetine, c/a-delta -prep amplitude ratios were significantly decreased after both hand and foot stimulation, and as for numerical rating scale (nrs) scores as the intensity of pain. patients with less pain relief showed the tendency of higher c/a-delta prep amplitude ratios before treatment compared to patients with better pain relief. the correlation between reduction of c/a-delta prep amplitude ratios and nrs reduction did not reach statistical significance. this pain-related sep study demonstrated that abnormal cortical response in patients with neuropathic pain could improve after the treatment with duloxetine, this might reflect the cortical hyperexcitability as a central sensitization. this study is funded by shionogi & co., ltd. the sponsors played no role in the design and management of the study, collection and analysis of data, interpretation of the results, or the writing of the writing of the report. peripheral nerve injury is commonly associated with traumatic injury which is often amenable to surgery. despite improved methods in surgical repair, functional recovery remains a challenging clinical problem that often leads to significant morbidity in patients. alternative therapies that could augment surgical repair may be beneficial in functional outcomes. neuroinflammation is a complex pathway with different cellular components and cytokines that are activated following peripheral nerve injury. macrophages are responsible for the breakdown of debris following injury as well as promotion of regenerative signals. macrophage polarization is the process by which macrophages take on phenotypically distinct functions based on the local environment and signaling cues. exercise has been shown to drive macrophage polarization from a pro-inflammatory m phenotype towards an anti-inflammatory m phenotype in numerous tissues, but remains uninvestigated in the peripheral nervous system. the purpose of our study was to identify how exercise affects macrophage polarization, motor and sensory function, and neuroregeneration following sciatic nerve crush. c bl/ mice underwent sciatic nerve crush injury and were then given access to running wheels (exercised) or not given access to running wheels (sedentary) for weeks. exercised mice ran an average of . km per night. injured exercised mice were protected from the development of thermal hyperalgesia when compared to injured sedentary mice. exercised mice had fewer paw slips on beam walk testing compared to sedentary mice. no differences were measured in mechanical sensitivity or motor coordination and balance assessed by rotarod. while motor nerve conduction velocities were significantly reduced for injured mice compared to uninjured controls, motor nerve conduction velocities from injured exercised animals were significantly higher than injured sedentary animals suggesting improved nerve recovery with exercise. injured sciatic nerves from exercised mice demonstrated increased m macrophages compared to sciatic nerves from injured sedentary mice. the behavioral changes and altered macrophage polarization correlated with increased epidermal nerve fiber density, improved myelination, and increased in vitro neurite outgrowth from injured exercised animals. therefore, exercise alters macrophage polarization towards an anti-inflammatory phenotype which improves repair and recovery of the injured peripheral nerve. jack mm , shah k , everist b , reyna j , hylton p . university of kansas medical center, kansas city, ks, usa. diffusion tensor imaging (dti) has long been used to evaluate the location and integrity of white matter tracts in the brain. dti uses quantitative data and directionality of water diffusion to determine axonal connectivity of the nervous system. the technology has only recently been utilized in limited settings in the peripheral nervous system due to challenging technical factors and lack of widespread availability. magnetic resonance (mr) neurography or peripheral neurography is a technique which uses diffusion to differentiate between intraneural and perineural tissues. it allows for fascicle patterns to be visualized particularly in the setting of peripheral nerve sheath tumors. peripheral nerve sheath tumors of various pathologies cause surrounding nerves to be involved or displaced in a range of directions. this technique helps determine the anatomic location of these nerve fibers in relation to the mass, which is particularly helpful at distinguishing neuromas from schwannomas. this data is invaluable to the surgeon to ensure a safe and low morbidity operation. while this technology has benefit particularly with surgical planning, it has been underutilized due to the challenges of requiring complex software to produce fiber tracks and the inability to translate these images into the operating room. here, we utilized brainlab software that is commonly available and utilized in surgical suites to produce images of the radial nerve fiber tracts with an associated peripheral nerve sheath tumor prior to surgical resection. while the software is commonly used in the central nervous system, it has not been reported to have been used in the peripheral nervous system. this software offers a high usability and produces anatomically correct and reliable fiber tracts. this technique overcomes the reliance on highly specialized software and extensive training required for use that most other tractography software has. utilizing peripheral neurography in this case allowed for complete surgical resection without postoperative deficits. this data offers clinicians an option to investigate peripheral nerve fibers in various pathologic states, to plan appropriate operative trajectories to peripheral nerve pathology, and to improve surgical outcomes for patients with peripheral nerve sheath tumors. jacobsen b , parry g , allen j , walk d , muley s , ortega e . barrow neurological institute, phoenix, az, usa; university of minnesota, minneapolis, mn, usa. chronic inflammatory demyelinating polyneuropathy (cidp) is an immune mediated neuropathy that is responsive to immunomodulatory agents such as glucocorticoids, intravenous immunoglobulin (ivig) and plasma exchange (pe). the specific immunopathogenic mechanisms of cidp remain unclear but there is increasing interest in nodal proteins as a site of the immune attack. even though the majority of patients respond to one of the aforementioned immunomodulatory agents there are some who are unresponsive or incompletely responsive to these first line agents and other more aggressive treatments may be necessary. cyclophosphamide and stem cell transplantation may be effective but are associated with considerable morbidity. anecdotal reports suggest that rituximab may be beneficial for some patients that fail first-line therapy, especially if they have antibodies to nodal proteins. we present four patients with intractable cidp who responded to rituximab. one of the three patients had diabetes. disease duration prior to starting rituxan was short ( months) in two patients and longer and months in the other two. all patients had failed treatment with glucocorticoids and ivig, and in two, plasma exchange and ivig-pe were also ineffective. two of the four patients were quadriparetic and non-ambulatory. two gm doses two weeks apart of intravenous rituximab were instituted in all patients. all patient tolerated the treatments well without adverse effects. all patients responded within four weeks and continued to improve at six months. other immunomodulatory agents were successfully tapered but not totally discontinued. it remains unclear whether antibodies to nodal proteins were present in these patients. in conclusion, although rituximab efficacy remains uncertain on the basis of randomized controlled clinical trials, it may be beneficial in selected patients otherwise intractable to first-line treatments. further studies are necessary to better understand which patients may benefit most from rituximab and where in the treatment algorithm rituximab should be applied. neuropathic pain is a chronic condition seen in patients suffering a direct injury to the peripheral or central nervous system or an indirect injury due to, e.g., diabetes or multiple sclerosis. current treatment options fall short of preventing or completely relieving patients of their pain. for years, research has focused on understanding the role of neurons in neuropathic pain pathogenesis while overlooking the role of supportive cells in general and satellite glial cells (sgcs) in the dorsal root ganglion in particular. these cells not only buffer the neuronal microenvironment they are also involved in controlling the electrical activity flowing through the neurons and in neuropathic pain pathogenesis. the aim of this project is to understand the role of sgcs in neuropathic pain development and thereby aid the identification of new drug targets. to purify the sgcs from adult mice we optimized a fluorescently activated cell sorting (facs) protocol. the success of our purification method was confirmed using qrt-pcr and visual inspection of the sorted cells. finally, we are running rna sequencing on sgcs after peripheral nerve injury to compare their transcriptome to that of uninjured cells at different time points. the results from our study are likely to deepen our understanding of how sgcs contribute to the development and maintenance of neuropathic pain. guillain-barrésyndrome (gbs) is an immunemediated disorder in the peripheral nervous system (pns) triggered by molecular mimicry against nerve gangliosides. one of the cell surface receptors (fas)-ligand (fasl) interaction transmits apoptotic signal to eliminate the auto-reactive b and t-cells, which generates cross-reactive antibody against nerve cells. host genetic polymorphism of fas and fasl may alter their expression and induce aberrant apoptotic response to develop gbs. therefore, we determined the single nucleotide polymorphisms (snps) of both fas receptor (− g/a and - a/g) and fasl ligand (− c/t) in gbs patients (n= ) as well as healthy controls (n= ) using the lightcycler technique. serum level of soluble form of fas and fasl was measured using commercially available sandwich elisa kit. comparison of genotype, allele and haplotype frequencies was done with the gbs subgroups based on the clinical and serological data. ag heterozygote (p= . , or= . , % ci= . - . ) and polymorphic g-allele (p= . , or= . , % ci= . - . ) of fas receptor - a/g promoter snps were significantly associated with anti-ganglioside (gm ) antibody positive gbs patients. in addition, − g-allele (p= . , or= . , % ci= . - . ) and - g/- g haplotype (p= . , or= . , % ci= . - . ) were predominantly associated with the axonal variant of gbs patients. serum soluble form of sfas (median levels pg/ml vs. pg/ml, p= . ) and sfasl (median levels pg/ml vs. pg/ml, p= . ) were found to be elevated in anti-gm antibody positive gbs patients compared to anti-gm negative patients. no significant association was found in genotypic distribution between gbs patients and healthy controls. in conclusion, fas/fasl promoter snps are not a susceptible factor for gbs but could be one of the influencing factors to develop cross-reactive anti-ganglioside antibodies in gbs patients in bangladesh. furthermore, functional studies with a larger sample size (using cohort like international gbs outcome studies-igos) are required to explain the immune pathogenic role of these snps for gbs patients. jang sy , yoon ba , shin yk , yun sh , jo yr , park ji , shin kj , kim jk , park ht . dong-a university, busan, south korea; inje university, busan, south korea. myelination is essential for the proper function of the nervous system. schwann cells, which form the peripheral myelin sheath, have the unique ability to dedifferentiate and to destroy the myelin sheath under various demyelination conditions. during schwann cell dedifferentiation-associated demyelination in wallerian degeneration after axonal injury, schwann cells exhibit myelin and junctional instability, down-regulation of myelin gene expression and autophagic myelin decomposition. however, in inflammatory demyelinating neuropathy, it is still unclear how schwann cells react and contribute to segmental demyelination before myelin scavengers, macrophages, are activated for myelin clearance. here, we show that schwann cell dedifferentiation-associated demyelination is a mechanism involved in the initial demyelination observed in a mouse model of inflammatory demyelinating neuropathy using ultrastructural, biochemical and microarray analyses. myelin uncompaction and myelin membrane instability generated by dedifferentiated schwann cells lead to autophagolysosome-dependent cytoplasmic amputation between the axon-containing myelin sheath and the schwann cell body, resulting in the formation of the "myelin corpse", thereby allowing macrophages to phagocytose the myelin corpse in the end stage of segmental demyelination. we found myelin corpse formation in inflammatory demyelination to be a process similar to the myelin rejection during wallerian degeneration, which appeared to be dependent on schwann cell autophagolysosome activation since schwann cell-specific atg knockout mice exhibited delayed myelin rejection following nerve injury. finally, lysosome inhibition in schwann cells not only prevented segmental demyelination but also delayed the progression of clinical stages by suppressing the myelin corpse formation in inflammatory demyelinating neuropathy. thus, our findings indicate that demyelination by schwann cells and macrophages might be part of a process that includes sequential divisions of labor with respect to myelin rejection and digestion, respectively. in conjunction with previous studies showing schwann cell dedifferentiation and autophagy in toxic and hereditary neuropathies, the concept of "schwann cell dedifferentiation-associated demyelination" provides insight into the development of possible therapeutic strategies to prevent schwann cell demyelination in peripheral demyelinating neuropathies. methods: electrophysiological data, the charcot marie tooth exam score (cmtes), and bmi from patients with known cmt a were obtained and analyzed. results: when controlled for age, bmi does not affect studies of ulnar motor nerve conduction in ctm a patients, but rather specific components of the cmt exam scores (cmtes, loss of pinprick sensation and motor strength in the lower extremities). discussion: bmi and clinical components of the cmtes are correlated, but it is uncertain which is the primary effect -i.e., whether the reductions in pinprick sensation and motor strength in the lower extremities lead to a higher bmi, or higher bmi results in these signs. introduction: charcot-marie-tooth disease type c (cmt c) is a rare, dominantly inherited neuropathy caused by mutations in the lipopolysaccharide-induced tumor necrosis factor (litaf) or small integral membrane protein of the lysosome/late endosome (simple) gene. methods: we present a case series comprised of patients in whom cmt c is caused by a gly ser substitution in the encoded protein. we focus on clinical presentation, electrodiagnostic analyses, and our findings in the context of previously described cases. results: the gly ser mutation causing cmt c is a mild form of cmt, as patients walked on time, had less weakness than those with charcot-marie-tooth disease type a (cmt a), had a charcot marie tooth neuropathy score (cmtns) indicative of mild disease, and had faster ulnar and median motor nerve conduction velocities compared to those with cmt a. discussion: the g s mutation in litaf seems to be clinically indistinguishable from a mild presentation of cmt a. jha mk , russell k , lee y , rothstein jd , morrison bm . johns hopkins university, baltimore, md, usa. peripheral nerves are highly dependent on metabolic energy to maintain both basic cellular functions such as axon transport, na+/k+ ion gradients, and myelination, as well as to support regeneration following injury. though glucose certainly provides some metabolic support, our recent studies have shown that monocarboxylates, such as lactate, pyruvate, and ketone bodies, also contribute to recovery from peripheral nerve injury. monocarboxylate transporters, particularly mct , are the predominate transporters for monocarboxylates in the peripheral nerve. in a recent publication, we found that mct heterozygous null mice, which express % less mct in all cells, have slowed nerve regeneration and reduced myelination following sciatic nerve crush. this study was limited by the global reduction of mct , which is widely expressed in schwann cells (sc), dorsal root ganglia (drg) neurons, endothelial cells, macrophages, and perineurial cells within the regenerating peripheral nerve. to better understand the mechanism by which mct contributes to normal nerve function and nerve regeneration, we produced and validated conditional mct null (mct loxp) mice that allow selective deletion of mct from scs, drg neurons, endothelial cells, or macrophages through mating to cell-specific cre lines. we are currently quantifying peripheral nerve development, aging, and regeneration in each of these mouse lines. following sc-, but not drg-, specific mct knockdown, sensory peripheral nerves develop demyelination by months of age, manifest by reduced myelin thickness, increased g-ratio, and reduced conduction velocity. studies are ongoing in cultured scs to determine the mechanism for demyelination. neither sc nor drg knockdown of mct impairs nerve regeneration following sciatic nerve crush. these results suggest that sc-specific mct is critical for maintaining myelin in sensory, but not motor, peripheral nerves as they age. they also suggest that mct expression in peripheral nerve cell types, other than sc and drg, is important for nerve regeneration. ongoing studies are determining the contribution of mct in other peripheral nerve cell types, particularly endothelial cells and macrophages, to normal development and regeneration following injury. our results will clarify the role of lactate and its transporter, mct , in peripheral nerve function, potentially suggesting novel targets for demyelinating neuropathies or nerve injuries. small fiber neuropathy (sfn) is a condition in which the smallest nerve fibers are affected, characterized by neuropathic pain and autonomic dysfunction. according to international criteria, sfn diagnosis is based on clinical symptoms in combination with abnormal temperature threshold testing (ttt) and/or reduced intraepidermal nerve fiber density (ienfd) in skin biopsy. skin biopsy is moderately sensitive, invasive and the process is time consuming and expensive. ttt is a widely available diagnostic tool, but lacks specificity. previous studies introduced stimulated skin wrinkling (ssw) as an objective, non-invasive diagnostic tool to detect sympathetic nerve dysfunction in sfn by means of a categorical assessment. however, our unpublished data has shown that inter-observer reliability of categorically assessed ssw is quite low. in this current study we will use a new digital method for ssw quantification: the digit wrinkle scan© (dws©). the primary study objective is to define normative values for dws© expressed as total wrinkle length per fingertip surface (mm/mm ). subsequently we investigate the applicability of dws© in patients with definite sfn, based on abnormal iefnd and/or ttt, determining the dws© sensitivity and specificity, as well as its validity. for this cross-sectional study, we will include healthy participants and patients diagnosed with sfn. eligibility is based on meeting the inclusion and exclusion criteria and providing written informed consent. skin wrinkling is induced by emla (eutectic mixture of local anesthetics) cream© application and captured by taking pictures. the primary outcome measure is total length of wrinkles per mm as shown on the photographs, which will be calculated by a new software program. patients are stratified according to age and gender. based on the results of healthy participants, normative values will be defined. inter-and intra-observer reliability will be determined. in the sfn group, additional correlation analysis will be conducted to determine the correlation between dws© and different outcome measures (sfn-symptom inventory questionnaire, visual analogue pain scale, neuropathic pain scale, sfn-rasch-built overall disability scale, ienfd and ttt). we expect to provide digitally quantified ssw (dssw©) normative values that can be used in clinical practice in the diagnostic workup for sfn. cmt , characterized by axonal degeneration, is an inherited motor and sensory neuropathy accounting for about % of total cmt patients. the cmt subtype shows on its own, a vast genetic heterogeneity with more than mutations in known genes rendering the identification of relevant drug targets and therapies very challenging. so far, only hdac inhibitors have shown promising results in mouse models for hspb mediated axonal cmt (cmt f), albeit such a single gene approach may have a limited relevance at clinical levels owing to the limited number of patients per genotype. in this study, we investigated common causative molecular players of cmt associated axonal degeneration. for this, an itraq based proteome analysis was performed on five patient's derived lymphoblasts bearing different cmt causal mutations alongwith respective age and gender matched unaffected controls. software-assisted interpretations of the obtained data led us to identify two proteins which were significantly downregulated in cmt patients compared to controls. these two proteins were then validated using western blotting and qpcr on patient derived lymphoblasts and fibroblasts. our results prompted us to unveil whether these two proteins can be used as potential biomarkers for identifying cmt patients. therefore, through a europe-wide collaboration, we constructed a cohort of cmt patients and healthy controls. these two proteins exhibited significant downregulation in this cohort suggesting a potential new role of these proteins as cmt biomarkers. remarkably, we were also able to validate the significant decrease in ineurons (neurons differentiated from patient derived ipscs) strengthening the importance of our finding and also suggesting the relevance of these proteins in the pathogensis of axonal cmt. this will be the first study involving multiple cmt causal genes at once, thereby holding the potential to offer new drug targets and potential biomarkers with wider application both clinically and pharmaceutically. mesenchymal stem cells (mscs) represents a valuable source of stem cell therapy, can differentiate into various cell types. we investigated of the neuromuscular potential of human tonsil-derived mscs (t-mscs) for neuromuscular regeneration in trembler-j mice that is considered to be a model for charcot-marie-tooth disease type a (cmt a diseases), which is involving hereditary motor and sensory peripheral neuropathies. the t-mscs differentiated toward skeletal myocytes, as evidenced by increased expression of skeletal muscle-related markers (including troponin i type , and myogenin) and the formation of myotubes in vitro. in situ transplantation of t-msc-derived myocytes (t-myocytes) into gastrocnemius in trembler-j mice, a mouse model of cmt a, enhanced motor function, as identified with recovery by a compound muscle action potential (cmap) amplitude. and the regenerated shape of the sciatic nerve and skeletal muscle by immunochemistry, without the formation of teratomas. furthermore, the expression levels of nerve growth factor (ngf) and glial cell line-derived neurotrophic factor (gdnf) were significantly increased in t-myocyte compared with t-mscs in vitro. these results indicate that the transplantation of t-myocyte can be a therapeutic option of cell therapy for the neuromuscular regeneration in hereditary peripheral neuropathy, comprising cmt a disease. kagiava a , karaiskos c , richter j , tryfonos c , lapathitis g , sargiannidou i , christodoulou c , kleopa ka , . neuroscience laboratory, the cyprus institute of neurology and genetics, nicosia, cyprus; department of molecular virology, the cyprus institute of neurology and genetics, nicosia, cyprus; neurology clinics, cyprus school of molecular medicine, the cyprus institute of neurology and genetics, nicosia, cyprus. x-linked charcot-marie-tooth disease (cmt x) is a common form of inherited demyelinating peripheral neuropathy resulting from mutations affecting the gap junction protein connexin (cx ). using a cx knockout (ko) mouse model of the disease, we have shown that targeted expression of virally delivered cx results in morphological and functional improvement. since patients with cmt x express mutant forms of cx in schwann cells, that could potentially interact with virally delivered wild type (wt) cx through dominant-negative effects, we also treated mutant mice expressing the t i, r w and n d mutations associated with cmt x on a cx ko background. all three mutants were localized in the perinuclear compartment of myelinating schwann cells consistent with retention in the er (t i) or golgi (r w, n d) with loss of physiological expression in non-compact myelin areas. following intrathecal delivery of the human gjb gene we could detect the virally delivered wt cx correctly localized in the non-compact myelin areas only in t i/cx ko mutant mice, but not in the other two mutants, suggesting dominant effects of the r w and n d mutant but not of the t i mutant. gjb treated t i/cx ko mice showed improved motor performance, along with lower ratios of abnormally myelinated fibers and reduced numbers of inflammatory cells in all tissues examined compared to mock-treated animals. in contrast, gjb treated r w and n d mutant mice showed only slight but not statistically significant improvement. this study provides additional proof of principle for a clinically translatable gene therapy to treat cmt x even in the presence of endogenously expressed cx mutants, since at least one er-retained cx mutant did not interfere with the expression of virally delivered cx allowing a therapeutic benefit similar to cx ko mice. however, golgi-retained mutants may interfere with virally delivered wt cx and other approaches besides gene addition may be needed for effective treatment. funding: muscular dystrophy association (grant mda to kak). kaida k , kadoya m , koike h , iijima m , takazaki h , ogata h , moriguchi k , shimizu j , nagata e , takizawa s , chiba a , yamasaki r , kira j-i , sobue g , ikewaki k . national defense medical college, tokorozawa, japan; nagoya university graduate school of medicine, nagoya, japan; kyushu university, fukuoka, japan; university of tokyo, tokyo, japan; tokai university school of medicine, isehara, japan; kyorin university, tokyo, japan. antibodies to a glial protein, neurofascin (nf) have recently been identified in approximately % of patients with chronic inflammatory demyelinating polyneuropathy (cidp), which are igg -predominant. igg anti-nf -associated cidp may be a distinct subtype from typical cidp in terms of clinical features and response to immunotherapy. however, a diagnostic criterion of anti-nf -associated cidp has not established yet. to develop optimal criteria and design the best treatment plan for the anti-nf -associated cidp, procedures for determining anti-nf antibodies should be simplified, prevalent, and reproducible, as well as being accurate. cell-based assay (cba) has hitherto been utilized for determining antibodies to nf in sera from patients, results of which have been confirmed by immunohistochemistry (ihc) using teased nerve fibers from rodents. these methods are the most reliable techniques, while not necessarily easy-to-use and easy to maintain in most laboratories. in the present study, we aimed to validate the diagnostic utility of a conventional enzyme-linked immunosorbent assay (elisa) for determination of anti-nf antibodies and the igg subclass. sera from patients with efns/pns criteria-met cidp were examined with elisa using human recombinant nf . to verify elisa results, ihc on rat sciatic nerves, western blot (wb) and cba using nf -transfected and naive hek cells were conducted. the human nf -based elisa clearly distinguished between anti-nf antibody-positive and -negative sera. fifteen cidp patients ( %) were igg anti-nf antibody-positive, which were confirmed by wb, ihc and cba studies. none of disease controls or healthy subjects had positive results. twenty-five sera randomly selected from anti-nf -negative cidp sera were also negative on cba. the anti-nf activities on elisa were significantly positively-correlated with those on cba (p < . ). analyses of clinical and laboratory findings showed that anti-nf -associated cidp was characterized by younger onset, distal dominant phenotype, tremor, sensory ataxia, higher protein levels in cerebrospinal fluid, and poor response to ivig, which were consistent with those in previous studies. this elisa combined with determination of the igg subclass is a simple and reliable method for initial screening for anti-nf antibodies. the genetic abnormality responsible for x-linked charcot-marie-toothy neuropathy subtype cmtx was recently identified by whole genome sequencing to be a kb insertion into chromosome xq . . the clinical profile of cmtx in childhood is not well described. we reviewed the clinical characteristics, neurophysiological profile and cmt pediatric scale (cmtpeds) assessments of children with genetically confirmed cmtx . cmtx was characterized by early onset, and early and progressive hand weakness. most affected children were symptomatic within the first two years of life. the most common presentation was with equinovarus foot deformity in the first year of life. cmtpeds analysis in these children revealed that cmtx progressed more rapidly ( . ± . points/ year, n= ) than cmt a and cmtx . grip strength in the second decade of life in most affected males was two standard deviations below age-and sex-matched normative reference values. the most severely affected individual was wheelchair bound at years of age and two individuals had no movement in the small muscles of the hand in the second decade of life. there was only a single symptomatic female identified and she had mild signs. nerve conduction studies showed a demyelinating sensorimotor neuropathy with motor conduction velocity in eight children while one child had a length-dependent sensorimotor axonal neuropathy. understanding the unique phenotype of cmtx is essential for directing genetic testing, as the cmtx insertion will not be detected on the snp microarrays, multi-gene panels or whole-exome sequencing currently used for the diagnosis of cmt. the early onset of disease coupled with rapid progression means that many children with cmtx will have severe disability within the first two decades of life and hence early diagnosis is needed for early commencement of rehabilitation. kapoor m , catania s , sarri-gonzales s , lunn mp , manji h , reilly mm , carr as . centre for neuromuscular diseases, national hospital for neurology and neurosurgery, london, uk; department of neurophysiology, national hospital for neurology and neurosurgery, london, uk. the conventional dosing of ivig in cidp and mmn is based on treatment trials that used bolus and maintenance dosing of ivig between - . g/kg. there are rare published articles reporting the efficacy of higher maintenance ivig doses. we present three cases of inflammatory neuropathies, who are currently stabilized on ivig doses of mg- mg/kg of ivig per month, refractory to standard dose ivig and other immunosuppressants. the first case is a year-old-lady with cidp who presented with episodes of ascending sensory disturbance, weakness, and diplopia. she had activity related fluctuations and pre-dose deterioration on g/kg/month ivig. she then had an acute deterioration with mrc sum score dropping from to even with additional plasma exchange. her bilateral foot drop (mrc grade - ) and fluctuations persisted with an increase of ivig to . g/kg/month. she is now clinically stable (ankle dorsiflexion mrc grade - , mrc sum score ) on mycophenolate and g ivig weekly ( . g/kg/month). case is a -year-old male fitness instructor with mmn and sjogren's syndrome. he presented with recurrent proximal and distal weakness that responded to g/kg of ivig and deteriorated with iv methylprednisolone. he had peri-dose fluctuations, intermitted proximal weakness, and persistent foot drop (ankle dorsiflexion mrc grade - ) at . mg/kg/month, worsening to mrc grade - on . g/kg/month and fluctuating between mrc grade - on . g/kg/month. an increase of ivig to g weekly ( . g/kg/month), has resulted in mmn rods scores of / , improved distal power and return to full capacity at work. case is a -year-old man with predominantly upper limb cidp. he received g/kg ivig without any benefit, had no response to doses of plasma exchange, doses of cyclophosphamide or dose of rituximab between and . since , he has received g/kg/month ivig with improvement of mrc sum score from to . these cases highlight that some patients require a much higher than conventionally prescribed dose of ivig, and that these doses are tolerated over years without serious adverse events. idiopathic rapid eye movement sleep behaviour disorder (irbd) has been identified as a precursor of alpha-synucleinopathies, such as parkinson's disease, dementia with lewy bodies, multiple system atrophy. several studies linked changes in cutaneous innervation with central nervous system pathology in neurodegenerative disorders. recently small fiber neuropathy and alpha-synuclein deposition in the skin found to be a potential biomarker in parkinson's disease. we evaluated the epidermal innervation of irbd patients and age and sex-matched controls from skin punch biopsies from the distal leg using pgp . immunohistochemistry. furthermore, a battery of clinical examinations were performed on patients and controls alike, including structured interviews, clinical motor and non-motor questionnaires and rating scales (e.g. unified parkinson's disease rating scale [updrs], non-motor symptoms questionnaire [nms-quest] and beck depression inventory, epworth sleepiness scale, evaluation of cognitive and olfactory functioning as well as blood samples. irbd patients, compared to controls, showed a significant reduction in intraepidermal nerve fiber density (p = . ), whereas the axon swelling ratio, did not differ between groups. patients with irbd reported non-motor symptoms more frequently than controls (updrs i, nms-quest). olfaction and daytime sleepiness differed between both groups, whereas there were no differences regarding cognition. these in vivo findings demonstrate small fiber neuropathy in irbd patients that are associated with non-motor symptoms indicating that peripheral abnormalities may occur early in irbd. they warrant larger scale longitudinal studies in order to investigate their prognostic value. katz j , lewis r , spatafora d . california pacific med center, san francisco, usa; cedars-sinai medical center, los angeles, usa; neuropathy action foundation (naf), santa ana, usa. multifocal motor neuropathy (mmn) is a rare condition that affects . in every , individuals worldwide and is associated with motor dysfunction and moderate to severe disability. the neuropathy action foundation conducted a global survey to determine the impact of mmn on patient quality of life (qol) and gaps in patient/provider educational needs. the first global mmn qol survey was an item internet questionnaire available between january and july , . the survey focused on three primary areas: timely and accurate diagnosis, the efficacy of treatment, and the impact of the disease on patients qol. the survey was completed by patients from countries. the majority of respondents said they were diagnosed between the ages of and years ( . %), more than % reported that it took more than one year to be diagnosed and more than % reported that it took - years or longer to be accurately diagnosed. with respect to treatment options : . % reported receiving intravenous immune globulin and . % reported receiving subcutaneous immune globulin therapy. other therapies being used to treat mmn were gabapentin ( . %), and pregabalin ( %). almost half ( . %) said that mmn often impacts their overall schedule. half of the participants reported that mmn often or always interferes with their employment; % had difficulty typing on a computer or using a telephone, . % had trouble concentrating, and . % said they had to work really hard to pay attention or else they would make a mistake. this is the first assessment of mmn from a patient's perspective. the survey highlighted critical issues relating to the diagnosis, management, and impact on the qol of individuals with mmn. the data also identified gaps and insights in provider education relating to proper diagnoses and management of the condition from a patient's perspective. katz j , levine t , dimachke m , barohn r . forbes norris center, san francisco, ca, usa; phoenix neurological institute, phoenix, az, usa; kansas university medical center, kansas city, ks, usa. in the united states, cidp cases are submitted to insurance companies to determine whether ivig therapy is appropriate. this is done using specified diagnostic criteria, which reduce diagnosis to a boolean analysis, where a disease can only be present or absent. this leaves no room for uncertainty, even when it truly exists. boolean criteria are useful for clinical trials, but fall short where real decisions are made under uncertainty and based on perceived cost/benefit analysis. this project attempts to elucidate root causes of uncertainty and to find solutions to this dilemma. we asked cidp experts to select a single diagnosis in cases where ivig was approved using the submitted case records. while there was agreement on many cases, in the five most "uncertain" cases no more than reviewers agreed on a single condition, who chose up to four separate entities. among these, at least three reviewers diagnosed an immune neuropathy in all five. the root cause of the disagreement, to a large degree was unclear documentation (aunts) which consisted of pasted, missing, and disorganized data. reviewers missed useful information, admitting it was too difficult to fully parse records. to resolve uncertainty, reviewers admitted to discounting certain reported datum to help fit the entity they suspected, such as reported therapeutic responses or certain electrodiagnostic/exam findings. other disagreement, however, reflected the complexity of neuropathy diagnosis, such as knowing if improvement was due to natural history or treatment, unawareness of rare presentations, or analyzing a true uncle (complex case). reviewers used bayesian (select most likely diagnoses from a list) and fuzzy logic (compare best fits to base cases). when the "best" diagnosis did not fit the base case, they had to re-interpret the data. improving review procedures requires eliminating aunts by collecting all key information and simplifying how records are presented. it also needs more advanced data methods to analyze common and rare borderline presentations (uncles like mama v pma, cidp v cspn, etc..), developing diagnostic algorithms that address real uncertainty, educating prescribers and patients on process, and creating systems that measure outcomes longitudinally after induction or tapering of therapy. keisuke y , miyuki m , motoi k , susumu k . department of neurology, faculty of medicine, kinki university, osaka, japan. anti-ganglioside antibodies are closely associated with clinical phenotype and specific symptoms in acute immune-mediated neuropathies. igg anti-gq b antibodies are specifically associated with miller fisher syndrome (mfs), bickerstaff brainstem encephalitis (bbe) and guillain-barré syndrome (gbs) with opthalmoplegia (gbs-op). in addition, ganglioside complexes (gscs) containing gq b also can be targets in such diseases, and might be associated with the clinical features. however, factors regulating clinical phenotype in those gq b-associated antibodies-positive diseases have not yet been known. for investigating the differences of antibody reactivities among those diseases, we examined, using combinatorial glycoarray, igg antibodies to ten individual glycolipids [gm , gm , gm , gd a, gd b, gq b, galactocerebroside (gal-c), lactosylceramide (laccer), ga , sulfatide] and glycolipid complexes consisting of two of the glycolipids listed above in sera from patients with gbs-op who were positive for anti-gq b antibody by elisa (gbs-op-gq b), patients with mfs with the clinical triad (opthalmoplegia, ataxia, and areflexia), and patients with bbe. by combinatorial glycoarray, overall sensitivity of antibodies to gq b and gscs containing gq b was . % ( / ) in gbs-op-gq b, . % ( / ) in mfs, and . % ( / ) in bbe, respectively. there were no significant differences in antibody reactivities between mfs and bbe. it is notable that antibodies to gscs containing gd b were more frequently found in gbs-op-gq b patients than in mfs or bbe patients (e.g., gd b/sulfatide: p= . and p< . , respectively). presence of the antibody reactivities to gscs containing gd b may possibly be related with clinical features of gbs-op-gq b, including frequent need of artificial ventilation. kennedy r , , , carroll k , , paterson k , ryan mm , , , mcginley jl , . royal children's hospital, parkville, australia; university of melbourne, parkville, australia; murdoch childrens research institute, parkville, australia. problems with walking and footwear fit are often reported by children and adolescents with charcot-marie-tooth disease (cmt). a cross-sectional, case controlled study of gait was conducted in children with cmt and typically developing (td) children. gait was assessed barefoot and in two types of the participants' own typical footwear; optimal (e.g. athletic shoes) and suboptimal (e.g. slip-on footwear). the aims were to determine differences in spatio-temporal (s-t) gait variables between children with cmt and td children; and to investigate the effect of footwear choices. twenty-nine independently ambulant children aged - years with confirmed genetic or clinical diagnoses of cmt, and age and gender matched td children participated (mean age . years; males). exclusion criteria included developmental disorders, other neuromuscular conditions or musculo-skeletal diseases that could affect gait, and lower limb injury or surgery in the preceding months. assessment included s-t gait patterns, footwear characteristics, metre run, and cmtpeds. gait was assessed at self-selected speed with an electronic walkway (gaitrite™), with trials for each condition. the primary gait variable assessed was speed; other variables included step length, step time, cadence, base of support width and step-to-step gait variability. across all footwear conditions children with cmt walked more slowly ( regeneration of cutaneous unmyelinated axons is known to be slowed in dm-patients and after -months, the density of intraepidermal nerve fibers (ienf) does not return to baseline levels after chemical or mechanical axotomy. however, the long-term outcome of regeneration in dm or control subjects is not known. additionally, it is not clear if the regeneration of sensory distal axons is length-dependent. here we measured the rate of axonal regeneration -months after chemical denervation using a capsaicin model in dm patients (n= / dm /dm ) without neuropathy, and controls. dm skin punches were performed at distal thigh at baseline, -hours post-capsaicin, and at , , and days. blood glucose and hgba c were measured at baseline, , and days. healthy controls had skin punches at both distal leg and proximal thigh at baseline, after capsaicin chemical axotomy, and days , , and . regeneration rate was significantly higher at the thigh in healthy controls ( . fibers/mm/day ( % ci: . - . fibers/mm/day) compared to dm (p= . ), but no difference between dm ( . fibers/mm/day % ci: . - . fibers/mm/day) or dm ( . fibers/mm/day % ci: . - . fibers/mm/day) (p= . ). comparing regeneration rate at different time intervals, showed that regeneration was significantly slowed between day and dm patients, while it continued with the same rate in controls. blood glucose or hga c had no effect on regeneration rate. ienfd returned to baseline in controls by -months ( % of baseline) while it is did not in dm subjects, %/ % (dm /dm ) of ienfd baseline, (p= . dm vs. controls). there was no difference in regeneration rate ienfd %-baseline by -months at distal leg and proximal thigh in controls (p= . ). these results suggest that the rate and outcome of regeneration is independent of the length of the axon. additionally diabetic patient have incomplete nerve regeneration after months regardless of diabetes type or the level of glycemic control. regeneration of axons slowed down over time in patients with dm and reached a plateau after days. kiessling p , lledo-garcia r , watanabe s , langdon g , tran d , bari m , christodoulou l , price g , smith b , byrnes w , brock m , jolles s . ucb pharma, monheim, germany; ucb pharma, slough, uk; ucb pharma, braine-l' alleud, belgium; ptx solutions ltd, london, uk; ucb pharma, raleigh, durham, usa; university hospital of wales, cardiff, uk. ucb is a humanised high-affinity monoclonal igg antibody developed to bind human neonatal fc receptor (fcrn), selectively inhibiting igg salvage and recycling. conditions such as myasthenia gravis (mg) are characterised by pathogenic igg autoantibodies; inhibition of fcrn may provide a suitable therapeutic approach. this phase i, double-blind, dose-escalating, first-in-human study (nct ) evaluated the safety and pharmacology of ucb . forty-nine healthy adults (mean age years, range - ) were randomised and received a single dose of intravenous (iv) or subcutaneous (sc) placebo (n= and n= , respectively), or a single dose of iv or sc ucb ( , or mg/kg; n= per dose, per administration). subjects were followed up until day . one placebo iv subject did not complete the study. twenty-seven of subjects ( %) receiving ucb , and / ( %) receiving placebo, reported ≥ treatment-emergent adverse event (teae) of mild/moderate intensity. severe teaes occurred in four subjects, all in the ucb mg/kg iv group (headache [n= ], back pain [n= ]). no serious aes occurred. incidence of infections was similar with ucb and placebo. the most frequently reported infection was nasopharyngitis. treatment-related teaes were reported by % of subjects receiving ucb and % receiving placebo: the most common in the ucb -treated groups were headache ( / ; %) and vomiting ( / ; %); these occurred more frequently with the iv than sc route. non-linear increases in ucb plasma concentration-time profile with increasing dose were observed with ucb . serum igg was reduced in a dose-depended manner with ucb iv and sc: decreases from baseline to day with ucb iv were . %, . % and . % for , and mg/kg doses, respectively, and . %, . % and . %, with ucb sc doses, respectively. these data indicated that the fcrn inhibitor ucb effectively reduced serum igg, with sc administration generally better tolerated than iv. further to these observations, the efficacy, safety and tolerability of ucb sc for chronic-intermittent treatment of moderate-to-severe mg are being evaluated in an ongoing phase ii, multi-centre, randomised, double-blind, placebo-controlled study (eudract - - middle east respiratory syndrome (mers) has a high mortality rate and pandemic potential. however, very little information has become available on this syndrome since it first erupted in . this study aimed to evaluate the frequency of neurological complications and their clinical presentations in mers. we reviewed the medical records of all patients who were diagnosed with laboratory-confirmed mers coronavirus (cov) infections and subsequently admitted to a single reference center for mers treatment during the outbreak in korea. in total, patients ( . %) reported neurological symptoms during or after mers-cov infection. the potential diagnoses in these cases included bickerstaff's encephalitis overlapping with guillain-barré syndrome, critical illness polyneuropathy or other toxic or infectious neuropathies. neurological complications did not appear concomitantly with respiratory symptoms, but were instead delayed by - weeks. neuromuscular complications were not rare in mers-cov-infected patients, and they may have previously been underdiagnosed. understanding neurological manifestations is important in an infectious disease like mers, because evaluation is frequently limited during treatment, but it can interfere with prognosis and sometimes require modification of treatment. kim hj , hyun jk , kim tu . dankook university, cheonan, south korea. the diagnosis of carpal tunnel syndrome (cts) is based on clinical symptoms, physical examinations and supported by nerve conduction study (ncs). ultrasonographic examinations can be performed to assess peripheral nerves with less discomfort and the surrounding anatomic structures. while the usefulness of ultrasonography (usg) in the cts has been reported, no study to date has compared the diagnostic utility of various usg findings for cts. we investigated the correlation of various usg findings to the clinical symptoms/signs and ncs findings in patients with cts. twenty-eight hands ( patients) with cts based on electrodiagnostic criteria and clinical symptoms such as tingling sensation or pain in the first to third fingers, burning sensation, paresthesia and weakness of hand grip power. all subjects were examined with usg. cross-sectional area (csa) and flattening ratio (fr) of the median nerve was calculated at level of radio-ulnar joint, pisiform and hamate. swelling ratio of the median nerve and palmar displacement of the flexor retinaculum was also calculated. clinical assessment was conducted using the boston carpal tunnel questionnaire (bctq) scale and historical-objective (hi-ob) scale. the analysis of correlation between usg findings and clinical symptom scales/ncs findings was performed using correlation analysis. the csa of the median nerve at level of radio-ulnar joint was significant correlated with bctq scale, hi-ob scale, distal motor latency, and conduction velocity (cv). the csa of the median nerve at level of pisiform was significantly correlated with hi-ob scale, distal motor latency, and cv. the fr of the median nerve at level of radio-ulnar joint was significantly correlated with bctq scale, hi-ob scale, distal motor latency, and cv. the swelling ratio of the median nerve was also significantly correlated with distal motor latency and cv. in patients with cts, csa of the median nerve at level of radio-ulnar joint was most closely related to ncs findings and clinical symptoms. so, csa of the median nerve at radio-ulnar joint might be a complementary tool for the diagnosis of cts. kitaoji t , tsuji y , ashida s , yamada t , ishii r , tanaka a , mizuno t . department of neurology, graduate school of medical science, kyoto prefectural university of medicine, kyoto, japan. the case was a -year-old woman. first, she noticed paresthesia in the right plantar eight months before admission and in the left plantar four months before admission. three months before admission, she developed muscle weakness in her feet. the muscle weakness and paresthesia extended to the lower legs in a few months. twenty days before admission, she experience difficulty in walking. on admission, the muscle weakness was observed in the legs, especially in the right tibial anterior muscle (ta). there was severe sensory disturbance and loss of deep tendon reflex in the legs. she had trouble walking due to the weakness and sensory aphasia. in nerve conduction study (ncs), conduction block was observed between the ankle and popliteal in both tibial nerves. the blood level of angiotensin converting enzyme (ace) was elevated. cerebrospinal fluid analysis was normal. there was no enhancement in the lumbar nerve roots shown on mri. gallium- scintigraphy showed hot spots on bilateral hilar lymph nodes and mediastinal nodes and biopsy of mediastinal nodes showed non-caseating epithelioid granuloma. therefore, we diagnosed her with sarcoid peripheral neuropathy by sarcoidosis. by using the nerve ultrasound, partial spindle-shaped nerve enlargement was observed at the part of conduction block in the left tibial nerve. we started the treatment with methyl prednisolone ( mg, days) and oral prednisolone therapy ( mg/kg/day). after treatment, the paresthesia and muscle weakness in the legs had gradually improved. the partial enlargement in the left tibial nerve also improved on the -hospital day. in ncs, the conduction block improved, however, the compound muscle nerve potential of tibial nerve decreased because of axonal damage. this partial spindle-shaped nerve enlargement by using ultrasound has never been reported in sarcoid peripheral neuropathy before. the nerve ultrasound may be useful for evaluation of therapeutic effect of sarcoid peripheral neuropathy. peripheral nerve injuries are still debating problems in the world because of poor recovery. there is absolutely a need for new therapeutic agents to improve outcome by altering nerve regeneration. there are some studies in the literature about some therapeutic agents that used in the cases of peripheral nerve injuries. despite these studies, an agent with clinical use has not been presented yet. in this experimental study, we aimed to analyze the effects of curcumin (cur) in the cases of peripheral nerve injuries. forty rats were randomly and equally divided into four groups. the first group was control group. rats in this group were not operated. right sciatic nerve injuries were performed to the other groups. the second group was operation group with no therapeutic agent. the third group was operation and local cur applied group. the fourth group was operation and systemic cur applied group. electrophysiological evaluations were performed with electroneurography (enog) before and after the surgeries. systemic use of cur although caused improvement in the enog values but could not make a positive contribution to the nerve regeneration statistically. additionally local use of cur made negative effect to the nerve regeneration statistically. according to our statistical results we could not recommend cur as a nerve protective agent. klein cj , wu y , jentoft me , mer g , spinner rj , dyck pjb , dyck pj , mauermann ml . mayo clinic, rochester, usa. intraneural perineurioma is a hypertrophic peripheral nerve tumor having immunoreactivity to epithelial membrane-antigen, negative for s- . the origin of perineurial cells is debated to be similar to meningeal cells. ip does not metastasize, but motor deficits accumulate over time from tumor growth in nerve and plexus. after schwannomas and neurofibromas, perineuriomas are the most common nerve tumor of young adults. a chromosome q deletion has been reported in one patient. we identified ip cases from our previously published clinical cohort with available flash frozen ip tissue for dna isolation. wes with cnv analysis and cgh microarray analysis (agilent x k superprintg ) were performed on extracted dna; had available germline dna (lymphocytes and buccal tissue). we compared the exome data against online and in-house control data (∼ , ) examining variants less than . frequencies, predicted damaging or nonsynonymous. wes identified three novel, heterozygous, damaging mutations in tumor necrosis factor receptor-associated factor (traf ) in of ( %) cases; p.l p (n= ), p.h r (n= ) and p.s r (n= ). mutations were within the wd domain, p.l p, p.h r within exon and p.s r within exon , and mapped to a limited region of traf with protein structure modeling. two of cases ( . %) showed macroduplications/deletions on multiple chromosomes, including chromosome , confirmed with cgh microarray analysis and cnv results from exome data analysis. four of ( %) had no discovered mutation. age of onset or severity did not correlate with type of mutations. this study provides strong evidence that traf is a specific tumor driver of ip. mutations in traf are also linked to benign intracranial meningiomas suggesting a shared pathogenesis and close origins of perineurial and meningeal cells. study supported by: mayo foundation and the mayo center of individualized medicine. klein i , , bobylev i , , lehmann hc , . university hospital cologne, cologne, germany; center for molecular medicine cologne (cmmc), cologne, germany. peripheral neuropathy is a common side effect of paclitaxel. clinical evidence suggests that the delivery mechanism of paclitaxel formulations influence time course and severity of paclitaxel induced peripheral neuropathy. in a preclinical model we studied access, distribution and toxicity of two paclitaxel formulations (nanoparticle albumin-bound paclitaxel (nab) and solvent-based paclitaxel) in the peripheral nervous system (pns). c bl/ mice were treated with mg/kg or mg/kg of nab-paclitaxel or solvent based paclitaxel. kinetics of paclitaxel in neurons was assessed by a newly established immunostaining technique. neurotoxicity was evaluated by functional assays and nerve morphology. paclitaxel accumulated mostly in dorsal root ganglia, whereas distal nerve segments showed only low uptake of paclitaxel. treatment of mice with the two paclitaxel formulations resulted in paclitaxel uptake mostly in nf + larger fiber neurons. in ib +, and cgrp+ small fiber neurons, paclitaxel was less frequently detected. nab-paclitaxel was incorporated more rapidly compared to solvent-based paclitaxel but neurons also showed a faster clearance of nab-paclitaxel compared to solvent based paclitaxel. functional assays and nerve conduction studies indicated that nab-paclitaxel was less neurotoxic compared to solvent-based paclitaxel. this is the first study that characterizes in detail the access of nab-paclitaxel and solvent based paclitaxel into the pns. our findings have important implications to understand the pathomechanisms of paclitaxel induced neurotoxicity and to develop neuroprotective strategies by preventing access of paclitaxel to the pns. the aim of our study was to investigate the etiology of neuropathy in patients with rheumatoid arthritis (ra). subjects were neuropathy patients with ra admitted to our department. laboratory investigations, nerve conduction studies (ncs) and sural nerve biopsy were performed. mean patient age was . years (range, - years), and mean disease duration was . years (range, - years). clinical diagnosis for neuropathy was rheumatoid vasculitis (rv) in patients, rv with acute motor axonal neuropathy (rv-aman) in and chronic inflammatory demyelinating polyneuropathy with ra (ra-cidp) in . rheumatoid factor ( / ) and rheumatoid arthritis particle agglutination ( / ) was high and c tended to be lower in the rv group (rv and rv-aman). anti-ganglioside antibodies were examined in patients, with positive results in . an rv-aman case was diagnosed with motor-dominant clinical presentation and the presence of anti-galnac-gd a immunoglobulin (ig)g antibody. no other cases with rv were examined for anti-ganglioside antibodies. positive results for anti-gm and gm igg antibody were seen in one ra-cidp patient. we evaluated sural/median (s/m) ratio) for sensory nerve action potential (snap). s/m ratio was low in rv cases ( / ) and high ( / ) in immune-mediated cases, suggesting a so-called normal sural abnormal median pattern in immune-mediated neuropathies. the rv-aman case showed a moderate value in s/m ratio. nerve biopsy revealed thinly myelinated nerve fibers in ra-cidp cases compatible with demyelination, while the rv group showed the typical pathology for necrotizing vasculitis. rv cases were treated with prednisolone (psl), intravenous methylprednisolone, intravenous cyclophosphamide and increased psl dose. ra-cidp and rv-aman were treated with intravenous ig. in conclusion, neuropathy in ra can be divided into vasculitic and immune-mediated groups. ncs, and the s/m ratio of snap with some laboratory parameters in particular, may be of use in differential diagnosis and deciding treatment strategies. koh s , wong shj , loh kw , chng ysk , pawa c , ei ma , lee bjh , subramaniam t , umapathi t . national neuroscience institute, singapore, singapore; lee kong chian school of medicine, nanyang technological university, singapore; yong loo lin school of medicine, national university singapore, singapore; tan tock seng hospital, singapore; khoo teck puat hospital, singapore. treatment-induced neuropathy of diabetes mellitus (dm) (tind) is an acute painful autonomic neuropathy that develops with abrupt improvement in glycaemic control. typically, type or dm patients on insulin or oral hypoglycaemic agents (ohga), present with painful neuropathy and autonomic dysfunction within weeks of rapid improvement in glucose control. current emphasis to achieve good glycaemic control rapidly may inadvertently increase incidence of tind, hence the impetus to understand risk of over-zealous glycaemic control. we therefore set out to study the occurrence of tind in a dm cohort of a tertiary hospital. we screened all patients who had two hba c measurements between and . during this period, approximately patients were seen per year. we found patient-encounters that showed hba c decrease of ≥ % over months or ≥ % over months. we then used a structured checklist of tind symptoms to shortlist cases. these case-encounters were scrutinised and classified as; 'probable tind': acute painful neuropathy and acute dysautonomia with temporal relationship to the decrease in hba c; 'possible tind': acute painful neuropathy or acute dysautonomia or uncertain temporal relationship to decrease in hba c; unlikely tind: alternative explanation exists for symptoms. only one case was deemed 'probable tind'-a middle-aged man with newly diagnosed type dm who presented to emergency department with palpitations and worsening 'frozen feet' sensation that disturbed sleep. his hba c decreased by . % in weeks. his symptoms improved within a month with neuropathic pain medications and resolved months later. he also developed maculopathy and proliferative retinopathy. ten months later, he developed significant proteinuria. four other cases were classified as 'possible tind' while the remaining were unlikely tind. our study is limited by retrospective design and reliance on hospital records. nevertheless, our findings suggest that tind is uncommon in a general cohort of dm patients. on the other hand, the number of patients with painful neuropathy and acute dysautonomia symptoms contemporaneous with rapid decline in hba c raises the intriguing possibility that forme fruste of tind exists and one should interrogate the rate of hba c decline in dm patients with these symptoms. koike h , kadoya m , kaida k , ikeda s , kawagashira y , iijima m , kato d , ogata h , yamasaki r , matsukawa n , kira ji , katsuno m , sobue g . nagoya university graduate school of medicine, nagoya, japan; national defense medical college, tokorozawa, japan; nagoya city university graduate school of medical sciences, nagoya, japan; kyushu university, fukuoka, japan. we investigated the morphological features of chronic inflammatory demyelinating polyneuropathy (cidp) with autoantibodies directed against paranodal junctional molecules, particularly focusing on the fine structures of the paranodes. sural nerve biopsy specimens obtained from cidp patients with anti-neurofascin antibodies and patient with anti-contactin antibodies were assessed. these antibodies were examined using sera obtained from patients with cidp who fulfilled the criteria of the european federation of neurological societies/peripheral nerve society. thirteen cidp patients without these antibodies were also examined to compare pathological findings. characteristic light and electron microscopy findings in transverse sections from patients with anti-neurofascin and anti-contactin antibodies indicated a slight reduction in myelinated fiber density, with scattered myelin ovoids, and the absence of macrophage-mediated demyelination or onion bulbs. teased-fiber preparations revealed that segmental demyelination tended to be found in patients with relatively high frequencies of axonal degeneration and was tandemly found at consecutive nodes of ranvier in a single fiber. assessment of longitudinal sections by electron microscopy revealed that detachment of terminal myelin loops from the axolemma was frequently found at the paranode in both anti-neurofascin and anti-contactin antibody-positive cidp patients compared with antibody-negative cidp patients. patients with anti-neurofascin antibodies showed a positive correlation between the frequencies of axo-glial detachment at the paranode and axonal degeneration, as assessed by teased-fiber preparations (p < . ). in conclusion, paranodal dissection without classical macrophage-mediated demyelination is the characteristic feature of patients with cidp with autoantibodies to paranodal axo-glial junctional molecules. koike h , ikeda s , takahashi m , kawagashira y , iijima m , misumi y , ando y , ikeda si , katsuno m , sobue g . nagoya university graduate school of medicine, nagoya, japan; kumamoto university, kumamoto, japan; shinshu university hospital, matsumoto, japan. peripheral neuropathy is the cardinal feature of familial amyloid polyneuropathy (fap), but its mechanism has not been fully elucidated. we used electron microscopy to examine schwann cells and endoneurial microvessels. sural nerve biopsy specimens from fap patients with transthyretin val met mutation were assessed. patients were consisted of early onset cases from endemic foci and late onset cases from non-endemic areas. loss of nerve fibers with or without neighboring amyloid deposition was a common feature. the amount of amyloid deposition was greater relative to the extent of nerve fiber loss in early onset cases than in late onset cases. the atrophy of schwann cells, particularly nonmyelinating cells, that were apposed to amyloid fibrils was more conspicuous in early onset cases than in late onset cases. the numbers of endothelial cell nuclei, endothelial cell profiles, and occluded microvessels were significantly increased in the fap patients compared with patients with nutritional/alcoholic neuropathies (p < . , . , and . , respectively). findings suggestive of the disruption of blood-nerve barriers, such as the loss of tight junctions and the fenestration of endothelial cells, were also more frequently found in the fap patients (p < . ), irrespective of the presence or absence of amyloid deposition. in conclusion, these findings suggest that direct insult of amyloid fibrils causes schwann cell damage resulting in the predominant loss of small-fiber axons characteristic of early onset cases. in addition, vasculopathy may also participate in the pathogenesis of neuropathy, particularly in late onset cases. kolb n , smith ag , singleton jr , beck s , howard d , dittus k , karafiath s , mooney k . university of vermont, burlington, vt, usa; university of utah health, slc, ut, usa. chemotherapy induced peripheral neuropathy (cipn) is a major cause of morbidity due to numbness, pain, and gait instability. this prospective study compares the current standard care for cipn symptom management to a new care delivery model which utilizes an automated symptom tracking program paired with a nurse practitioner led intervention triggered by moderate to severe symptoms. all participants beginning taxane or platin based chemotherapy called a telephone based automated symptom tracking program daily (symptom care at home -sch) to report chemotherapy related numbness and tingling. sch tracked the presence and severity of neuropathic symptoms and their interference with activities of daily living (adls) on a - scale. participants were randomized to two groups. the usual care (uc) group was advised to call their oncology provider for recommendations on symptom management. in the nurse practitioner (np) group, when symptom severity was ≥ participants received automated self care strategies and a call from a nurse practitioner to provide treatment recommendations based on consensus guidelines. patients participated in the study. mean duration of follow up was . ± . days with . ± . calls. the np group had fewer days with any neuropathic symptom ( . % ± . vs. . % ± . , p= . ), with moderate to severe neuropathic symptoms ( . % ± . vs. . % ± . , p< . ) or days of distress from neuropathic symptoms ( . % ± . vs. . % ± . , p= . ). on days with moderate to severe symptoms participants also reported burning ( . ± . %), weakness ( . ± . %), balance problems, ( . ± . %), and tripping ( . ± . %). there was no significant difference between groups in the interference in adls (np . ± . vs. uc . ± . , p= . ). overall the automated telephone system effectively identified neuropathy symptoms and their severity. compared to usual care in which patients must independently reach out to their care team for symptom management, sch is effective in decreasing symptom prevalence, severity and distress. kouton l , kremer l , tard c , morales r , kuntzer t , attarian s , boucraut j , delmont e . referral centre for als and neuromuscular diseases, marseille, france; neurology department, strasbourg, france; neurology department, lille, france; neurology department, montpellier, france; neurology department, lausanne, switzerland; immunology laboratory, marseille, france. antibodies against proteins of the node of ranvier have been recently described in severe chronic inflammatory demyelinating polyradiculoneuropathies (cidp). they target paranodal proteins, namely contactin (cntn ) and neurofascin (nf ). cell-based assay and elisa are available in research, but no gold standard technic is admitted for the detection in routine of these antibodies. our objective was to evaluate if flow cytometry analysis is an efficient technic to detect antibodies against ctn and nf in a large cohort of cidp patients. flow cytometry analysis were performed on a bd facs-diva. human embryonic kidney (hek) cells were transfected either with nf or cntn . sera were diluted / . antibodies anti-cntn or nf were revealed using fitc conjugated anti human igg antibodies. delta mfi (mean fluorescence intensity) was calculated as mfi of transfected cells less mfi of non-transfected cells. measures were normalized using positive controls and negative controls from healthy blood donors. sera of cidp patients from different french neuromuscular referral centres were analysed with flow cytometry. respective delta mfi were (standard deviation ) and (standard deviation ) for antibodies against nf and cntn in cidp antibodies negative patients. antibodies against nf were found in patients (respective mfi , , ) and against cntn in two other patients (respective mfi and ). isotype of these antibodies was igg in patients and igg and igg in the remaining patient. all the patient had severe cidp. four patients had poor response to intravenous immunoglobulins (ivig) and have been treated with immunosuppressive drugs. as usually reported, the patient with anti-nf antibodies had postural tremor. flow cytometry seems effective to detect antibodies against nf and cntn . compared to other assays, benefits of flow cytometry are: to analyse a large number of sera in the same time and to give objective numerical results expressed in mfi that can be compared to the results of other samples. further studies are needed to confirm that flow cytometry can be the best test to assess antibodies against cntn and nf in routine. the mechanism by which intravenous immunoglobulins (ivig) improves peripheral nerve function in multifocal motor neuropathy (mmn) is unknown. the rapid clinical improvement following ivig could be related to blocking complement deposition on gm epitopes, change in ion-channel properties of affected motor axons, or both. the present study investigated median nerve motor excitability parameters at ∘ c just before ivig administration as well as at the peak of clinical improvement in patients with mmn. the investigated nerves were characterized either by conduction block (n= ), demyelinative slowing without block (n= ), or motor axon loss (n= ). the results of motor excitability testing in mmn showed no difference between pre-and post ivig recordings. clinical assessment of apb muscle showed increase in mrc score in patients and decrease in patient after ivig administration. in patients mrc score of the apb remained the same. those findings indicate that clinical changes following ivig administration are not related to excitability parameters of affected motor axons in mmn. where impulse conduction is blocked or markedly slowed in motor axons but is normal in sensory axons. sensory symptoms or signs are usually absent but have occasionally been reported in skin areas innervated by nerves with prominent motor axon loss. although the mechanism of selective motor involvement in mmn is unresolved, it may be related to differences in antigenic properties between motor and sensory axons or differences in biophysical properties. the objective of the present study was to compare ion-channel activity in both motor and sensory axons of nerves affected by mmn. affected nerves had to have motor conduction block, demyelinative slowing on motor ncs, or motor axon loss, whereas sensory ncs had to be normal. we performed excitability tests of motor and sensory axons in affected median nerves of mmn patients and healthy controls at ∘ c. conditioning and test stimuli were delivered at the median nerve at the wrist; cmaps were recorded from the thenar muscle and snaps from the rd digit. results of motor excitability testing in mmn showed fanning-out of threshold electrotonus, decreased i/v slope, and increased superexcitability, all compatible with persistent hyperpolarization of resting membrane potential in motor axons. sensory excitability testing in mmn showed decreased subexcitability but was otherwise normal. this may indicate minimal involvement of sensory axons in mmn. krarup c , , moldovan m , , alvarez s , ciano c , pisciotta c , pareyson d . university of copenhagen, copenhagen, denmark; rigshospitalet, copenhagen, denmark; fondazione irccs istituto neurologico c. besta, (incb), milan, italy. mutations in the gene coding for myelin protein zero (mpz, p ) are associated with different forms of charcot-marie-tooth (cmt) disease. we describe a family harboring a frameshift mutation (c. dela / p.asp thrfster ) in the p gene, predicted to result in a nonfunctional p truncated very early in the extracellular domain. this offered the rare opportunity to assess the consequences p deficiency in absence of the potential gain-of-function effects of the mutations itself. conventional conduction studies and multiple measures of nerve excitability by "threshold tracking" were carried out in heterozygote parents (aged and ) and their two homozygote sons (aged and ). in the homozygous patients, all distal limb cmaps and snaps were absent. for neurophysiological assessment, the spinal accessory nerve was stimulated at the neck and cmap was recorded over the upper trapezius muscle. eight normal subjects, mean age , were used as control. the two sibs showed a severe phenotype with early onset, severe scoliosis, complete loss of distal movements and relevant proximal weakness, cmt examination score (cmtes) - / ; both heterozygous parents had very mild adult-onset neuropathy with cmtes < / . control subjects had a trapezius cmap with a latency of . ms and an amplitude of . mv. heterozygotes had a mild cmt type b phenotype, with a cmap latency of . ms and an amplitude of . mv whereas the homozygotes had a severe neuropathy with a cmap latency of . ms and an amplitude of . mv. consistently, the homozygotes had a more severe impairment in excitability with a rheobase of . ma as compared to . ma in the heterozygote and . ma in controls. deviations in excitability measures were similar to our previous reports in p +/− and p −/− mice. mathematical modeling, indicated both altered passive cable properties due to dysmyelination and depolarizing features with increased na+ currents. our data suggest that p deficiency is associated with impaired axonal na+ channel function, arguing for the translational value of na+ channel blocker treatments as found in p null mouse models. krishnarajah s , divino v , mallick r , dekoven m . csl behring, king of prussia, pa, usa; quintilesims, fairfax, va, usa. chronic inflammatory demyelinating polyneuropathy (cidp) is a rare neurological disorder of the peripheral nervous system. the objectives of this retrospective real-world study were to compare demographic and clinical characteristics among cidp cases and matched controls and to assess cidp treatment utilization. adults newly diagnosed with cidp between / / and / / were identified in the quintilesims pharmetrics plus health plan claims database (first diagnosis date termed the index date). eligibility requirements were: confirmation of cidp (second cidp diagnosis or initiation of cidp therapy) within year of initial diagnosis, continuous health plan enrollment in the months prior to diagnosis (the pre-index) and the years following diagnosis (the follow-up), and no cidp diagnosis or use of cidp therapy in the pre-index. a total of , cidp cases met the study eligibility criteria. cases were direct-matched to controls based on age, gender, region, health plan, and payer type at index, and pre-index charlson comorbidity index score. the final sample consisted of cases matched to controls (both: mean [sd] age . [ . ]; . % male; . % commercially-insured). alternative pre-index diagnoses among cases included inherited neuropathies ( . %) and chronic acquired polyneuropathies ( . %). in the pre-index, neuropathic pain ( . % vs. . %), back pain ( . % vs. . %), and use of opioids ( . % vs. . %) and anti-convulsants ( . % vs. . %) were significantly higher among cases compared to controls (p< . for all). median total pre-index healthcare costs were . x higher for cases than controls ($ , vs. $ , , p< . ). over the follow-up, median total healthcare costs were . x higher for cases than controls ($ , vs. $ , , p< . [mean $ , and $ , ] ). cidp-related therapy costs accounted for . % of total healthcare costs for cases. the majority of cases ( . %) initiated cidp therapy over the follow-up, in a mean of . ( . ) days from initial diagnosis. half ( . %) of cases initiated treatment with corticosteroids only, while . % initiated ivig only. over the follow-up, . % of cases used any corticosteroid, while . % used any ivig. our findings suggest a substantial clinical and economic burden of cidp compared to matched controls. corticosteroids and ivig were most commonly used to treat cidp. kronlage m , baeumer p, pitarokoili k , schwarz d , schwehr v , godel t , heiland s , gold r , bendszus m , yoon ms . department of neuroradiology, heidelberg university hospital, germany; department of neurology, st. josef hospital, ruhr university of bochum, germany. objective: to evaluate large coverage magnetic resonance neurography (mrn) in chronic inflammatory demyelinating polyneuropathy (cidp). methods: in this prospective study patients with cidp and healthy controls were examined by a standardized mrn protocol at tesla. lumbosacral plexus was imaged by a t -weighted d-sequence ( mm isotropic voxel size); peripheral nerves of the upper and lower extremity by axial t -weighted turbo-spin-echo sequences ( . x . mm in-plane resolution). lesions were characterized by nerve cross sectional area (csa) and t -weighted signal (nt ). additionally, t -relaxometry of the sciatic nerve was performed using a multi-spin-echo sequence. all patients received a complementary electrophysiological exam. results: patients with cidp exhibited increased nerve csa and nt compared to controls (p < . ) in a proximally predominating pattern. roc analysis revealed best diagnostic accuracy for csa of the lumbosacral plexus (auc = . ) and nt of the sciatic nerve (auc = . ). csa correlated with multiple electrophysiological parameters of demyelinating neuropathy (f-wave latency, nerve conduction velocity) of sciatic and median nerve, while nt only correlated with f-wave latency of sciatic and not median nerve. t -relaxometry indicated that mr-signal increase in cidp was due to increase in proton-spin-density (p < . ), and not increase in t -relaxation time. conclusion: both nt and csa might aid in diagnosis of cidp, but csa correlates more robustly with electrophysiological parameters. since best diagnostic accuracy was shown for proximal nerve locations, mrn may be a useful complementary tool in select cidp cases. kühnemund j , wetzel c , bégay v , moshourab r , lewin gr . mdc & bih, berlin, germany; mdc, berlin, germany; charité, berlin, germany. damage of peripheral sensory nerves due to diabetes, herpes zoster infection, chemotherapy or trauma can cause chronic neuropathic pain. common symptoms include increased pain sensation (hyperalgesia), touch-induced pain (allodynia), paresthesia and spontaneous pain. we currently have poor understanding about the underlying molecular mechanisms at the peripheral level and treatment of patients suffering from neuropathic pain is inadequate. recent meta-analysis studies show that common first-line medications only yield nnts (numbers needed to treat) between . to . . it is still unclear to what extent allodynia can be attributed to changes in the physiological properties of intact sensory afferents. in this study, we aimed to elucidate whether changes occur in intact sensory afferents that innervate the plantar skin of the hind-paw following induction of neuropathic symptoms. this question was of particular interest considering that blocking mechanotransduction in the skin can alleviate mechanical hypersensitivity in neuropathic pain models (wetzel et al nature neuroscience ( ): - ). we used the chronic constriction injury (cci) model in mice which is behaviourally characterized by robust mechanical hypersensitivity. we made electrophysiological recordings from primary afferent neurons which had intact axons passing through the constriction to innervate the plantar skin using an ex-vivo skin-nerve preparation. we were able to record from myelinated afferent fibers and unmyelinated c-fibers with receptive fields in the control uninjured plantar skin as well as plantar skin of cci mice. we will present evidence that changes in the mechanosensitivity of sensory fibers innervating the glabrous skin may contribute to the symptoms of neuropathic pain. neuropathology, national hospital for neurology and neurosurgery, london, uk; irccs foundation "carlo besta" neurological institute, milan, italy; neurogenetics unit, national hospital for neurology and neurosurgery, london, uk; nuffield department of clinical neurosciences, oxford, uk; department of clinical neurophysiology, norfolk and norwich university hospital, uk. hsn secondary to sptlc / is a rare slowly progressive neuropathy resulting in marked sensory loss, especially nociception and significant motor deficit. despite most of the patients having the same c w mutation in sptlc , there is marked heterogeneity in the phenotype. l-serine oral supplementation has been suggested as potential therapeutic candidate however the lack of outcome measures is a major limiting factor in the initiation of a clinical trial. we undertook a natural history study to identify outcome measures that are responsive enough to be used in a clinical trial. the assessments used were cmt neuropathy score (cmtns version and cmtns version rasch modified), mri of calves and thighs, computerised myometry, quantitative sensory testing (qst), comprehensive neurophysiological assessment, proximal thigh skin biopsy for intra-epidermal nerve fibre density (ienfd), plasma dsl levels and patient based questionnaires (neuropathic pain symptom inventory and sf- v ). standardised response mean, srm (mean change/standard deviation of change) was used to compare responsiveness between tests. patients were recruited: with sptlc (c w) and with sptlc mutations. when analysed as a whole cohort, proximal calf mri fat fractions showed the most significant change over months. ienfd, plasma dsl levels, npsi and sf- v showed minimal change or the change was not in the clinically expected direction. for subsets of the remaining assessments which showed the highest responsiveness, the cohort was sub-divided into mild-moderate (cmtns ≤ ) and severe (cmtns> ) subgroups. in the mild-moderate subgroup, the greatest improvement in responsiveness was seen in computerised myometry (ankle plantarflexion: srm=− . and ankle eversion: srm=− . ). in the severe subgroup, qst (vibration detection thresholds on hands: srm=− . and face: srm=− . and pressure pain threshold on the face: srm= . ) and proximal calf mri fat fractions (srm range= . - . ) showed the greatest improvement. focusing on subgroups classified according to disease severity improved the responsiveness of some tests into the highly responsive range with mri still being the best outcome measure. this will reduce the number of participants required to power a clinical trial and might be a possible solution for designing a clinical trial for a rare, slowly progressive disease with a heterogeneous phenotype. kugathasan u , clark aj , suriyanarayanan s , laurá m , wilson e , , kalmar b , greensmith l , , hornemann t , reilly mm * , bennett dlh * . hsn secondary to sptlc / mutation is a slowly progressive sensory motor neuropathy leading to profound sensory loss with variable but often severe motor deficit. the genes sptlc and encode for the essential enzyme serine palmitoyltransferase (spt) which catalyses the rate limiting step in the sphingolipid de-novo biosynthesis. mutations in these two genes alter the substrate specificity of spt leading to the synthesis and accumulation of atypical metabolites called -deoxysphingolipids ( -deoxysl). plasma levels of -deoxysl are raised in hsn patients. deoxysphingolipids have been shown to be toxic in avian and by our group, in mammalian primary drg and motor neuron cultures. firstly, this study looked at the effects of -deoxysl on the survival and neurite integrity of human ipsc derived sensory neurons following exposure to different concentrations of deoxysphingolipids. later, we determined if there was autonomous -deoxysl production in hsn patient ipsc derived sensory neurons. sensory neurons were differentiated from human ipscs using a combination of small molecular inhibitors. deoxysphingolipids were found to be neurotoxic in this model after hours of treatment. in control lines, there is a significant reduction in neuronal survival following treatment with both -deoxysphinganine ( -deoxysa) and -deoxymethylsphinganine ( -doxmethsa). a clear dose-dependent increase in the expression of the axonal injury marker, atf , is seen with both -deoxysphngoid bases. in both instances, -deoxysa is more neurotoxic than -doxmethsa, which is similar to the findings in avian and mammalian primary neuronal cultures. autonomous -deoxysl production is seen in ipsc derived neurons obtained from three different hsn patients with the levels being significantly greater than that seen in multiple control lines. this is the first study to demonstrate that human ipsc derived sensory neurons can be used as an in-vitro model for hsn , providing a great opportunity both to probe the pathomechanisms mediating deoxysphingolipid toxicity and to test potential therapeutic agents. recent studies have demonstrated an association between autoantibodies directed against antineurofascin- (anti-nfasc ) and a subpopulation of cidp patients characterized by sensory ataxia, tremor and poor response to ivig treatment. here we report on the clinical features of three patients who developed acute changes in their phenotype during the course of their neuropathy, a potential clue to recognize a neuropathy with predominantly humoral dysimmunity. case reports: our index patient had developed sensory changes over weeks, followed by irregular locomotion, and muscle weakness with general areflexia. a first run of ivig improved the patient in a week, but he relapsed within days. a second ivig course with prednisone again normalized deficits within days. ivig runs and rituximab were still necessary to treat a nd , and then a rd relapse before obtaining complete improvement. the course of the neuropathy was months. two other patients were encountered with more chronic courses but in whom periods of worsening or improvement suggested a relapsing-remitting neuropathy, either spontaneously or following immunomodulating treatments. in all three, extensive work-ups were negative, anti-nfasc igg were positive, and detailed repeat nerve conduction studies demonstrated fluctuating conduction blocks. discussion: our report underscores that in chronic cidp patients a relapsing-remitting course could be encountered as a key feature of anti-nfasc neuropathy. this not yet described characteristic course could be of value when deciding using rituximab instead of immunomodulating treatments. kusunoki s , morikawa m , kuwahara m , ueno r , samukawa m , hamada y . faculty of medicine, kindai university, osaka-sayama, japan. anti-glycolipid antibodies are often detected in sera from patients with autoimmune neuropathies, such as guillain-barré syndrome (gbs), chronic inflammatory demyelinating polyradiculoneuropathy (cidp), and multifocal motor neuropathy (mmn). not only individual glycolipid antigens but also mixtures of two different glycolipids (glycolipid complexes) are sometimes recognized by serum antibodies. to investigate antibody activities against large number of glycolipid complexes in serum samples from patients with gbs, mmn, and cidp, we examined igm and igg antibodies against glycolipids [gm , gm , gd a, gd b, gq b, galnac-gd a, lm , galactocerebroside (gal-c), asialo-gm (ga ), and sulfatide] and glycolipid complexes consisting of two different glycolipids listed above, by using combinatorial glycoarray. serum was obtained from patients with gbs, patients with cidp and patients with mmn, all in the acute or relapsing phase. serum was also obtained from healthy controls and patients with other neurological diseases. we investigated the relationships between the clinical features and presence of those antibodies. high titers of igg antibodies were detected almost exclusively in gbs patients. in contrast, igm antibodies were frequently present in mmn and gbs. among the anti-glycolipid complex antibodies in gbs, anti-gm /sulfatide, anti-ga /sulfatide, anti-gm /gd a, and anti-gq b/sulfatide igg antibodies were common ( , , , and patients, respectively). igg antibodies against antigens containing gm were significantly correlated with pure motor gbs (p < . ) and those against antigens containing gq b were significantly correlated with gbs with ophthalmoplegia (p < . ). in seven of the patients with anti-gq b/sulfatide complex antibodies, the antibodies were specific to the gq b/sulfatide complex rather than the individual gq b and suldfatide antigens. moreover, four patients did not have antibodies other than those to the anti-gq b/sulfatide complex. in patients with mmn, igm antibodies to antigens containing gm or galnac-gd a were present in % and . %, respectively. glycoarray is efficient for detecting antibodies against numerous glycolipid complexes in immune-mediated neuropathies. we need further investigations on other immune-mediated diseases using larger number of antigens. kuwabara s , misawa s , sekiguchi y , susumu kusunoki and the jet-gbs study group . department of neurology, chiba university, chiba, japan; department of neurology, kindai university, osaka, japan; japanese eculizumab trial for guillain-barré syndrome, chiba, japan. guillain-barré syndrome is a monophasic immune-mediated neuropathy, but a substantial number of patients with severe disease have poor recovery, even if treated with immunoglobulin. recent studies suggest that complement activation plays a pivotal role in gbs-associated axonal degeneration, and eculizumab is a monoclonal antibody that specifically binds to complement component and inhibits complement activation. jet-gbs is an investigator-led, phase , randomized, placebo-controlled trial conducted in hospitals, this trial aims to investigate the safety and efficacy of eculizumab for treatment of severe gbs. patients were randomly assigned ( : ) to treatment with immunoglobulin plus either eculizumab ( mg/day; n= ) or placebo (n= ) once weekly for weeks. the primary outcome measures are safety and efficacy (the proportion of subjects who regain their ability to walk independently at week ). the secondary outcome measures included the proportion of subjects who were able to walk independently at week , and other measures such as mrc sum scores, nerve conduction parameters. enrollment for the trial began in august , and follow-up of the last patient was completed in october . analyses will be made in april , and the results will be presented at this meeting. this trial is registered with clinicaltrials.gov identifier: nct , and funded by the japanese agency for medical research and development, and alexion pharmaceuticals inc. neurofascin , a paranodal protein in peripheral nerve, is a target antigen for autoantibodies in a subset of chronic inflammatory demyelinating polyneuropathy (cidp). anti-neurofascin antibody-positive cidp is characterized by onset at younger age, tremor, and refractoriness to ivig. we have treated four patients with anti-neurofascin antibody-positive cidp at kindai university hospital. anti-neurofascin antibody was detected by both elisa and cell-based assay in those patients. igg subclass was predominantly igg in all four cases. the median age of the patients at admission was . years [range: - years]. among the four patients, three had tremor, and two had severe cerebellar ataxia. cerebrospinal fluid protein levels were remarkably increased [median: . mg/dl, range: - mg/dl]. although ivig treatment was administered in all four patients, the responses were poor or partial. in contrast, plasma exchange (pe) was performed in all four patients and the clinical symptoms dramatically improved in two of them. corticosteroids were also effective in those two patients. sural nerve biopsy was performed in all four patients. although sensory nerve action potentials of the sural nerves from those patients were not evoked, the transverse semithin sections of sural nerves from three patients revealed only slight or mild loss of myelinated fibers. partial paranodal demyelination was observed in teased-nerve fibers from three patients. in addition, abnormal paranodal lesions such as loss of the transvers bands were observed by electron microscope in all four patients. those electron microscopic findings were not observed in control patients with anti-neurofascin antibody-negative cidp. anti-neurofascin igg antibody-positive cidp shows distinctive clinical and pathological features. labeyrie c , besson f , vandendries c , cauquil c , beaudonnet g , not a , durand e , adams d . neurologie adulte, chu bicêtre, le kremlin bicêtre, france; médecine nucléaire, chu bicêtre, le kremlin bicêtre, france; clinique bizet, paris, france; unité de neurophysiologie clinique, chu bicêtre, le kremlin bicêtre, france. study of the proximal portion of the peripheral nervous system (pns) is difficult because less accessible to electrophysiological exploration and biopsy. the indications of mri are increasing in proximal neuropathies to analyse morphology of the roots, plexus and proximal nerves: integrity of the nerve bundle, inflammation or infiltration of these structures. pet-computed tomography (pet-ct) can detect infiltration of the proximal segments of the pns, with hypermetabolism of roots, plexuses and large nerve trunks. pet-ct is also useful in detection of solid neoplasm which can infiltrates pns and primary nerve sheath tumors. however, spatial resolution of pet -ct scan is limited to explore the roots and plexuses for moderate hypermetabolisms. we believe that a mild hypermetabolism could be highlighted in inflammatory neuropathies or in mild tumor infiltrations. we performed a fusion of whole body pet (performed because of suspected neoplasia) and plexus mri (pet-mri) images in ten patients with a peripheral neuropathy for which we suspected proximal involvement. the pet-ct and mri were first read separately, then with merge of the images by a nuclear physician and a neuroradiologist. five out of ten patients presented with hypermetabolism of pns (hmpn+) on pet-mri: root (n= ), and/or dorsal root ganglia (n= ). median sul of lesions was . . among the hmpn+ patients: hypermetabolism was already apparent on pet-ct in cases, the merge invalidated abnormalities seen on pet-ct in patients and mri detected additional lesions, not visible on pet-ct, in one patient. all hmpn+ patients had hypersignal and or hypertrophy of pns on mri either diffuse (n= ), or multifocal (n= ). among hmpn-(no hypermetabolism) out of ( %) had abnormal mri ( multifocal and diffuse). gadolinium enhancement was found in all patients receiving gadolinium in the hmpn+ (n= ) and only in / patients in the hmpngroup. final diagnoses of hmpn+ patients were neurolymphoma in one ( %), and idiopathic cidp in the others ( %) with histological proof on nervous root biopsy in one of them. final diagnosis in hmpn-patients was cidp in out of and sequelae of neuropathy in relation to lymphoma without relapse for the last one. cidp was more disabling (onls≥ ) in hmpn+ than in hmpn-group % vs. %. pet-mri could be helpful to detect a proximal inflammation of the pns, and to plan further testing. further studies are needed to evaluate the prognostic value of hmpn+. lancaster e , li j , liem r , scherer ss . perelman school of medicine, university of pennsylvania, philadelphia, pa, usa; columbia university school of medicine, new york, ny, usa. heterozygous nefl n s/+ mutant mice are the first animal model of a charcot-marie-tooth disease e (cmt e). people with this mutation have a severe, early onset axonal neuropathy. axons in the mutant mice have reduced number of neurofilaments and decreased diameters. they also show early onset of tremor and abnormal hindlimb clasping behavior. we measured the compound action potentials (caps) from tails every weeks from the same cohort of nefl n s/+ mutant mice and their wt littermates from to weeks. even at the age of weeks, the amplitude of mutant caps was only ∼ % of wt caps ( ± . microv, n= vs ± . microv, n= ), and the caps stayed substantially smaller than those in wt mice for weeks ( ± . microv, n= vs ± . microv, n= ). the conduction velocity and the duration of caps in mutants were slower and wider compared to those of wt. separate cohorts of mice (n= mutants and n= wt) were sacrificed at different time points for analysis by light microscopy. in caudal nerve of mutant mice, the number of axons was significantly reduced compared to that of wt at weeks and, by weeks, it was reduced more than %. this model system may be useful for preclinical studies of treatments for cmt e since the animals show progressive neuropathy over weeks, which can be objectively measured electrophysiologically and anatomically. despite more than cmt genes identified today, at least % of cmt patients do not carry a mutation in any of these genes. progress in gene identification in recent years suggests that there are still many more cmt disease genes to be discovered; however, it has become rare to gather support from multiple large families that allow for conclusive linkage analysis. we have studied multiple extended dominant cmt families with linkage support for a gene at chromosome p . . originally a czech family yielded a two point lod score of . at this locus and a family from southern italy showed a lod score of . . whole exome sequencing of multiple family members identified missense mutations in the gene atpase na+/k+ transporting subunit alpha (atp a ). atp a has not been associated with human diseases thus far. in expression studies on teased fiber preparations we already confirmed predominant expression in the nodes of ranvier of peripheral nerves. through collaborative efforts in the inherited neuropathy consortium and beyond we identified five additional multigenerational families via exome or sanger sequencing resulting in a total of seven unique segregating missense changes: leu arg, ile thr, ala thr, asp phe, pro ala, pro thr and asp ala. five of these mutations fall into a remarkably narrow motif associated with the sodium binding structure of atp a , flanking the flexible hinge motif. functional studies in different model systems (mammalian cell lines, xenopus oocytes, patient fibroblast lines) are underway to determine whether a loss or a dominant gain-of-function represents the disease mechanism. taken together, we show strong support for a major new dominant cmt gene, atp a . this finding represents a new pathway and an attractive new target for therapy development in axonal cmt. laurá m , ramdharry g , , singh d , kozyra d , skorupinska m , reilly mm . mrc centre for neuromuscular diseases, ucl institute of neurology, london, uk; school of rehabilitation sciences, st george's university of london/kingston university, uk; royal national orthopaedic hospital, stanmore, uk. charcot-marie-tooth (cmt) disease is the most common inherited peripheral neuropathy. foot deformities are frequent complications and orthopaedic surgery is often required. however there are no evidence based guidelines on the type or timing of the surgery. only few studies have described the long-term results of surgical procedures and evidence regarding optimal surgical management of these patients is lacking. we prospectively studied surgical management of cmt patients attending our centre. we collected data and assessed cmt patients before and after surgery. data included: history of ankle instability, pain, skin condition, details of physiotherapy and orthotic management, assessment of lower limb strength, charcot-marie-tooth examination score (cmtes), foot posture index, ankle dorsiflexion range of movement and specific questionnaires (foot index and manchester-oxford foot questionnaire, modified fatigue severity scale and modified falls efficacy scale), details of surgical procedures. patients were assessed yearly after surgery. so far patients ( males and females, age range - ) have been evaluated prior to surgery. all patients but one had genetically confirmed cmt ( cmt a, cmtx, cmt a). patients have been assessed after year, patients after years, patients after years and patient after years from surgery. a wide range of surgical procedures were performed by one dedicated orthopaedic surgeon. preliminary results showed reduction of number of falls in / ( %) patients and improvement of callosities in / ( %) patients at year follow up. there was also significant improvement of alignment of the operated foot (p= . ) and pain (p= . ). there were no significant changes in measures of strength and ankle range of motion. further analysis on a larger number of patients will be important to determine the long-term outcome of surgery. data acquired from this study will help develop orthopaedic intervention guidelines and identify areas for further research. lavigne-moreira c , oliveira mf , marques vd , onofre ptbn , dos santos acj , nascimento ojm , barreira a , marques w jr . division of neuromuscular diseases and neurogenetics, department of neurosciences and behaviour sciences, clinical hospital of ribeirão preto, university of são paulo, ribeirão preto, brazil; department of neurology, fluminense federal university, rio de janeiro, brazil. fap associated to ttr mutations is defined as a length-dependent axonal sensory and motor polyneuropathy that at early stages affects mainly the small nerve fibers, associated to autonomic and thermo-algesic sensations. the expected emg pattern was that of an axonal sensory and motor polyneuropathy, but in fact several unexpected patterns may be found. in this study we present the results found in a brazilian population with ttr mutation. patients were divided in three groups: ttr-met of early onset, ttr-met of late onset and ttr non-met . in the first group (ttr-met of early onset), ( . %) examinations were normal, ( . %) were axonal, were demyelinating fulfilling pidc criteria, one suggested a predominately motor polyneuropathy and the final one presented a lower motor neuron disease pattern. in the second group (ttr-met of late onset), ( . %) had an axonal pattern, ( . %) had an intermediate cv, ( . %) had a demyelinating pattern, had a lumbossacral pattern and the final one had no definite pattern. among the non-ttr met , two patients had the ttr -asp tyr, one presented an axonal pattern, while the second presented initially an axonal pattern, that changed to a demyelinating pattern in the second examination. patients with ile val mutation presented an axonal neuropathy associated to cts. most patients with demyelinating or intermediate pattern were treated with corticosteroids or ivig, with no satisfactory results. this small series of patients shows clearly that fap-ttr is associated to several emg patterns in addition to the expected sensory and motor axonal polyneuropathy. this variability is present in the same family and in the same patient in different occasions. clinicians should be alert to these possibilities to do not delay diagnosis and treatment. identifying the mechanisms involved in this variability could improve our knowledge of this intriguing disease. lavin tm . greater manchester neurosciences centre, salford royal hospital, manchester, uk. subcutaneous immunoglobulin (scig) has evidence from small trials for its use following intravenous igg (ivig) loading in chronic inflammatory demyelinating polyneuropathy (cidp). potential advantages of scig include stable igg levels and reduced complication rates. scig de novo can be considered as igg levels gradually rise with subcutaneous delivery and maybe beneficial for patients for whom complications are a concern. we present cases of cidp who were initiated on subcutaneous immunoglobulin g ( g/kg/month, % scig, hizentra, csl behring) following corticosteroids; without intravenous igg loading. patient is year old with a history of ihd with a progressive lower limb sensory changes and sensory ataxia over years. neurophysiology confirmed sensory demyelinating changes. he did not respond to corticosteroids. concerns were raised about the potential risk orf adverse cardiac events with iv immunoglobulin. scig therapy was introduced at g weekly. initial therapy has been well tolerated and objective measures (rods-cidp score, jamar grip strength, hole peg) have remained stable through initiation, and at months. patient is yr old male with a past history of branch retinal vein thrombosis. he developed a sensorimotor polyneuropathy over years with neurophysiology fitting efns criteria for cidp. given concerns regarding previous thrombosis, a trial of scig was given at g/week, with resolution of sensory ataxia and improvement in objective markers (jamar grip strength, rods-cidp score, hole peg) at months. unfortunately an adverse event occurred of an urticarial skin reaction. patient is a yr old male who presented with motor predominant cidp. there was no response to corticosteroids but improvement following plasma exchange. due to a past history of transient ischaemic attack, scig was started. there has been a positive response after months with improvement in walking distances and stable objective markers (rods-cidp score, grip and pinch strength). our cohort remained neurologically stable during initiation of scig, with adverse reaction. our experience would support further trials in this area regarding the efficacy of scig compared to ivig in both the short and long term. diabetic peripheral neuropathy is the most common and debilitating complication of diabetes and it is associated to neuropathic pain and non-traumatic amputations. despite the economic burden and human costs, nowadays there is not a specific treatment to cure diabetic peripheral neuropathy. hyperglycaemia and dyslipidemia-mediated oxidative and endoplasmic reticulum stress has been linked to diabetic peripheral neuropathy, among other altered pathways. in order to study in more detail the molecular mechanisms that lead to the development of diabetic peripheral neuropathy we set up in vitro models of the disease using nsc- (motoneurons) and med . (sensory neurons) cell lines or primary cultures of dorsal root ganglia and motoneurons exposed to saturated fatty acids. it has been reported that palmitic acid is able to induce endoplasmic reticulum stress and oxidative stress in multiple cell types including schwann cells and myenteric neurons, resulting in a good model of dyslipidemia-mediated stress. here we found that palmitate induces upregulation of the molecular chaperone bip/grp and the ccaat-enhancer-binding protein homologous protein (chop) mrnas and a dose-dependent downregulation of the apoptosis marker bcl- in primary cultures. moreover, palmitic acid induces loss of neuron dendrites and cell death at high doses and upregulation of heme oxygenase (ho- ) and chop at lower doses in the nsc- cell line. we are currently characterizing multiple molecules implicated in oxidative stress, endoplasmic reticulum stress and inflammation pathways in these cell types to find new therapeutic targets to treat type diabetic sensorimotor polyneuropathy, that will be subsequently validated in the db/db mouse model by pharmacological or gene therapy strategies. lee bjh , ohnmar o , wong j , koh sj , umapathi t . lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore. treatment-induced neuropathy of diabetes mellitus (dm) (tind) is an acute painful peripheral neuropathy and autonomic dysfunction that occurs within weeks of tight glycaemic control. therapy with either insulin or oral hypoglycaemic agents (ohga) may result in tind. the quantum and rate of decline in hba c predicts development and severity of tind. both type i and ii dm patients are prone to this iatrogenic complication. besides the expected morbidity associated with painful somatic and autonomic neuropathy, tind patients also develop life-threatening eye complications such as maculopathy. with greater awareness, we picked up at least typical cases of tind in recent few months. however, there was a fourth possible tind case, whom we feel deserves special attention. the circumstances surrounding this case are common and may go under-recognised in acute hospitals. a year-old man with -year history of poorly controlled type ii dm was admitted with a partial left middle cerebral artery stroke. one month before admission, his hba c was . %. his hospitalization was prolonged because of his considerable disability that required rehabilitation. he stayed weeks. his blood sugar was difficult to control with hyperglycaemic episodes requiring ohgas and insulin. the highest capillary blood glucose recorded was mmol/l. he also had episodes of hypoglycaemia. three weeks into hospitalization, he developed severe orthostatic hypotension. he came to our attention two months later when he was sent for autonomic screening tests for severe postural hypotension, in spite of therapy with fludrocortisone and midodrine. his hba c was . %. he had marked orthostatic hypotension suggestive of sympathetic dysfunction. we were not able to discern if he had a painful neuropathy because of his aphasia. there was no recent ophthalmology review. we suggested a diagnosis of possible tind. he was discharged with lower doses of ohga. however, day after discharge, he attended the emergency department for a syncopal episode. this case raises intriguing questions on the safety of glucose control paradigms commonly employed in patients with acute stroke and myocardial infarction as well as the possible role of major fluctuations in blood glucose in the development of tind. lee hs , kim sm . presbyterian medical center, jeonju, korea; yonsei university college of medicine, seoul, korea. the occurrence of peripheral neuropathy by a tumor within or compressing a nerve in neurofibromatosis (nf) type and is relatively well known. however, nf presenting with demyelinating polyneuropathy unrelated to tumor masses is rarely reported. we report a rare presentation of an nf patient with demyelinating polyneuropathy of subacute onset. a -year-old woman was referred to our hospital with paresthesia on both limbs months before. she was healthy and had no family history. the symptoms worsened over the next months, tremor and weakness in both limbs occurred. although she treated as a chronic inflammatory demyelinating polyneuropathy (cidp) with intravenous steroid and immunosuppressant in another hospital, symptoms were getting worse. bifacial numbness and left eyeball pain occurred weeks ago and she visited our hospital. on neurologic examination, bilateral facial sensory hypoesthesia and symmetrical both limbs weakness (mrc grade iv+) were observed. decreased touch and pinprick sensation on both limbs were observed like stocking-glove distribution. the reflexes were sluggish. on nerve conduction study, sensorimotor demyelinating polyneuropathy with conduction blocks and temporal dispersions was observed. bilateral r , r responses were prolongation on facial blink test. mri of cervical spine and brain revealed contrast enhancing tumor-like enlargement of multiple nerve roots and cranial nerves. nerve biopsy was performed on right supraorbital nerve, neurofibromatous changes were observed. the symptom deterioration was stopped without treatment. gene test dose not revealed nf type and . on review of the literature, polyneuropathy in nf patient can result from tumor masses within the proximal nerve roots, or along the peripheral nerve, or in the extramedullary lesion affecting neighboring nerve roots. thus, axonal type polyneuropathy and focal amyotrophy have been reported in nf. demyelinating polyneuropathy has not been reported before in our knowledge. although the etiology of demyelinating polyneuropathy in nf requires further clarification, some authors claim that unknown local toxic or metabolic influences of the endoneurial pathological cells on adjacent nerve fibers. in this patient, atypical symptoms were observed in cidp such as cranial nerve involvement and stocking-glove sensory distribution. in this case, a nerve biopsy can be helpful the accurate diagnosis. lee jy , yoo jh , kang dk , bae js . department of neurology hallym university, seoul, korea. acute disseminated encephalomyelitis (adem) is an uncommon post-infectious inflammatory demyelinating disorder of central nerve system, while guillain-barre syndrome (gbs) is a prototype of acute post-infectious peripheral neuropathy. previous reports regarding the coexistence of these relatively rare diseases suggest that certain immunogenicity within central and peripheral nerves may share a common autoimmune process during the disease course. a previous healthy years old man was admitted because of fever, headache, nausea and myalgia in department of infectious disease. two weeks before admission, he suffered from watery diarrhea for days and spontaneously recovered. at initial presentation, he had high fever( ∘ c), headache and myalgia and intermittent horizontal diplopia. a few days after admission, he began to complain of drowsy mentality, bilateral extremities weakness, especially lower limbs, dysarthria, bilateral facial paralysis, urinary retention, dyspnea. on neurologic examination, he had mildly drowsy mentality, symmetric muscle weakness scoring of on bilateral hip, knee flexion and finger extension, but he had no sensory symptoms. he showed gazed evoked nystagmus with no extraocular muscle palsies. deep tendon reflexes were not present. pulmonary function test revealed a severe restrictive pattern. csf studies disclosed a dissociative increase of protein contents ( mg/dl) without pleocytosis. anti ganlioside antibody assay identified an anti-gt a igg positivity in his serum. nerve conduction study (ncs) showed prolonged motor terminal latencies and slow motor conduction velocity on multiple nerves. in contrast, sensory ncs revealed no abnormal findings. imaging studies unexpectedly revealed apparently symmetrical lesions across bilateral brainstem and basal ganglia suggesting a diagnosis of adem. after both ivig and high dose steroid treatment, he remarkably recovered from disturbed mental state and motor weakness. about month after the symptom onset, he could walk with assistant aid and discharge to other hospital for rehabilitation. our case suggest that certain component of autoimmunity simultaneously result in both cns and pns inflammation. specific immunological mechanism is remained to be elucidated. although we could not conclude whether cellular component or humoral component is dominant for our case, the presence of anti gt a antibody suggest a role of humoral mechanisms. the aim of this study is to evaluate whether peripheral neuropathies and headaches affect the same subgroups of patients with ibd. since , we have established a cohort study to evaluate the prevalence and incidence of neurological diseases in patients with ibd. over a period of years, all patients with ibd (either crohńs disease or ulcerative colitis) were invited to participate in a study designed to evaluate the risk factors for the presence of headaches and peripheral neuropathy in ibd. a separate group of control patients (age-matched relatives of ibd patients) was also formed. after a clinical interview and neurological examination, patients were invited to undergo skin wrinkling test (swt) to evaluate small fiber function and/or electromyography. headaches were present in . % of the patients with ibd, and were more common in patients with ulcerative colitis than in control patients (p< . ). migraine comprised . % of all cases of headache and was more prevalent in patients with crohńs disease than control patients (p< . ). tensional headaches were also common affecting . % of the ibd patients. electromyography was abnormal in . % of the ibd patients tested ( / ). swt was abnormal in . % of the ibd patients tested ( / ). . % of the ibd patients had abnormal swt but had no neuropathy symptoms. patients with abnormal swt or emg were not more likely to have headaches (p= . and . , respectively). overall, patients with symptomatic polyneuropathy were not more likely to have headache (p= . ). patients with abnormal swt or emg were also not more likely to have migraine (p= . and . , respectively). patients with abnormal swt or emg were also not more likely to have tension-type headache (p= . and . , respectively). in summary, although highly prevalent in this population of brazilian ibd patients, primary headaches and neuropathy do not affect the same subgroups of ibd patients. further studies are necessary to understand the mechanisms of both conditions in ibd patients. leitao amf , , araújo df , marques h , pamplona l , , souza mh , , nbraga ll , , gondim faa , . the aim of this study is to evaluate whether peripheral neuropathies and headaches affect the same subgroups of patients with ibd. since , we have established a cohort study to evaluate the prevalence and incidence of neurological diseases in patients with ibd. over a period of years, all patients with ibd (either crohńs disease or ulcerative colitis) were invited to participate in a study designed to evaluate the risk factors for the presence of headaches and peripheral neuropathy in ibd. a separate group of control patients (age-matched relatives of ibd patients) was also formed. after a clinical interview and neurological examination, patients were invited to undergo skin wrinkling test (swt) to evaluate small fiber function and/or electromyography. headaches were present in . % of the patients with ibd, and were more common in patients with ulcerative colitis than in control patients (p< . ). migraine comprised . % of all cases of headache and was more prevalent in patients with crohńs disease than control patients (p< . ). tensional headaches were also common affecting . % of the ibd patients. electromyography was abnormal in . % of the ibd patients tested ( / ). swt was abnormal in . % of the ibd patients tested ( / ). . % of the ibd patients had abnormal swt but had no neuropathy symptoms. patients with abnormal swt or emg were not more likely to have headaches (p= . and . , respectively). overall, patients with symptomatic polyneuropathy were not more likely to have headache (p= . ). patients with abnormal swt or emg were also not more likely to have migraine (p= . and . , respectively). patients with abnormal swt or emg were also not more likely to have tension-type headache (p= . and . , respectively). in summary, although highly prevalent in this population of brazilian ibd patients, primary headaches and neuropathy do not affect the same subgroups of ibd patients. further studies are necessary to understand the mechanisms of both conditions in ibd patients. the classic guillain barré syndrome (gbs) is characterized by motor weakness, hyporreflexia, but limited sensory deficits. sensory variants involving either small or large fibers or both are unusual and represent a diagnostic challenge. we described patients presenting with the sensory variant of gbs and retrospectively analyzed the clinical and electrophysiological findings of patients fulfilling the criteria for sensory gbs according to oh et al. criteria. six patients were identified (mean age years: range - years). four had a previous infection. they all consulted due to distal painful paresthesias and allodynia. on examination the patients presented normal strength and normal cranial nerves through the course of the disease with reduced knee and ankle reflexes in patients. distal hyperesthesia to pinprick was identified in and one of them additionally had hyperhidrosis and constipation. two additional patients presented hypoesthesia to pinprick and temperature. one patient had distal proprioceptive sensory loss with sensory ataxia. csf albumin cytological dissociation was present in patients. nerve conduction studies (ncs) identified a sensory motor demyelinating neuropathy in patients. among the with normal ncs, had abnormal cold and warm threshold in their qst evaluation. all patients received symptomatic treatment for the neuropathic pain and only two ivig therapies. longstanding pain, fatigue or both were persistent findings in patients after a mean follow up of months. in conclusion the sensory variant of gbs is both an infrequent presentation and a diagnostic challenge. longstanding pain and fatigue are common persisting findings. the epidemic of zika virus (zikv) throughout the americas and asia, and the subsequent rise in reported cases of guillain-barré syndrome (gbs) caused worldwide concern. as of january , countries have reported evidence of mosquito-borne zikv transmission and in of these countries, a sudden increase of gbs has been reported. moreover, case studies and a case-control study further indicate that zikv may trigger gbs. however, accurate diagnosis of both zikv and gbs in many of these studies is disputed, and a comprehensive description of the clinical phenotype of gbs related to zikv is lacking. the international gbs outcome study (igos) is a prospective observational study on the factors determining the onset, clinical course and outcome of gbs. at present, a research consortium of centers from countries has included patients in igos. our aim is to investigate zikv-related gbs in igos as is already occurring in colombia. in igos-zika, data on clinical features and ancillary investigations will be collected in zikv endemic areas according to the igos protocol with some modifications. first, igos-zika has a case-controlled study design to define the association between gbs and zikv and other arboviruses. second, urine samples will be collected and additional questions on preceding events will be asked, focusing on arbovirus infections. third, a more limited follow-up is required. our aim is to recruit additional centers via the inflammatory neuropathy consortium (inc) and centers in all arbovirus endemic regions that are willing to participate. the focus of igos-zika will be on the accuracy of the diagnosis of both gbs and zikv and on defining the associated clinical phenotype, course and outcome. igos-zika provides the opportunity to combine data and biobanks from various geographical regions using a standardized protocol and to compare these data with data and biosamples already collected in igos. studying these cases will help to optimize diagnostics and care for gbs patients in arbovirus endemic countries and provides a unique opportunity to further understand the pathogenesis of gbs. moreover, this study design and network can be used to adequately respond to other future viral epidemics related to gbs. lerat j , cintas p , dzugan h , , magdelaine c , , sturtz f , , lia as , . service de biochimie et génétique moléculaire -chu de limoges, limoges, france; service de neurologie et d'explorations fonctionnelles -chu de toulouse, toulouse, france; ea -université de limoges, limoges, france. pharc syndrome is an autosomal recessive neurodegenerative pathology leading to demyelinating polyneuropathy, hearing loss, cerebellar ataxia, retinis pigmentosa and early-onset cataract. these various symptoms can occur at different ages, so that pharc syndrome can be a differential diagnosis of charcot-marie-tooth disease (cmt) associated with deafness. only abhd mutations have been reported in patients. we described the th mutation and compared our results to the literature data. we analysed by next generation sequencing (ngs) strategy using a targeted cmt and associated neuropathies -gene panel the dna of a -year old male who has suffered from demyelinating sensory and motor polyneuropathy and ataxia since the age of . bilateral sensorineural deafness was diagnosed at the age of five. bilateral congenital cataracts were operated on at the age of . a new large complex homozygous mutation, with one deletion of seven base pairs and one insertion of base pairs, was detected. by analyzing our patient data and those of the literature, we evaluated that, in pharc syndrome, sensorineural deafness always occurs as the first feature in late teens. the ophthalmological symptoms are cataracts that occur at a mean age of yo and then retinis pigmentosa at a mean age of . demyelinating sensory-motor polyneuropathy is the most variable characteristics, which occurs in the thirties. we report the first large complex homozygous mutation in pharc syndrome, which is certainly under-diagnosed. therefore, it seems interesting to include abhd in the panels of the five symptoms, especially deafness ones. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is a chronic disabling disease that often improves with immune therapy. to date, the most reliable diagnostic criteria for cidp are the efns/pns revised criteria, with a reported sensitivity of % and specificity of %. we implemented a web-based database to collect data from patients with cidp followed by italian centers with expertise on cidp to determine the frequency and characteristic of cidp and it variants, the diagnostic criteria used for their diagnosis, the possible evolution into typical cidp, the association with specific anti-nerve antibodies, and their response to therapy. all the patients were evaluated at the time of inclusion and will be followed for two years to monitor their outcome and response to therapy. by february we included patients with cidp and variants ( men, women), aged - years (median ) with a mean disease duration of . years (range . - years). based on clinical symptoms, cidp was defined as typical in % and atypical in %. the diagnosis of typical cidp fulfilled efns/pns criteria in % of the patients while nerve conduction studies were not diagnostic in % (grouped as clinical cidp) or not available in %. we analyzed the frequency of supportive criteria for the diagnosis of cidp in patients with clinical cidp and found that increased csf proteins, demyelination or cell infiltration on nerve biopsy and imaging abnormalities consisting with cidp on us or nmr were present in %, % and % of the patients, respectively. a relapsing course was present in % of patients with clinical cidp, increasing the reliability of the diagnosis for cidp. in addition an improvement after one or more therapies was reported by % of the patients, with a positive response to ivig in %, steroids in % and plasma exchange in %, similarly to what observed in patients fulfilling efns/pns criteria. in % of the patients with clinical cidp two or more supplementary criteria for cidp were present. this study on a large population of patients is providing useful information that may help to revise the current diagnostic criteria for cidp. li j , cannell m , suragani r , pearsall r , kumar r . acceleron pharma inc, cambridge, usa. charcot-marie-tooth (cmt) is the most common hereditary peripheral neuropathy and is characterized by demyelination and/or axonal damage of peripheral nerves and muscle weakness. foot drop, steppage gait, and foot deformities are a typically seen in cmt patients. consequently, falls are commonly reported in these patients. improvement of dorsiflexor muscle function to prevent falls may improve quality of life and activities of daily living in patients with cmt. ace- , a locally-acting ligand trap that binds growth and differentiation factors (gdfs) and activins, has previously been shown to increase muscle mass and force in both duchene muscle dystrophy (dmd) and amyotrophic lateral sclerosis (als) mouse models. in the current study, we evaluated the therapeutic effects of ace- to improve muscle strength in the trembler (tr-j) mouse model of cmt a. these mice harbor a mutation in the peripheral myelin protein (pmp ) known to cause cmt a. seven-month old (b .d -pmp tr-j /j) mice were administered ace- ( g, twice weekly) intramuscularly to one of the unilaterally tibialis anterior (ta) muscle for weeks. the contractility of the ta muscle was evaluated during isometric contraction. all data were compared to the uninjected contralateral control hind-limb. after weeks of ace- treatment, ta muscle mass was increased by % (p< . ) and its physiological cross-sectional area was increased by % (p< . ). the increase in muscle mass correlated with an increase in strength, with maximum tetanic force and twitch force improved by % (p< . ) and % (p< . ), respectively. in addition, temporal properties during isometric contraction, such as maximum rate of contraction and relaxation, were accelerated by % and % respectively (p< . ) in the ace- -treated ta muscle compared to its contralateral hind-limb. pathological and biochemical assessment of ace- -treated mice showed enlarged myocyte area (+ %, p< . ) and reduced atrogin- mrna expression (− %, p< . ). together, these results demonstrated that ace- attenuates the degree of muscle atrophy and also improves muscle function in a mouse model of cmt a. the current study provides proof of concept for the use of ace- as a therapy for cmt to improve dorsiflexor muscle function and alleviate foot drop. we have generated a rat model of charcot-marie-tooth disease a (cmt a) harboring the p.arg trp mfn mutation, whose human counterpart results in a severe, early-onset axonal neuropathy. the mutation was made using zinc finger nuclease-mediated genome editing in fertilized rat eggs. a large cohort of mutant and wt littermates were characterized behaviorally and found to develop multiple motor deficits that worsened over time. nerve conductions of the tail (caudal nerve) was performed on a separate cohort of mutant (n= ) and wt littermates (n= ) every weeks from to weeks. mutant rats showed a progressively decline in the amplitude of the compound action potential after weeks, whereas the amplitude progressively increased in their wt littermates. separate cohorts of rats were sacrificed at , , and weeks and analyzed by light microscopy. in mutant rats, there was a reduced density of myelinated axons and active axonal degeneration in distal but not proximal nerves, and in the fasciculus gracilis of the cervical spinal cord at and weeks. these findings were not present in the -week-old cohort of mutant rats, or in wt rats at or weeks. a genetically authentic animal model of cmt a that develops a progressive, length-dependent axonal neuropathy will be a valuable tool for examining the pathogenesis and treatment of cmt a. lindborg ja , niemi, jp , defrancesco a , zigmond re . case western reserve university, cleveland, usa. traditionally the role of immune cells in nerve degeneration and regeneration has focused on the infiltration of inflammatory monocytes into the distal nerve after nerve injury and the phagocytosis by the resulting macrophages of myelin and axonal debris, thereby clearing a path for regenerating axons. therefore, it was surprising when we discovered that in ccr knockout (ko) animals, in which the entry of these inflammatory monocytes does not occur, that wallerian degeneration precedes normally. we now report that the reason for this is that neutrophils and schwann cells compensate for the decrease in macrophage accumulation. furthermore, nearly complete depletion of circulating neutrophils by systemic injection of an antibody to ly g leads to an inhibition of myelin clearance both in ccr ko and in wild type animals. on the other hand, we have demonstrated a second site of macrophage accumulation in wild type animals, namely around axotomized sensory neurons in dorsal root ganglia (drgs). blockade of that accumulation, for example as occurs in ccr ko animals, leads to a dramatic impairment of nerve regeneration. to examine the relationship between macrophages and regeneration further the monocyte chemokine ccl was overexpressed in drg neurons in intact animals by viral infection using an aav containing the ccl coding sequence. the resulting overexpression of ccl led to the accumulation of macrophages in drgs even though no injury had taken place and subsequently to an increase in the intrinsic growth capacity of the sensory neurons. examination of changes in gene expression in the drgs in these animals revealed increased expression of the cytokine leukemia inhibitory factor and an increase in its downstream signaling pathway that involves the phosphorylation and nuclear translocation of stat . strikingly, pharmacological blockade of stat activation inhibited the increase in the neurons' growth capacity produced by the virus. these results reveal unexpected interactions between immune cells and neurons facilitating nerve degeneration and regeneration and could lead to therapies to improve regeneration after injury or in disease. lin j , , qiao k , , huang j , , zhao cb , , lu jh , . institute of neurology, fudan university, shanghai, china; department of neurology, huashan hospital, fudan university, shanghai, china. we used terminal latency index (tli) as a tool in differentiation between poems syndrome and chronic inflammatory demyelinating polyradiculoneuropathy (cidp). comparison of median and ulnar nerve conduction studies including motor conduction velocity (mcv), distal motor latency (dml) and terminal latency index (tli) were studied in poems patients, matched cidp patients and normal controls. in this cohort, the average age at evaluation was . ± . years old in poems group and that of cidp patients was . ± . years old. except the ulnar terminal latency index in cidp group, poems and cidp patients demonstrated prolonged distal latencies, low conduction velocities and increased terminal latency indexes compared with the normal group. reduced conduction velocities and higher terminal latency indexes in poems group than in cidp group was found. increased tli was demonstrated in . %(median nerve) and . %(ulnar nerve) poems and that in cidp patients was . %(median nerve) and . %(ulnar nerve). decreased tli was found in . %(median) and . %(ulnar) cidp patients and none in poems. temporal dispersion (td) and conduction block (cb) were more often seen in cidp patients with increased tli than that in poems. compared with cidp and poems showed greater slowing of the intermediate nerve segments and relatively more uniform demyelination. about % cidp demonstrated more distal conduction slowing and more td and cb especially in those with increased tli. terminal latency index combined with td and cb may be helpful in differentiating poems from cidp. lin y , sung j , chang t , jowy t . department of neurology, taipei municipal wanfang hospital, taipei, taiwan. the purpose of our study is to exam whether electrophysiology changes could be detected in prediabetes patients and to discover the possible mechanism of nerve injury in prediabetes stage. we analysis and compare the nerve excitability test data between prediabetic patients and age-matched normal control subjects. prediabetes is defined by american diabetes association (ada) as one of the three following: hba c . % to . %, fasting glucose mg/dl to mg/dl, and hour oral glucose tolerance test to mg/dl. patients with radiculopathy, myelopathy, entrapment neuropathy such as carpel tunnel syndrome, and polyneuropathy were excluded. the strength-duration time constant (sdtc) and superexcitability showed significant difference (p< . ) between two groups. we also find increased threshold electrotonus in depolarization (ted) and reduced relative refractory period (rrp) and refractoriness in . msec. these early changes in prediabetic patient are similar in nerve excitability feature of diabetic patients. however, the above changes are not found in motor axonal excitability test. our data supports that nerve excitability test may be a useful, non-invasive, and less time dependent tool to detect peripheral nerve injury in prediabetic stage. the sensory axons are more vulnerable than motor axons. superexcitability is the most sensitive parameter in prediabetes. transthyretin-related familial amyloid polyneuropathy (ttr-fap) is an autosomal dominant disorder caused by mutations of ttr gene and is associated with variable penetrance. ttr-fap is rare, except for endemic areas. this is a retrospective study of ttr-fap patients diagnosed at our center between - . we identified four families with different ttr mutations. in one family with v a mutation, nine family members over four generations diagnosed with ttr-fap was followed since . in the other families the index cases with different mutations were identified between - . affected family members with v a mutation developed severe progressive polyneuropathy with cachexia, with onset of the disease between ages and . three patients presented with marked visual symptoms (one patient underwent vitrectomy). nine patients died to years after disease onset. two patients (sisters) underwent liver transplantation -one died after years of disease at age , second is years old and wheelchair-bound as her symptoms continue to progress. the ttr mutations diagnosed in the index cases of three other families are: d v, f l and v m. they all presented with similar clinical picture of late-onset ttr-fap with predominant progressive axonal sensory, motor and autonomic polyneuropathy. all three index cases were men, the onset of symptoms was between - years with numbness and paresthesia in the feet followed by weakness and autonomic dysfunction. all had excessive weight loss resulting in cachexia and were diagnosed with cardiomyopathy. no patient suffered from visual symptoms. all three patients progressed to stage ii of ttr-fap -walking with assistance. time to diagnosis was . - years. due to advanced stages of their disease these patients were not suitable for therapy with tafamidis or the liver transplantation. the ttr d v (p.d v) was confirmed as a de novo mutation, which is uncommon in ttr-fap. in the remaining families carriers of ttr mutations were identified and are followed up. pedigree analysis of the family with f l mutation revealed affected members who with high probability died from ttr-fap. our study suggests that patients with ttr-fap in poland exhibits clinical and genetic heterogeneity. liu x , fan d . department of neurology, peking university third hospital, beijing, china. objective: to identify the gene mutation of chinese charcot-marie-tooth pedigrees and investigate the correlation among the clinical manifestation, electrophysiology and mechanism of different genotype. methods: we included pedigrees with cmt enrolled in our hospital from january, to december . we recorded clinical features, cmtns and electrophysiological data at diagnosis. the patients underwent mutation analysis of pmp , cx , mpz, mfn , hspb , hspb using mlpa, dhplc and sanger gene sequencing. results: we found pmp duplication pedigrees ( . %), cx pedigrees ( . %), mfn pedigrees ( . %), mpz pedigrees ( . %) conclusions: in chinese han population, the proportion of pmp duplication is relatively low, the majority of clinical manifestation is classical cmt. axonal cmt can show isolated lower extremity injury, with central nervous system involvement. hmn may be an underestimated clinical types, the identification should be done with caution in differential diagnosis. liu y , liu b , sebastian b , wozniak km , wu y , slusher b , polydefkis m . johns hopkins school of medicine, baltimore, usa. chemotherapy-induced peripheral neuropathy (cipn) is a common dose-limiting toxicity in the treatment of many cancers. most cipn studies preferentially focus on sensory fiber loss and dysfunction. here, we compared the structural and functional recovery of autonomic fibers in sweat glands (sweat gland nerve fiber density, sgnfd) and sensory fibers (intra-epidermal fiber density, ienfd) in mouse footpads after exposure to a maximum tolerated dose (mtd) of several common chemotherapy agents. additionally, we assessed footpad sweat production as a functional correlate to sgnfd reductions. female balb-c mice ( -animals/group) were treated with a mtd of four anti-tubulin drugs: paclitaxel (pca, mg/kg), ixabepilone (ixa, mg/kg), eribuline (erib, . mg/kg), vinoelbine (vino, mg/kg), or corresponding placebo given intravenously, mwf for two weeks. recovery was assessed at -hours, , , , , and weeks following the last dose. footpads were processed to visualize epidermal nerve fibers using pgp . and autonomic nerve fibers with tyrosine hydroxylate and pgp . . ixabepilone-treated mice experienced significant reductions in sgnfd at hrs, while ienfd nadir occurred at a later time point, -weeks. the recovery to baseline levels occurred more quickly for ienfd ( -weeks) than sgnfd ( -weeks). in contrast, vinorelbine and eribuline treated mice experienced a maximum deficit in sgnfd and ienfd at hrs and sgnfd recovery was slower ( -weeks) compared to ienfd ( -weeks). pca-treated animals showed more severe ienfd and sgnfd deficits compared to the other agents with both ienfd and sgnfd not recovering completely until -months. reductions in th-sgnfd were comparable or more pronounced to decreases in pgp . -sgnfd for all agents and timepoints. pca-treated animals demonstrated reductions in footpad sweat droplet number thereby providing a functional correlate. together, these data indicate that in mouse models of cipn, autonomic nerve fibers are affected more severely than sensory nerve fibers, and also recover more slowly than intraepidermal nerve fibers. autonomic dysfunction may be an important and under-appreciated consequence of chemotherapy exposure. the pi -kinase vps (pik c ) synthesizes phosphatidylinositol -phosphate (pi p), a lipid critical for both endosomal membrane traffic and macroautophagy. human genetics have implicated pi p dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating charcot-marie-tooth (cmt) peripheral neuropathy. here, we investigated the role of vps , and pi p, in mouse schwann cells by selectively deleting vps in this cell type. vps -schwann cell knockout (vps scko ) mice show severe hypomyelination in peripheral nerves. vps −/− schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. upon reaching the promyelinating stage, vps −/− schwann cells are significantly impaired in the elaboration of myelin. nerves from vps scko mice contain elevated levels of the lc and p proteins, indicating impaired autophagy. however, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in vps scko mice is caused by impaired autophagy. endosomal membrane traffic is also disturbed in vps −/− schwann cells. we investigated the activation of the erbb / receptor tyrosine kinases in vps scko nerves, as these proteins, which play essential roles in schwann cell myelination, are known to traffic though endosomes. in vps scko nerves, erbb was hyperphosphorylated on a tyrosine known to be phosphorylated in response to nrg exposure. the overall level of erbb was also decreased during myelination. our findings suggest that the loss of vps alters the trafficking of erbb / through endosomes. abnormal erbb / signaling may contribute to the hypomyelination observed in vps scko mice. lombardi r , devaux j , cortese a , dacci p , benedetti l , demichelis c , lauria g . irccs foundation "carlo besta" neurological institute, milan, italy; aix-marseille université, marseille, france; irccs c. mondino national neurological institute, pavia, italy; university of genoa and irccs aou san martino-ist, genoa, italy. the recent identification of igg anti-neurofascin (nfascin) antibodies in a group of patients has widened the spectrum of presentation for chronic inflammatory demyelinating polyradiculoneuropathy (cidp). these patients can be distinguished by disabling tremor, poor response to intravenous immunoglobulin and distal and sensory disturbances. cell-adhesion molecule nfasc and cntn are expressed on the paranodal junction (pnj) of nodes of ranvier, and play key roles on sodium channel clustering and glia-axon interactions. quantification of unmyelinated intraepidermal nerve fibers (ienf) is a useful parameter employed in small nerve fiber pathology diagnosis. in addition the immunohistochemistry evaluation of dermal nerve fibers allows to examine morphological changes of myelin sheath and ranvier nodes structure. we performed immunofluorescent colocalization studies using antibodies to visualize axons (protein-gene-product . , neurofilament, tubulin), sheath of myelin (myelin-basic-protein) and specifically nodal/paranodal/juxtaparanodal structures (pannfascin, nfascin , nfascin , caspr, cntn , potassium and sodium channels) in skin tissues from seronegative and seropositive cidp patients. we analyzed axon and myelin sheath damage, abnormal nodal-paranodal-juxtaparanodal architecture and morphometric parameter as internodal length of ranvier nodes. our results on skin biopsies from three igg nfasc -positive cidp patients revealed complete loss of nfasc staining at the paranodes, asymmetrical paranodes and widening of the nodes of dermal myelinated nerve fibers. one igg cntn -positive cidp patient showed abnormal nodal/paranodal immunostaining with different features as compared with igg nfasc -positive patients suggesting specific changes. however, such alterations were not found in four seronegative cidp patients. our data support the hypothesis that examining specific axonal and myelin markers could provide diagnostic and prognostic clues on nodo-paranodopathies. the goal of this study is attempt a possible correlation between the presence of serum autoantibodies and structural changes in nodal/paranodal regions of dermal nerve fibers in cidp patients. knowledge of autoantibodies expression in the peripheral myelinated nerves of cidp patients could serve for stratifying patients and potentially guiding personalized treatments. lunati a , lerat j , dzugan h , , rego m , magdelaine c , , bieth e , calvas p , cintas p , gilbert-dussardier b , goizet c , journel h , magy l , toutain a , urtizberea j , sturtz f , , lia as , . charcot-marie-tooth disease is one of the most frequent inherited peripheral neuropathies ( / ). so far, mutations in more than genes have been identified causing either the demyelinating form (type ) or the axonal form (type ). duplication of pmp gene is the most frequent cause of autosomal dominant demyelinating form. autosomal recessive demyelinating form is often due to sh tc gene mutations. patients suffer then from early severe neuropathy starting in the first decade. scoliosis and deafness are often observed. we analysed patients suffering from peripheral neuropathy, by multiplex-ligation-dependant-probe-amplification (mlpa), followed by targeted next-generation-sequencing (ngs) using a -gene custom panel designed for the diagnosis of charcot-marie-tooth and associated neuropathies. mutations of interest were verified by sanger sequencing. diagnosis was positive for patients. as expected, the most frequent mutation was the pmp duplication detected in patients. deletion of pmp was observed in patients and pathogenic point mutations were detected in patients. sh tc gene appeared to be the most frequently mutated with nine patients diagnosed. associated with known mutations, four new mutations have been identified: two nonsense mutations and two missense mutations. all these patients presented deafness and/or scoliosis. sh tc appears to be an important gene involved in charcot-marie-tooth disease, often associated with deafness and /or scoliosis. it is important to pay attention to these associated symptoms in charcot-marie-tooth patients in order to guide their diagnosis and to improve their medical care. lupo v , , frasquet m , , sánchez-monteagudo a , , barreiro m , alberti ma , casasnovas c , quintáns b , , , camacho a , domínguez c , sedano mj , pelayo al , pardo j , sobrino t , sobrido mj , , , sevilla t , , espinós c , . mme (membrane metalloendopeptidase) mutations, inherited in an autosomal recessive fashion, have been recently identified in japanese probands (higuchi et al. ) . thus, mme has been included in the list of cmt genes as a new autosomal recessive axonal form, cmt t (mim ), and moreover, it is considered a strong candidate for the genetic diagnosis of unsolved late-onset cmt cases. in fact, few months later auer-grumbach et al. ( ) reported european probands with autosomal dominant late-onset cmt and mutations in mme. we have investigated a clinical series of patients diagnosed of motor or sensory-motor peripheral neuropathy using an updated version of our custom gene panel, which includes mme. in this study, we report probands with cmt , intermediate cmt or dhmn/cmt and homozygous or compound heterozygous mutations in mme, and proband with cmt and a heterozygous mutation in mme. we have identified different type of mutations: novel splice donor variant, frameshift, nonsense and missense mutations. the two nonsense changes, p.trp * and p.arg *, and the splice donor variant c. + g>a, were detected in homozygous or compound heterozygous state in patients, while the frameshift mutation, p.pro leufs* , was detected in homozygous or heterozygous in the remaining patients. strikingly, the two nonsense and the frameshift mutations had been previously reported as causative for autosomal-dominant cmt t (auer-grumbach et al. ) . out of three missense mutations, one is novel (p.his tyr), and two are reported in control databases (p.asn lys and p.arg trp). this study shows that the autosomal recessive cmt t is common in spanish population, and moreover, it suggests that screening of mme using gene panel testing could help to improve diagnosis of unclarified inherited peripheral neuropathies cases. funds: isciii (pi / , pi / ); fundació per amor a l' art. inherited peripheral neuropathies (ipns) encompass a group of disorders highly heterogeneous, clinically and genetically. charcot-marie-tooth (cmt) disease is closely related to distal hereditary motor neuropathy (dhmn) or distal spinal muscular atrophy (dsma), and some patients show additional signs associated with amyotrophic lateral sclerosis (als). targeted gene panel and exome sequencing are considered to be powerful and cost-effective tools for diagnosis of these disorders. we have investigated a clinical series of patients diagnosed of motor or sensory-motor peripheral neuropathy: families were investigated by exome sequencing and, cases were tested using different updated versions of a gene panel . each version comprises , or ipn genes, respectively and it shows a high coverage performance: percentage of analyzable target base with > coverage was , %. both exome and gene panel capture libraries were based on sureselect capture technologies (agilent technologies), and sequencing was performed in miseq or hiseq illumina equipment. we have identified novel genes and novel mutations in known genes, broadening the phenotypical spectrum associated with ipns. exome sequencing has allowed us to identify causative gene in % of familiar or sporadic cases: morc , aars, bscl , kif a, gars, egr , fig , dnajb , drp , ighmbp , dao, sod , fig . gene panel testing has been mostly performed in sporadic cases, and it has allowed us to identify either disease-causing or candidate mutations in % of cases: kif a and bicd were the most common genes mutated. update gene panels neuro and neuro have revealed novel mutations in genes recently associated to cmt and cmt disease: mme and pmp , respectively. in sum, both strategies have helped us to achieve a more accurate clinical and genetic reclassification of these disorders, an impossible challenge using conventional sequencing methods. our study expands the clinical phenotype previously associated to known ipn causing-gene, and emphasizes that gene panels should be considered as a first diagnosis method for unclarified ipn patients. funds: isciii (pi / , pi / , pi / ); fundació per amor a l' art. magy l , mathis s , goizet s , tazir m , vallat j-m . department and laboratory of neurology, national reference center for rare peripheral neuropathies, chu limoges, france; department of neurology, chu bordeaux, france; department of medical genetics, chu bordeaux, france; department of neurology, chu algiers, algeria. charcot-marie-tooth (cmt) disease is a hereditary neuropathy with a relatively homogeneous phenotype but is genetically heterogeneous. moreover, nerve conduction studies distinguish different forms, adding another level of complexity. the current classification of cmt being difficult to understand for physicians, scientists and patients, we presented and published a proposal for updating this classification, based on inheritance, nerve conduction findings and gene/mutation involved. inputs from colleagues prompted us to conduct a survey in order to try to reach some consensus about our proposals. we conducted an internet survey between october and december . the link to complete the survey was sent several times by email with an introduction to more than people. participants were contacted through the emailing list from the last cmt meeting in venice (september ) and additional physicians and scientists who are involved in cmt care and research were contacted as well. one hundred seven people from various countries (mainly france, italy and the usa) answered the survey. most ( %) of the participants were between and years of age, % being physicians and % being scientists. the vast majority ( %) considered the proposal constituted an improvement over the historical classification whereas % wanted to keep the old one. about the order of information, % of participants thought the mode of inheritance should come first, whereas % felt the phenotype should be placed at the beginning. ninety-one percent of people thought cmt should be kept as a generic name for hereditary sensory and motor neuropathy. for pure sensory neuropathy, % favoured hsn over hsan although % thought the opposite and % of participants felt dhmn should be kept for distal motor neuropathy. about nerve conduction findings, % of participants thought the intermediate phenotype has to be kept and % favoured our proposal to replace " " by "de" (for demyelinating) and " " by "ax" (for axonal). finally, % of responders thought that genetic information should be included in the classification of cmt. overall, our proposal of a new classification received a very good appreciation from physicians and scientists implicated in the care of patients with hereditary neuropathy. mallik r , hubsch a , gaida a , barnes d . csl behring, kop, usa; csl behring, bern, switzerland; csl behring, ottawa, canada. the risk of hemolytic events (hes) with intravenous immunoglobulin (ivig) therapy appears to be linked to the isoagglutinin (anti-a and anti-b) level of the specific ivig product. using published anti-a and anti-b titers for seven ivig products and corresponding he rates reported to the eudravigilance database, we developed a mathematical model to predict the risk of he to patients receiving ivig products of given anti-a and anti-b levels. modeling was performed separately for the risk to patients with blood groups a, b, ab and o and the overall population risk was estimated assuming a blood group distribution of % a, % b, % ab and % o. applying the prediction model, we calculated the he risk for an ivig product produced via a chromatographic process (privigen ® , csl behring) a) without any isoagglutinin reduction measures ( ) ( ) ( ) ( ) ( ) ( ) ( ) , b) with an anti-a donor screening program eliminating approximately % of donors with high anti-a titers ( ) ( ) ( ) , c) incorporating an anti-a/anti-b specific immunoaffinity chromatography (iac, igisolo tm ) step in the manufacturing process (since ) and d) with both measures (b and c) combined; as well as for an ivig product produced with a cohn-like cold ethanol fractionation process (carimune ® nf/sandoglobulin ® , csl behring). isoagglutinin titers in ivig products, measured by european pharmacopoeia direct assay, were provided by dr c bellac, swissmedic, bern, switzerland. the predicted risk was highest with the chromatographically purified ivig without isoagglutinin reduction ( . cases expected per kg ivig used). anti-a donor screening reduced the predicted risk to . cases/ kg. a greater risk reduction was predicted with the iac isoagglutinin reduction step ( . cases/ kg). the combination of both methods produced little benefit ( . cases/ kg) versus iac alone. the predicted hemolytic risk with ivig produced by cohn-like ethanol fractionation was low ( . cases/ kg). an observational cohort study to confirm these hemolytic risk reductions is in progress. at present, the observed hemolytic risk for anti-a donor screening appears consistent with the prediction calculated by the model; results for iac isoagglutinin reduction are expected in . the cmt infant scale (cmtinfs) is an outcome measure of functional ability for young infants and children aged < years. cmtinfs aligns with the cmt pediatric scale and cmt neuropathy score to measure disease severity across the lifespan. to measure gross motor and fine motor function, cmtinfs comprises of two subscales: gross motor (e.g. head control, crawling, walking, jumping and hopping) and fine motor function items (e.g. grasping, reaching, tearing paper and buttoning). overall and subscale-specific function is expressed as a z-score based on normative reference values (positive z-scores indicate poorer function). a total of controls aged - months (mean age , sd m) have been assessed across australia (n= ), thailand (n= ) and usa (n= ). total cmtinfs z-scores did not differ significantly between sites (australia vs thailand) (p= . ) or gender (p= . ). data collection is ongoing and infants aged < years are eligible for inclusion. to date, infants ( % male) aged - months (mean age , sd m) with a range of cmt subtypes ( cmt a, cmt d, cmt c, cmt x and unidentified gene) have been assessed with cmtinfs. mean total z-score for infants with cmt ( . , sd . , range: − . - . ) was significantly higher than controls ( . , sd . , range: − . - . , t=− . , p= . ). differences between affected infants and controls were larger in infants older than months. infants with cmt a (cmtinfs z-score . , sd . ) and cmt c (z-score . ) were less affected than cmtx (z-score . ) and cmt d (z-score . ). the gross motor function subscale differed significantly between cmt cases and controls ( . , sd . vs . , sd . ; p=. ) and a significant difference was also observed for the fine motor function subscale ( . , sd . vs . , sd . ; p= . ). reliability, factor and rasch analysis of the cmt-infs is underway to assess validity. initial results support the sensitivity of cmtinfs in distinguishing between infants with and without cmt. preliminary analyses also suggest the scale is sensitive to genetic subtype. with increased power, cmtinfs promises to become a useful outcome measure of disease severity and function in infants with cmt. manso c , querol l , mekaouche m , illa i , devaux j . aix-marseille université, marseille, france; universitat autónoma de barcelona, barcelona, spain. contactin- , contactin-associated-protein- (caspr ), and neurofascin- (nfasc ) are essential for the formation of paranodal axoglial junctions. igg autoantibodies to contactin- , caspr , and nfasc are associated with subsets of patients with chronic inflammatory demyelinating polyradiculoneuropathy (cidp) presenting with common clinical features. anti-contactin- igg autoantibodies have been shown to be pathogenic and to affect the paranodal axoglial junctions in vivo and in vitro. by contrast, the pathogenic effect of anti-nfasc igg have not been demonstrated. here, we purified anti-nfasc igg from cidp patients' plasma and investigated their effects after passive transfer. to determine whether these antibodies can pass the paranodal barrier, we performed intraneural injections of anti-nfasc igg autoantibody. by contrast to anti-contactin- igg , anti-nfasc did not penetrate the paranodal regions after intraneural injections, but bound to the surface of the schwann cell. to perform chronic exposure, lewis rats were implanted with intrathecal catheter and anti-nfasc igg were administrated in a daily manner during three weeks. igg to nfasc , but not control igg , induced progressive clinical deteriorations characterized by gait ataxia and hindlimb paraparesis. these deteriorations were associated with nerve activity loss in motor spinal nerves and with a selective loss of the paranodal specialization characterized by the disappearance of the caspr /contactin- /nfasc complex at paranodes. the passive transfer of anti-nfasc igg thus seem to induce similar pathogenic effects as the anti-contactin- igg . however, the pathogenic mechanisms leading to paranode disappearance appear different. our findings indicate that igg directed against nfasc are pathogenic and further show that these antibodies are reliable biomarkers of a specific subset of cidp patients. better fit the needs of the consortium and improve patient experience. these enhancements include creating mobile friendly webpages, updating enrollment form content, access to a customized dashboard, and the ability of registrants to explore their data in comparison to other registrants. we will review these enhancements in depth, and demonstrate their impact on the growth and development of the rdcrn inc contact registry. martinez c , hubsch a , watson dj , shebl a , wallenhorst c , simon tl . institute for epidemiology, statistics and informatics gmbh, frankfurt, germany; csl bering ag, bern, switzerland; csl behring llc, king of prussia, usa; csl bering gmbh, marburg, germany. hemolytic anemia (ha) is a complication of intravenous immunoglobulin (ivig) treatment, particularly in patients receiving high dose ivig for immune modulation, such as guillain-barré syndrome or chronic inflammatory demyelinating polyneuropathy. the primary mechanism for the increased risk is believed to be passive acquisition of anti-blood group a and b antibodies (isoagglutinins) from the ivig product. to reduce the quantity of isoagglutinins, an anti-a donor screening was implemented for the ivig privigen ® from - and donors with high titers were excluded from contribution to pooled plasma. anti-a donor screening was replaced since with an immunoaffinity chromatography step, which decreases isoagglutinins to a greater extent, but no data are available to test its clinical effectiveness. to test the effectiveness of the donor screening, two cohorts of patients treated with privigen ® before and after start of donor screening were identified from a hospital-based administrative database of us hospitals with in-and outpatient discharge diagnoses, procedures, drug utilization and laboratory tests between / and / (period ) and between / and / (period ). privigen ® dose per kg body weight was estimated from the daily quantity administered and age-and sex-specific us population body weight estimates. ha within days of privigen ® use was assessed from manual records review and the incidence rate of ha in the two periods compared. incidence rate ratios (irr) of ha were adjusted for sex, age, treatment setting, indication and dose per kg body weight using period as reference. the incidence rate of ha was . / , person-days ( % confidence interval: . - . ) in period ( has in , person-days) and . ( . - . ) in period ( has in , person-days). the adjusted irr was . ( . - . ). significantly less ha risk was found with high dose (≥ . g/kg body weight) privigen ® , irr . ( . - . , p= . ). we conclude that anti-a donor screening and exclusion of donors with high anti-a titers from plasma pools is associated with a decreased risk of ha with ivig. matsumoto a . department of neurology, keijinkai jozanki hospital, sapporo, japan. subacute myelo-optico neuropathy (smon) is the intoxication of clinoquinol with main clinical symptoms of paresthesia and spasticity of legs. these symptoms have been considered to be elicited by the disturbance of spinal cord and peripheral nerve as the intoxication of clinoquinol. however, as to the patients with smon who have been still living after the onset of disease, the examination of nerve conduction velocities are in normal ranges and the clinical symptoms of peripheral neuropathy are not observed now. in order to investigate whether the peripheral neuropathy were observed in the early stage of smon, we investigated the longtidunal changes of electrophysiological results in patients who could examine the nerve conduction studies from early stage of smon until the present time. as to the disturbance of pyramidal tract functions (myelopathy) in smon patients, the central motor conduction times were calculated by transmagnetic stimulation of motor cortex, cervical roots and lumbar roots. the peripheral nerve conduction velocities of sensory nerve were examined with the sural nerves. as the results, in patients with smon who could examine the electrophysiological examination from the early stages of smon until to years later, the central motor conduction times of leg muscles from motor cortex to lumbar roots were prolonged in the smon patients compared to the normal cases. these results suggest the presence of disturbances of conduction velocities of spinal cord. conduction velocities of sensory nerve velicities (sncvs) showed the delayed sncvs of sural nerves( - m/sec) at the first examination from the onset of - years. however from to years later, sncvs of these cases were covered to - m/sec. from these electrophysilogical examinations, it was suggested that the presenting main symptoms of smon were the disturbance of myelopathy, and the disturbance of peripheral nerve function had been recovered after onset of smon being elapsed a long time, natural killer (nk) cells are part of our innate immune system with regulatory and effector functions. they comprise the first line of defence in the recognition and destruction of virus-infected and pathologically altered cells. different studies suggest that the treatment with intravenous immunoglobulins (ivig) has an immunomodulatory effect on nk cells. ivig is a first-line treatment for various autoimmune diseases in particular in chronic inflammatory demyelinating polyneuropathy (cidp). the lack of a predictive marker for ivig responsiveness in cidp avoids the early preservation of non-responding patients. to better understand the effect of ivig in patients with cidp, we tested whether ivig treatment altered the nk cell status. additionally, we analysed if the alteration in the populations may serve as a surrogate marker in predicting the outcome of ivig treatment. using semi-quantitative pcr and flow cytometry in the peripheral blood of patients with cidp, we analysed the effects of ivig on the nk cell population before treatment initiation and h after first dose and correlated the changes with the reponsiveness to ivig. ivig administrations induced a reduction in the expression of several typical nk cell genes. interestingly, this ivig-induced reduction of nk cells was reversible four weeks after the ivig treatment. flow cytometry data revealed that ivig reduced the cytotoxic cd dim nk cell population, while regulatory cd bright nk cells remained almost unaffected or were even increased. interestingly, we found that the observed effects on nk cells almost exclusively occurred in cidp patients who responded to ivig therapy. correlation between the changes in the nk cell population and treatment efficiency suggests a crucial role for nk cells in the immunomodulatory mechanism of ivig. further studies are warranted to investigate whether the differences in the nk cell status of patients with cidp represent a reliable surrogate marker in predicting the outcome of ivig therapy. mccray b , sullivan j , woolums b , aisenberg w , lloyd t , sumner c . johns hopkins university, baltimore, usa. mutations in transient receptor potential vanilloid (trpv ), a calcium-permeable non-selective ion channel, cause charcot-marie-tooth disease type c (cmt c). trpv is unique in that it represents the only membrane-expressed ion channel in cmt and thus a potential therapeutic target. previous work has suggested that trpv mutations lead to gain of channel function and toxicity in cultured cells. neuropathy-causing mutations of trpv largely cluster in the cytosolic ankyrin repeat domain (ard) that is known to mediate protein-protein interactions, suggesting that pathogenesis may be related to disruption of such interactions. in order to identify trpv -interacting proteins, we performed two unbiased proteomics screens and identified multiple cytoskeletal-modifying proteins including syndapin- , a neuronal protein known to promote axonal outgrowth by influencing the actin cytoskeleton. in cultured cells, we have shown that trpv and syndapin- co-localize to highly dynamic actin-rich cellular processes and together stimulate robust neurite extension, but this facilitation of neuritogenesis is impaired by disease-causing mutations in trpv . we have also shown that over-expression of syndapin reduces trpv -mediated calcium influx in cultured cells and rescues toxicity of mutant trpv . in addition, syndapin over-expression suppresses mutant trpv phenotypes in a drosophila model of trpv -related neuropathy. further, we have demonstrated that treatment of drosophila with a specific trpv channel antagonist ameliorates trpv mutant toxicity. together, our data highlight the importance of trpv interaction with cytoskeletal proteins such as syndapin- in the pathogenesis of cmt c. specifically, our results suggest that mutations in trpv disrupt the normal role of trpv in regulation of cytoskeletal dynamics and that interactions with the cytoskeleton reciprocally modulate trpv channel function and influence toxicity of mutant trpv . mcgonigal r , yao d , barrie ja , crawford c , willison hj . university of glasgow, glasgow, uk. guillain-barré syndrome (gbs) is in part mediated by anti-gm ganglioside antibodies induced by preceding infections. anti-gm antibodies target plasma membrane gm that is extensively distributed in both glial and axonal membranes, particularly at the node of ranvier. antibodies deposited at this site in models of gbs are associated with complement deposition, conduction block, structural disruption of ion channels and macrophage infiltration. the wide distribution of the gm ganglioside target leads to unwanted complexity in ascribing pathological outcomes to injury of cell-specific membranes, in particular unravelling the consequence of paranodal schwann cell membrane injury on axonal function, and vice versa. to overcome this impasse, we have generated transgenic mice through glycosyltransferase manipulation that express gm exclusively in neurons or glia, thus allowing us to very specifically target and injure axonal or glial membranes with a single anti-gm ganglioside antibody. through this route we can create mouse models of both the axonal and demyelinating forms of gbs, induced by a single anti-gm antibody, thus creating otherwise highly comparable conditions. here, we show anti-gm antibody binding is restricted to the nodal axolemma in galnact −/− -tg(neuronal) mice and conversely to paranodal loops in galnact −/− -tg(glial) mice. when anti-gm antibody and a source of complement is added to a nerve-muscle ex vivo injury paradigm, there is a loss of axonal integrity (i.e. loss of neurofilament immunolabeling) when the neuronal membrane is targeted in galnact −/− -tg(neuronal). conversely, axonal integrity is maintained when the paranodal membranes are decorated by antibody and complement products ex vivo in galnact −/− -tg(glial) mice. in a passive immunisation model in vivo, galnact −/− -tg(neuronal) mice acutely develop weakness, respiratory dysfunction, associated complement deposition, and degenerative pathology in distal axons. in contrast, galnact −/− -tg(glial) mice have significantly fewer abnormalities under the same acute conditions. these data indicate the high vulnerability of axonal membranes to acute injury and underline the importance of developing specific axonal protection strategies. in summary, targeting the nodal axolemmal or glial membranes allows us to study associated nodal pathology, and determine the downstream consequences on function and axon fate, currently a major area in gbs clinical research. memon a , madani s , schultz l , grover k , arcila-londono x , sripathi n , ahmad bk . neuromuscular division, department of neurology, henry ford hospital, detroit, michigan, usa. objective: to differentiate sensory electrophysiology, tli and treatment response in patients with paraproteinemic cidp. background: low tli has been reported as a useful electrophysiological marker for mag-cidp. to our knowledge comparison of sensory electrophysiology and tli of paraproteinemic cidp subgroups have not been previously reported. methods: retrospective review(january -december ) of patients with cidp fulfilling electrophysiological criteria(aan ad hoc subcommittee and albers and colleagues).cidp patients with diabetes(n= ) were excluded. patients were divided into idiopathic (n= ) and paraproteinemic cidp(n= ). paraproteinemic cidp sub-groups: mag( ), non-mag( ) and igg( ) were compared to idiopathic cidp( ). these groups were compared for demographics, history of cancer, csf protein, sensory conductions, tli measurements and response to treatment using chi-square tests for binary and categorical variables and t-tests for continuous measures. results: there was a higher proportion of females in idiopathic-cidp compared to non-mag-cidp ( % vs %). idiopathic group having a higher proportion of patients on monotherapy( % vs %) and combination therapy( % vs %) compared to non-mag. higher mean csf protein compared to mag-cidp(p= . ) was seen in the idiopathic. the difference between idiopathic and igg-cidp was significant for overall rx response(p= . ) and rx response in patients with follow-up(p= . ). for both variables, patients in the idiopathic group had a higher proportion of patients on combination therapy and lower proportion of no treatment offered compared to patients in the igg-cidp. % of non-mag-cidp patients had a history of cancer vs % of mag-cidp. none of the other differences were significant. there were no group differences in sensory electrophysiology and tli. conclusions: sensory electrophysiology and tli may have no value in differentiating paraproteinemic cidp. csf protein is higher in idiopathic cidp compared to mag-cidp. idiopathic-cidp has a higher proportion of females compared to non-mag-cidp and a higher proportion of patients on combination therapy compared to igg-cidp. cancer screening should be considered in patients with non-mag-cidp. memon a , madani s , ahmad bk , schultz l , grover , arcila-londono x , sripathi n . department of neurology, henry ford hospital, detroit, michigan, usa. introduction: sensory electrophysiology and terminal latency index (tli) differences have been described in various cidp sub-groups. objective: evaluate electrophysiology, tli and treatment response in idiopathic and diabetic cidp. methods: retrospective review of patients with cidp who underwent electrodiagnostic evaluation (january -december . patients fulfilled electrophysiological criteria described by ad hoc subcommittee of american academy of neurology (aan) and albers et al. we excluded patients( ) with acute inflammatory demyelinating neuropathy, hereditary sensorimotor neuropathy, vasculitis and polyneuropathy with paraproteinemia. patients were divided into idiopathic( ) and diabetic( ) groups. these groups were compared for age, sex, history of cancer, csf protein, response to treatment, sensory response abnormalities and tli measurements using chi-square tests for binary and categorical variables and t-tests for continuous measures. all testing was at the alpha= . level. results: group differences for age, sex, history of cancer, csf protein and treatment response were not significant. comparing tli values in measurable responses, the difference between the two groups for tibial tli was significant (p= . ), with idiopathic group having a lower mean as compared to the diabetic. tli values differences for median, ulnar and peroneal nerves were not significant. the difference in abnormal rates of sensory responses was significant for the sural nerve with the idiopathic group having a lower rate compared to the diabetic group ( % vs %, p< . ). no differences were noted for the ulnar, median and radial nerves. tibial tli and sural sensory responses have some value in differentiating the two groups. larger prospective studies are needed to confirm our findings. ivig is an important treatment option for cidp. although recommended by treatment guidelines, little is known about the consequences of temporary ivig withdrawal to assess ongoing immunoglobulin need. path is a randomized, double-blind trial of the subcutaneous immunoglobulin (scig) igpro (hizentra ® , csl behring) in cidp. before scig randomization, subjects underwent periods of ivig withdrawal ( weeks or until pre-determined indications of clinical deterioration) and ivig restabilization (igpro ; privigen ® , csl behring). subjects not showing deterioration during withdrawal period were withdrawn from study. to proceed to scig randomization, subjects had to achieve "cidp stability" (no relevant change in incat score at last two restabilization visits and at least the same total score as at screening). subjects entered the ivig withdrawal period. ( %) of these qualified for igpro restabilization; subjects ( %) were not ivig dependent, and ( %) were withdrawn for other reasons. one subject withdrew consent before igpro dosing. at the end of the igpro restabilization period, cidp stability was achieved in % of subjects ( did not reach stability, were withdrawn for other reasons). post-study follow-up information was available for / subjects who did not reach stability: ( %) had improved to baseline clinical status and had not, meaning at least % of the subjects improved to their pre-study status. during the restabilization period, / subjects ( %) improved in at least one of the predefined outcome measures. on average, subjects improved by . points in incat total score, . points in i-rods centile score, kpa in mean grip strength (dominant hand), and . points in mrc sum score. improvement occurred with a median of days after the first igpro dose in one or more efficacy outcome measures and in % of cases after the third igpro maintenance infusion. headache and nasopharyngitis were the most frequently reported adverse events (aes) during restabilization. aes deemed causally related were mostly mild or moderate. no unexpected aes or laboratory or vital sign findings associated with igpro occurred during the study. in summary, igpro reversed neuromuscular disability and improved activity/participation after previous clinical deterioration during an ivig withdrawal period. pmp duplication is the most frequent cause of charcot-marie-tooth disease (cmt). since it discovery, more than genes have been identified to be potentially responsible for cmt disease. however only single nucleotide variations (snvs) or small indels have been described. this could be due to the new sequencing strategy (ngs), especially ngs by amplicon sequencing, for whose few convenient tools are available and easily usable to detect cnvs responsible for inherited disease. to overcome this problem, we designed "cov'cop", a user-friendly tool able to detect cnvs among amplicons sequencing data. using the run's coverage file provided by the sequencer, "cov'cop" simultaneously analyzes all the patients of the run using a two-stages algorithm containing correction and normalization levels and provides an easily understandable output, showing with various colors, potentially deleted and duplicated amplicons. we validated our method on several datasets, including those of our targeted ngs panel screening genes known to be involved in cmt and close pathologies. cov'cop detected easily pmp duplication and deletion in our patients, confirmed by mlpa. in addition, cov'cop permitted the detection of new cnvs different from the pmp duplication, in cmt patients. we confirmed these cnvs by quantitative pcr and cgh array. we present here one of these cnvs: the duplication of aars gene detected in cmt patients and we discuss the pathogenicity of this new cnv. additional cnvs responsible for cmt disease are probably still to be discovered and we believe that cov'cop will help molecular geneticists to rapidly identify them. poems (polyneuropathy, organomegaly, endocrinopathy, m-protein, and skin changes) syndrome is a rare cause of demyelinating neuropathy associated with plasma cell dyscraisia and vascular endotherial (vegf) overproduction. although number of therapeutic interventions for plasma cell dysorders have been applied to poems syndrome, there have been no randomized clinical trials. this phase / double blind, randomized, placebo comtrolled trial was performed to investigate the safety and efficacy of thalidomide for patients with poems syndrome who are not eligible for stem-cell transplantation. the primary endpoint was the reduction rate of serum vegf concentrations at weeks in intention to treat analysis. additional outcomes of long-term extension study included progression-free survival. twenty-five poems patients were randomly assigned to either thalidomide plus dexamethasone or placebo plus dexamethasone from nov , , to july , . one patient in the placebo group was excluded from analyses because of a protocol violation. the adjusted mean serum vegf reduction rate at weeks was . (sd, . ) in the thalidomide group compared with − . ( . ) in the placebo group (p= . ). the kaplan-meier rate of progression-free survival at months was . in the thalidomide group, as compared with . in the placebo group (hr, . ; % ci, . to . ). in the randomized study period, mild sinus bradycardia was more frequent in the thalidomide group than in the placebo group ( % vs %; p= . ). thalidomide suppresses serum vegf concentrations and lengthened pregression-free survival in poems patients who are ineligible for stem cell transplantation. although thalidomide treatment has a risk of bradycardia, the benefits would exceed the risk. this study is registered with the umin clinical trials registry, umin . montes-chinea ni , coutts m , vidal c , courel s , rebelo a , abreu l , zuchner s , saporta, ma , . department of neurology, university of miami, miami, usa; department of human genetics, university of miami, miami, usa. autosomal dominant mutations in synaptotagmin- (syt ), a synaptic vesicle protein that functions as a calcium sensor for neurotransmission, have been previously linked to presynaptic neuromuscular junction (nmj) dysfunction and motor neuropathy in two families. both pathogenic mutations (asp ala and pro leu) were located in the c b domain of syt , which is essential for neurotransmitter release at the nmjs. we report a family with a new missense mutation in the c b domain of syt and a similar phenotype characterized by a slowly progressive, predominantly motor neuropathy and evidence of presynaptic nmj dysfunction on nerve conduction studies. the index case is a year-old woman with gradually progressive weakness of her extremities. she had normal developmental milestones, but was found to have bilateral high arched feet and hammertoes and occasional falls around the age of . she gradually developed progressive leg weakness, worsening bilateral hand cramping, weak handgrip, and only mild paresthesias on distal extremities. family history is remarkable for similar symptoms reported by her maternal grandfather, two maternal uncles, her mother and a younger sister. her neurological exam revealed inability to walk on heels or toes, significant distal lower extremity weakness and absent ankle deep tendon reflexes. cranial nerve examination and coordination were normal and there were only non-specific sensory changes in the lower extremities. emg/ncs revealed normal sensory responses throughout; however, motor nerve evaluation demonstrated globally reduced amplitudes with a > % increment after brief isometric contraction. further electrophysiological evaluation with slow ( hz) repetitive nerve stimulation of the right ulnar motor nerve revealed a % decremental response in amplitude and a > % increase in amplitude immediately after a one-minute period of sustained muscle contraction, which rapidly extinguished after one minute. voltage-gated calcium channel (vgcc) antibodies and a chest ct were normal. targeted sanger sequencing revealed an ile lys mutation in syt , which is located in the c b domain and is predicted to impair protein function. syt -related neuropathy is a rare disease, but should be suspected in patients presenting with a combination of pre-synaptic nmj dysfunction (resembling lambert-eaton myasthenic syndrome) and a predominantly motor neuropathy, especially in the context of a positive family history. charcot-marie-tooth (cmt) disease affects roughly in , individuals and is described as an inherited peripheral neuropathy primarily affecting distal muscles. limited studies detail patient-reported impact of muscle weakness on functional activities. this anonymous survey was developed with input from clinical experts and patient interviews and aimed to better understand the prevalence and impact of various cmt clinical manifestations on patients' lives. the survey was administered online to the hereditary neuropathy foundation's (hnf) patient contact database and is ongoing through june . here we present preliminary data on patient characteristics and disease impact for cmt patients collected february - , . respondents were mostly female ( %) and mostly from the us ( %). median age (range) at symptom onset was years ( - years), at diagnosis was years ( - years), and at present was years ( - years). the sample was representative of all cmt types (cmt , , , ,and x). the most common physical and clinical manifestations of cmt were problems with balance ( %), ankle weakness/foot drop ( %), loss of feeling or abnormal sensation in the lower leg/foot ( %), and hand muscle weakness ( %). maintaining balance, walking long distances, and climbing up and down stairs were key challenges associated with ankle weakness/foot drop. foot drop was considered by % to be the primary factor contributing to falls, which averaged . falls to ground per month. of those with foot drop, % had bilateral weakness. a majority of respondents ( %) used some form of assistive device for mobility, including ankle-foot orthotics/below-the-knee leg braces ( %), canes/walking sticks ( %), and custom foot orthotics/inserts ( %). the most common drug therapy included pain and anti-inflammatory medications ( %). foot surgery was the most common surgical procedure received ( %) and toe surgery was the most common surgery considered ( %). key symptoms that affected quality-of-life "very much" included problems with balance ( %), ankle weakness (foot drop) ( %), and fatigue ( %). these data suggest a high prevalence of lower leg muscle weakness; therefore, therapies aimed at improving ankle weakness and the resulting foot drop and imbalance may be beneficial to patients' daily functioning and quality of life. morano m , , gambarotta g , ronchi g , , cillino m , fornasari be , , fregnan f , , tos p , cordova a , moschella f , geuna s , , raimondo s , . department of clinical and biological sciences, university of turin, orbassano (to), italy; neuroscience institute of the "cavalieri ottolenghi" foundation (nico), university of turin, orbassano (to), italy; plastic and reconstructive surgery. department of surgical, oncological and oral sciences, university of palermo, palermo, italy; hand microsurgery and surgery, gaetano pini hospital, milan, italy. nerve fiber regeneration and complete functional recovery after peripheral nerve injury do not always occur and can be influenced by many factors including patient age, gender, lesion site, injury severity, size of the gap between damaged nerve stumps and time interval that elapses before performing surgical repair. the poor outcome occurring after a long delay can be due to loss of the neuron ability to regenerate, loss of the schwann cell ability to support regeneration and, of course, progressive muscle atrophy. the aim of this study was to investigate the nerve regeneration after delayed repair and to study the degenerative processes of the denervated distal nerve stump and denervated muscle. in particular, the analyses were focused on the role of nrg/erbb system, that is expressed both in nerve and in muscle tissue, during degenerating and regenerating processes. functional recovery analysis performed after nerve repair showed that only the group repaired immediately and not the groups repaired with a delay of or months, recovered partially. nevertheless, morphological analyses demonstrated that, despite the delay, the nerve fibers are still able to regenerate, even if they are fewer and smaller than the immediate repaired group. moreover, the analysis of the nrg /erbb system showed a significant decrease of soluble nrg in both degenerating and delayed-repaired nerves. the poor outcome after delayed nerve regeneration might be explained by schwann cell impairment and the consequent ineffective support for nerve regeneration. as regards denervated muscle analysis, results showed that erbb receptors expression is related to the innervated state of the muscle, with an upregulation of erbb clearly associated with denervation state. interestingly, nrg isoforms are differently regulated depending on the type of nerve injury. future experiments will be needed to address the in vivo efficacy of different isoforms of nrg both in injured nerve and denervated muscle. we planned a multicenter, prospective, randomized, single blind, controlled study to evaluate the efficacy and safety of an innovative rehabilitation protocol based on the use of treadmill training in a cmt a population. the protocol required that subjects were blindly randomized into two treatment groups, spe (three months of respiratory, proprioceptive and stretching exercises) or trespe (the same treatment plus aerobic training at the treadmill). subjects were evaluated at baseline (t ), after three month of treatment (t ) and further three months of follow up free of therapy (t ). the full assessment included: -mwt (primary outcome measure), -mwt, walk- , short physical performance battery (sppb); lower limbs dynamometric strength evaluation; berg balance scale (bbs); cmt neuropathy score; medical outcomes study short form (sf ). a total of subjects (mean age of . ± . years) were recruited. at t we found a significant improvement in both groups in the -mwt (p< . ), -mwt (p< . ), bbs (p< . ) and sppb (p< . ), while at t only the -mwt was still significantly improved (p< . ) in the spe group. no significant differences between groups were observed for any of the outcome measures. performances on walk did not significantly change during follow-up (p= . ). concerning the sf , we did not observe consistent changes during follow-up or consistent differences comparing the two treatments. in conclusion, this multicenter, prospective, randomized, single blind, controlled study shows that the combination of respiratory, proprioceptive and stretching exercises has a positive impact on the performance of cmt patients, especially regarding walking tests. the aerobic exercise at the treadmill, is well tolerated but apparently does not add any further improvement to the conventional treatment. we speculate that the relatively low clinical severity of the patients, due to the selection criteria, may have prevented a positive effect of treadmill exercise. müller m , dohrn m , romanzetti s , reetz k , gess b . university rwth aachen, department of neurology, aachen, germany. muscle mri is increasingly used in neuromuscular patients to detect changes in muscle volume, muscle fat infiltration and edema. muscle mri are mostly analyzed by qualitative means as quantitative analysis is time-consuming and not well established. here, we developed a novel method for semi-automated segmentation of muscle mri data sets. based on axial t -weighted dixon mri stacks, muscle volumes were quantified by an adapted water-shed algorithm. muscle volumes of thighs and calves were determined separately and the ratio of thigh/calf was calculated. myopathy, neuropathy patients and healthy controls were included in the study. muscle volumes determined by semi-automated segmentation were very similar to manually segmented data sets, differences being < %. this was the case for patients as well as healthy controls. the time-saving effect of automated segmentation was very strong ( vs min. per patient). muscle volumes of the thigh and also of the calf of myopathy patients showed a highly significant difference (p< . ) compared to healthy subjects. in neuropathy patients there was a just significant difference (p< . ) of muscle volumes compared to healthy patients that did not sustain in bonferroni's multiple comparison test. the ratio of thigh/calf muscle volume was significantly different comparing patients with myopathy and neuropathy (p< . ). subgroup analyses of different groups of myopathy patients showed highly significant differences (p< . ) in myositis, limb-girdle-muscular dystrophy and metabolic myopathy, compared to healthy patients, but no significant differences in-between these groups. taken together, the data shows that automated segmentation of muscle mri allows for exact and fast quantification of muscle volumes in neuromuscular patients. higher patient numbers are necessary to test differences between specific disease groups. further studies should also address the possible use as a marker of disease progress for clinical studies or therapy monitoring. murakami t , nishimura h , nagai t , hemmi s , kutoku y , sunada y . department of neurology, kawasaki medical school, kurashiki, japan; department of pathology, kawasaki medical school, kurashiki, japan. familial amyloidotic polyneuropathy (fap) is an autosomal dominant hereditary systemic amyloidosis caused by mutation of transthyretin (ttr) gene, and usually shows sensory dominant polyneuropathy and autonomic neuropathy at the initial stage. the pathogenesis of neuropathy is not well understood, and explained by several mechanisms, including such as mechanical compression, vessel occlusion, ttr toxicity and schwann cell dysfunction. we describe a sporadic patient with late-onset fap due to ttr e k. she noticed dysesthesia first in the foot at age . the symptoms were slowly progressive, and abnormal sensations were extended up to the both upper arms and the both knees at age . distal muscle weakness and atrophy was also observed in the extremities. she noticed difficulties in walking and frequent diarrhea. echocardiogram revealed diffuse left ventricular hypertrophy, suggesting cardiac amyloidosis. amyloid deposits were not detected in the endoneurim or perineurium of the sural nerve years after the onset of the disease, but a marked loss of myelinaed and unmyelinated nerve fibers was observed in it. ttr-derived amyloid deposits were confirmed in the peroneous brevis muscle, salivary gland and heart tissue. dna analysis revealed the heterozygote mutation, p.e k (e k)/c. g>a, of ttr gene, and she was diagnosis as fap. these findings suggest that the proximal parts of peripheral nervous system might be strongly involved by ttr aggregates or amyloid fibrils. blood-nerve barrier in the distal part of peripheral nerves could be preserved until later in the patient. several biopsy sites other than nerve may be helpful and necessary for diagnosis of ttr amyloidosis in mild or late-onset fap as our case. amyloidgenic element (cae) encoded by the ' utr. this study also identified a de novo c. _ dup frameshift mutation predicting p.lys glnfs* in nefh from a cmt family with atypical clinical symptom of proximal dominant weakness. this mutation is located near the previously reported frameshift mutations, suggesting a mutational hot spot. these relatively frequent deletion/duplication events with this resign might be caused by the putative hairpin structure. patient's lower limb mri revealed a marked hyperintense signal changes in the hip muscles than those in the thigh or lower leg muscles. this study also observed an anticipation pattern of earlier onset ( yrs old for mother to yrs old for daughter) and more severe symptoms in later generation. therefore, this study suggests that the stop loss and translational elongations by the ' utr of the nefh mutations may be relatively a frequent genetic cause of axonal peripheral neuropathy with the specific characteristics of proximal dominant weakness and an anticipation pattern. neil j , haghi ashtiani b , choumet v , musset l , léger jm . department of immunology, pitié-salpêtrière hospital (ap-hp), paris, france; department of neurology, national referral center for rare neuromuscular diseases, pitié-salpêtrière hospital (ap-hp), paris, france; institut pasteur, emerging diseases epidemiology unit, paris, france. the myelin-associated glycoprotein (mag) is a transmembrane glycoprotein localized in periaxonal shwann cells and oligodendroglial membranes of myelin sheaths. mag contains a carbohydrate epitope (hnk- ) that is a target antigen in autoimmune peripheral neuropathy associated with monoclonal igm gammopathy. in these neuropathies, numerous studies report the absence of correlation between the titers of anti-mag antibodies and the disease course. anti-mag titers and igm level at diagnosis are not always associated with disease severity and there is not good correlation between pre-and post-treatment anti-mag titers in patients who respond clinically to immunomodulators. mag belongs to siglec- a family and the linkage of sialic acid to the underlying sugars is an important determinant of siglec binding. mag shows high affinity for alpha- , -linked sialic acid ( , -sa).moreover, human monoclonal igm possesses heavy chain glycosylation sites at asn , , , and with sialylated olidgosaccharides and high-mannose type oligosaccharides. igm may bind to mag via these glycan epitopes as an alternative and additional route of antigen binding other than through the fab v regions. this mag-glycans igm interaction may be clinically neutral but could lead to an overvaluation of the biological results. in this study, we analyzed sera from patients with igm reactivity against mag: of them had an anti-mag neuropathy with various degrees of severity, and the last one had igm monoclonal gammopathy, strong serum anti-mag reactivity but no neurological disease. igm were extracted and purified from these sera by affinity chromatography. for each batch, an aliquot was digested by jack bean alpha-mannosidase and anti-mag reactivity was performed by elisa and indirect immunofluorescence (iif), before and after demannosylation. these extracts, tested in an iso quantitative way with regard to the original serum, showed a decrease of activity (elisa) and intensity (iif) after demannosylation. furthermore, elisa anti-mag was carried out in sera from patients with igm monoclonal gammopathy without neurological impairment: of them ( . %) showed a significant biological response. taking into account the fact that anti-mag antibodies are pathogenic (in animals models), these results support the hypothesis of neutral intermolecular interactions between igm and mag. ng cjb , ng jph , tay lb, t , umapathi . yong loo lin school of medicine, national university of singapore, singapore; lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore. recent developments have validated non-invasive means of measuring central arterial blood pressure (casp) and have shown that casp and peripheral blood pressure (pbp) are unidentical entities. patients with postural orthostatic tachycardia syndrome (pots) have marked tachycardia with no associated decrease in pbp. we asked if the reflex tachycardia, which corresponds to postural dizziness in these patients, could be a result of a decrease in casp. two male patients, and years of age, with clinical features typical of pots went through a complete battery of autonomic screening tests. the sympathetic and parasympathetic responses were normal other than a heart rate increase of and beats per minute, respectively, on standing. there was no significant decrease or increase in pbp on standing for and minutes. casp was measured non-invasively by a device bpro r that imputes the measured radial waveform onto the brachial blood pressure to generate a pressure waveform from which a numerical casp value is derived. the casp measurements for both patients did not decrease on standing for and minutes. our preliminary observation suggests that the basis of tachycardia in pots patients may not be a decrease in central blood pressure. we are proceeding to systematically study more pots patients to corroborate the above observation. we are also trying to compare the difference between pbp and casp in pots and age, gender-matched normal controls. the major limitation of the study is the model-based mathematical derivation, rather than direct measurement, of casp. we are proceeding to systematically stud y more pots patients to corroborate the above findings. ng jph , ng cjb , t umapathi . lee kong chian school of medicine, nanyang technological university, singapore; yong loo lin school of medicine, national university of singapore, singapore; national neuroscience institute, singapore. recent developments have validated non-invasive means of measuring central arterial blood pressure (casp) and have shown that casp and peripheral blood pressure (pbp) are unidentical entities. orthostatic hypotension (oh) is a prominent component of autonomic dysfunction (ad), the consequent hypoperfusion of vital organs responsible for considerable morbidity and mortality. these structures are exposed to casp rather than pbp. we sought to understand the relationship of casp to peripheral blood pressure (pbp) in patients with autonomic dysfunction exposed to orthostatic stress. we reviewed autonomic function tests of patients tested at our laboratory over a -year period. the patients were divided into cohorts: ( ) no ad and no oh ( ) no ad, with oh ( ) mixed ad ( ) parasympathetic dysfunction ( ) sympathetic dysfunction. casp was measured non-invasively by a device, bpro r , that imputes the measured radial waveform onto the brachial blood pressure to generate a pressure-wave form from which a numerical casp value is derived. the difference and ratio of casp to pbp was recorded at rest and after minutes of standing. out of patients had complete data and a definitive final diagnosis. cohorts - had , , , , patients respectively. mean casp-pbp difference in cohorts - were − . , − . , − . , − . , − . respectively at minutes, and − . , − . , − . , − . , − . respectively at minutes. mean casp/pbp ratio in cohorts - were . , . , . , . , . , . respectively at minutes, and . , . , . , . , . respectively at minutes. there was no significant difference in the response of casp and pbp to orthostatic stress across abnormal cohorts to and in comparison with the "normal" cohort (p= . to . ). there was also no relationship to symptoms, namely postural dizziness. in conclusion, autonomic dysfunction does not seem to affect the casp-pbp relationship as measured by non-invasive means. the absence of true normal controls, the exclusion of significant number of patients because of incomplete data and the model-based mathematical derivation rather than direct measurement of casp are limitations that we aim to address in follow-on studies. niu jw , guan hz , cui ly , guan yz , liu ms . the department of neurology, peking union medical college hospital, chinese academy of medical sciences, beijing, china. we aimed to explore the correlation between afterdischarges in motor nerve conduction studies and clinical motor hyperexcitability in patients with voltage-gated potassium channels (vgkc) antibodies. six patients with positive serum antibodies to contactin-associated protein-like (caspr ) or/and leucine-rich glioma-inactivated protein (lgi ) were recruited, including with autoimmune encephalitis, and with cramp-fasciculation syndrome. electromyography (emg), nerve conduction studies (ncs) and f waves were performed, and afterdischarges were assessed. one patient was followed up. five patients had clinical evidence of peripheral motor nerve hyperexcitability (myokymia or cramp), and four of them had abnormal spontaneous firing in concentric needle electromyography. prolonged afterdischarges following normal m waves were present in all six patients, including the two patients who had no emg evidence of peripheral nerve hyperexcitability (pnh). in the patient who was followed up, afterdischarges disappeared after treatment with intravenous immunoglobulin (ivig). afterdischarges in motor nerve conduction study might be more sensitive than needle electromyography for detecting peripheral motor nerve hyperexcitability in patients with vgkc antibodies, and could disappear gradually in accordance with clinical improvement and reduction of antibodies. in our research, multiple sites measurement of cross sectional areas (csa) by ultrasound was performed to differentiate charcot-marie-tooth type a (cmt a) and chronic inflammatory demyelinating polyradiculoneuropathy (cidp). twenty-eight patients with cidp, patients with cmt a, and healthy controls (hc) were recruited prospectively. consecutive ultrasonography scanning was performed from wrist to axilla on median and ulnar nerves. csas were measured at predetermined sites of each nerve. cmt a had significantly larger csas at all sites of median and ulnar nerves (all p< . ). in cmt a, csas increased gradually and homogeneously from distal to proximal along the nerve, except potential entrapment sites. cidp displayed three different morphological patterns, including mild enlargement in patients, prominent segmental enlargement in , and slight enlargement in one, among which different treatment responses were observed. all patients with mild nerve enlargement treated with intravenous immunoglobulin (ivig) were responsive ( / ), while less than half of those with prominent segmental enlargement ( / ) were responsive (p< . ). the patterns of csa enlargement were different in cmt a and cidp patients. consecutive scan along the nerve and multiple sites measurement by ultrasound could supply more detailed morphological feature of the nerve and help to differentiate cidp from cmt a. guillain-barre syndrome (gbs) mainly consists of acute inflammatory demyelinating polyradiculoneuropathy (aidp) and acute motor axonal neuropathy (aman). in aman, conduction block (cb) could be reversible or followed by axonal degeneration. we aimed to identify the correlation between existence of cb and the functional outcome for patients with gbs. gbs patients were prospectively recruited for serial electrophysiological tests and disability evaluation. all patients received treatment of intravenous immunoglobulin (ivig), and their disabilities were evaluated on the hughes functional grading scale before and month after treatment. patients were classified into aidp, aman, equivocal or normal according to electrodiagnostic criteria described by rajabally et al. aman patients who had follow-up nerve conduction studies were further classified into three groups. group was typical aman without conduction block, group had reversible conduction block, group had conduction block and subsequential axon degeneration. electrophysiological study results showed aidp, aman, equivocal and normal. probable or definite conduction block was observed in aidp patients and aman patients. aman with cb had higher reduction of hughes grade at one month ( . ± . vs . ± . ,p= . ), and lower percentage of patients with slow recovery (unable to walk independently at six months) ( % vs %, p= . ) compared with aman without cb. there were no significant differences between aidp with cb and without cb, in the reduction of hughes grade at one month. among the aman patients who were followed up, were typical aman without cb (type ), had reversible cb (type ), had cb and subsequential axon degeneration (type ). hughes grades at nadir were similar, while patients with reversible cb (type ) had the largest hughes grade reduction at one month (type - . vs type - . vs type - . ). none of the patients with axon degeneration (type ) showed rapid recovery, while % of those with reversible cb (type ) had rapid recovery (improvement by two or more hughes grades within four weeks after onset). electrodiagnosis of aman with conduction block, especially reversible conduction block, might be a marker of good recovery. we report a follow-up study of nerve ultrasound in a patient with primary neurolymphomatosis. a -year-old female presented with -months history of asymmetric limb pain, paresthesia, and weakness. electrodiagnostic studies and spinal cord mri showed an axonal neuropathy involving cervical and lumbosacral root, brachial plexus and left median nerve. detection of malignant b lymphocytes by cytology and flow cytometry of cerebral spinal fluid confirmed the diagnosis of b-cell non-hodgkin lymphoma. nerve ultrasound showed dramatic enlargement of upper, middle and lower trunks of left brachial plexus (cross sectional area-csas mm , mm , mm respectively), middle trunk of right brachial plexus (csa mm ), and proximal part of left median nerve (csa - mm ). five months later, after five chemotherapy of rituximab and high-dose methotrexate, and intrathecal injection of cytosine arabinoside and dexamethasone, the patient had clinical improvement. nerve ultrasound also showed alleviation of nerve enlargement. the csas of upper, middle and lower trunks of left brachial plexus were mm , mm , mm respectively; the csa of middle trunk of right brachial plexus was mm ; the csa of proximal part of left median nerve was - mm . peripheral nerve ultrasound could help locate the distribution of nerve involvement, and reveals disease progression. nolano m , provitera v , stancanelli a , caporaso g , saltalamacchia am , borreca i , lullo f , califano f , lanzillo b , iodice r , manganelli f , barone p , santoro l . irccs "salvatore maugeri" foundation, institute of telese terme (bn), italy; "maugeri" clinical and scientific institutes irccs, institute of telese terme (bn), italy; center for neurodegenerative diseases (cemand), department of medicine and surgery, neuroscience section, university of salerno, italy. a peripheral nerve involvement has been demonstrated in pd with the evidence of a small fiber pathology as possible intrinsic feature of the disease and a higher occurrence of large fiber neuropathy in patients longtime treated with l-dopa. however the role that disease itself and ldopa have on small and large fiber pathology in pd is still debated. we studied morphology and function of cutaneous innervation, in idiopathic pd patients ( male, aged . ± . ), including naïve and l-dopa treated subjects without electrophysiological signs of neuropathy, with the aim to assess and characterize small and large fiber involvement and the effect of l-dopa on it. all patients underwent a screening to rule out potentially neurotoxic conditions such as glucose intolerance, dysendocrinopathies, vitamin e, b and folic acid deficiency, hepatic or renal failure, hiv or connective tissue disorders. skin biopsies were obtained from thigh, leg and fingertip from the more affected side and bilaterally from thigh and leg in patients. samples were processed with indirect immunofluorescence technique using primary antibodies to mark different sensory and autonomic fiber populations. density of intrapapillary myelinated endings (ime), meissner's corpuscles (mc) and epidermal nerve fibers (enfs) was obtained as well as a semi-quantitative assessment of sudomotor, pilomotor and vasomotor innervation. further evaluation included sympathetic skin response, quantitative sensory testing and dynamic sweat test. morphological and functional findings were compared with data extracted from our age and sex stratified normative dataset. ienf, ime, mc densities were lower (p< . ) compared to controls in both naïve and l-dopa treated patients without differences between them except for mc density that was lower in l-dopa treated subjects ( . ± . vs . ± . /mm ). a loss of autonomic nerves was also found in both groups compared to controls. significant abnormalities (p< . ) of thermal sensory thresholds, tactile thresholds, mechanical pain perception and reduced sweating output were present and similar in both groups. our work confirms in pd an intrinsic peripheral nerve pathology involving both small and large fibers. small fiber pathology isn't affected by l-dopa treatment while sensory large fibers involvement, already present in naïve patients worsens with ldopa treatment. noto y , garg n , li t , timmins hc , park sb , shibuya k , kiernan mc . brain and mind centre, the university of sydney, sydney, australia. the diagnosis of amyotrophic lateral sclerosis (als), a progressive, fatal neurodegenerative disorder defined by combined upper and lower motor neuron involvement, remains clinically based. the purpose of this study was to determine the ultrasound appearance of peripheral nerves in als patients, and to investigate whether parameters such as distal/proximal ratios of nerve cross-sectional areas (csas) may effectively differentiate disease mimics from als. nerve ultrasound of the median, ulnar, and tibial nerves was performed in als patients compared to mimic patients ( patients with peripheral nerve hyperexcitability syndromes (pnhs) and patients with multifocal motor neuropathy (mmn)). comparison of nerve and the distal/proximal ratios was undertaken by ultrasound and compared across clinical and neurophysiological parameters.compared to normal controls, csa of the median nerve at the upper arm was decreased in als (p < . ). in comparison to als mimic disorders, csa at the proximal site of the median, ulnar and tibial nerve and the forearm/upper arm ratio of the median and ulnar nerves had diagnostic values. in addition to csa of the median, ulnar, and tibial nerves, the median and ulnar nerve forearm/upper ratios may provide a useful marker in for the diagnosis of als. approximately in , individuals are diagnosed with charcot marietooth (cmt) disease, making it the most common hereditary peripheral neuropathy. there is no documented cure for cmt, however, many of those affected, report difficulty with mobility, imbalance, and weakness of the feet and hands. in the general population, patients reporting difficulty walking, falls and/or fear of falling, and poor strength are often referred to physical (pt) and occupational therapists (ot) to skillfully address the impairments and help restore function and quality of life (qol). for patients diagnosed with cmt this is unfortunately not the norm, often leaving patients without any skilled guidance on managing their functional impairments. patients ( males) with a mean age of . years ( - ), went through a progressive and skilled pt intervention over the course of months. the program included: therapeutic activities and exercise, neuromuscular reeducation, and manual techniques to address the documented deficits. patients were progressed through the program based on the borg scale. each patient was assessed prior to and post the commencement of the program with the berg balance scale, minute walk test, timed up and go, sit to stand in seconds, foot self selected and fast gait speed, the lower extremity functional scale, activities balance confidence form, upper extremity functional index, oswestry, and the sf- qol measure. the patients were seen by the same skilled pt - a week for weeks. all but patients improved in all measures taken, indicating an improvement in function and overall quality of life. participants reported a total of falls in the months prior to the initiation of the study and only fall was reported during the month pt intervention. this study makes a strong case for the utilization of skilled pt to address deficits in patients with cmt. additionally, the utilization of objective, valid and reliable outcome measures in this population may help healthcare practitioners establish baseline function and response to change. a large randomized control trial is recommended to study the effects of a specific pt intervention on outcome measures in patients with cmt. ogata h , fujita a , yamasaki r , matsushita t , kira ji . department of neurology, neurological institute, graduate school of medical sciences, kyushu university, fukuoka, japan. clinical features of chronic inflammatory demyelinating polyneuropathy (cidp) patients with autoantibodies against neurofascin (nf) , one of the paranodal proteins, have been elucidated while the relation between anti-nf antibody levels and long-term clinical course in these patients still remains elusive. we retrospectively collected clinical, electrophysiological and immunological data of three anti-nf antibody-positive cidp patients. they were all males and their ages at onset were , , and years old. their clinical severity was evaluated by deep tendon reflexes (dtrs), grip strength and hughes functional scale. anti-nf antibody levels were measured by flow cytometry using hek cell lines stably expressing human nf . after immunotherapies of various combinations, including intravenous immunoglobulin, plasmapheresis, corticosteroids and other immunosuppressants, were introduced, their clinical parameters were gradually improved. decreased or absent dtrs were normalized and grip strength was increased by more than kg. hughes functional scale scores were decreased by at least one point compared with those at nadir. ncs findings of all three patients also showed obvious amelioration. for example, their f wave latencies in the right ulnar nerve were improved from to ms, from to ms, and from to ms, respectively. anti-nf antibody levels after treatment were decreased in two patients whose pre-and posttreatment sera were available. when dose of oral prednisolone was being tapered, they experienced re-exacerbation of clinical parameters, especially dtrs and grip strength. their ncs findings and serum anti-nf antibody levels were also deteriorated. exacerbation of these laboratory data in one patient preceded his clinical fluctuation, which suggests that ncs and serum anti-nf antibody levels could be used as early disease activity markers. in this case series, not only clinical but also laboratory data support a notion that anti-nf antibody-positive cidp patients were reactive to combined immunotherapies including corticosteroids. even though various treatments were administered to them, efficacy of oral corticosteroids seemed to be dose-dependent. optimal disease activity markers and immunotherapies for long-term maintenance of remission in anti-nf antibody-positive cidp should be identified. evaluated by deep tendon reflexes (dtrs), grip strength and hughes functional scale, after starting immunotherapies, ohnmar o , kamyw , ng lfp , t umapathi . university of medicine , yangon, myanmar; singapore immunology network, a*star, singapore; national neuroscience institute, singapore. singapore's zika virus (zikv) outbreak started in late august . over a period of months, we studied patients enrolled into our institution's prospective guillain-barré syndrome (gbs) database for relationship to zikv infection. we also studied gbs controls that were seen before the established outbreak and non-gbs controls. the index cases tested negative for zikv pcr in blood and urine. we proceeded to test zikv igg, igm, dengue virus (denv) igg and igm, and neutralization assays against zikv and denv. one patient with anti-gq b igg positive miller fisher syndrome had detectable zikv igm and zikv igg. the serum showed low titre denv igm and denv igg. follow-up serum at about months showed increase in zikv igg. we believe this patient has zikv-gbs. one patient with acute motor sensory axonal neuropathy and another with acute inflammatory demyelinating polyneuropathy had high zikv igm but low denv igm and igg. another patient with mfs showed high levels of zikv and denv igm but low igg. the latter two patients had gbs before the zikv outbreak in singapore. we suspect these patients could have zikv-gbs, but are awaiting convalescent sera for confirmation. two patients seen during the outbreak had detectable levels of zikv igg but serial testing showed a decline after a period of - months. the initial and follow-up sera showed raised denv igm and igg levels in one and raised igg levels in the other. in addition, both had stronger neutralizing capacity against denv than zikv suggesting that the initially detectable zikv igg levels was due to cross reactivity with previous denv infection. four patients, gbs control and non-gbs controls also showed serological response consistent with previous exposure to denv. one normal control showed nil exposure to both viruses. in summary, using various overlapping serological methods we diagnosed definite and suspect zikv gbs cases. our findings highlight ) insensitivity of blood and urine pcr to diagnose zikv-gbs ) the problems of interpreting zikv serology from cross-reaction with denv ) serial serology increases diagnostic accuracy. okar sv , ergunay k , bekircan-kurt ce , , erdem-ozdamar s , , tan e , . department of neurology, hacettepe university, ankara, turkey; virology unit, department of medical microbiology, hacettepe university, ankara, turkey; neuromuscular disease research laboratory hacettepe university, ankara turkey. guillain-barré syndrome (gbs) is an acute monophasic immune-mediated polyradiculoneuropathy. it is believed that acute inflammatory demyelinating poliradiculoneuropathy is caused by t-cell mediated autoimmune response targeting peripheral nerve myelin. molecular mimicry plays a role in the pathogenesis of some gbs cases. this mechanism has been well demonstrated in the acute motor axonal neuropathy (aman) variant, in which autoantibodies to camphylobacter jejuni share epitopes with peripheral nerve gangliosides. this molecular mimicry mechanism can be attributed to some cases with atypical triggers such as zika virus or west nile virus infections. moreover there are accumulating clinical data for vector borne viral infections triggering gbs. we evaluated vector-borne viral infections in our gbs and aman patients. eight patients with gbs, two patients with aman and as a control group, seven patients with normal pressure hydrocephalus were included. gbs and aman was diagnosed with clinical, electroneuromyograpic and cerebrospinal fluid (csf) findings. cerebrospinal fluid serum and urine samples were examined for vector borne viral infections via generic flavivirus and phlebovirus pcr. we also documented our patients prognostic scores such as modified erasmus gbs outcome score (megos) and modified erasmus gbs respiratory insufficiency score (megris). the mean age of the patients was , ( - years) , six of them were female. all csf, serum and urine samples of our patients and control patients were negative for flavivirus and phlebovirus families. the preliminary results of our study in this our small cohort did not show any correlation between the vector-borne viral infections and gbs. further studies with broad number of patients are needed for more suggestive results. neuromyotonia (nmt) consists of spontaneous motor unit activity that reflects increased peripheral nerve excitability, leading to involuntary, persistent muscle activity, visible as muscle twitching at rest, with generalized, easily provoked cramps. since the first electrophysiological (emg) description of nmt by denny-brown and foley ( ) , there has been discussion about the origin of the abnormal electrical activity recorded in needle emg studies. we studied two patients. patient 's nmt is aggravated by cold, and he has an associated non-progressive mild polyneuropathy with demyelinating features. he is now -year-old and has been followed in our centre for years. he is negative for anti-vgkc antibodies. firstly, he was treated with carbamazepine and phenylhydantoin with poor response, but he has shown major improvement on intravenous immunoglobulin (ivig) during the last years. patient is a year-old man with nmt, followed for year, with high titters of anti-vgkc antibodies ( pmol/l; normal< ). he improved on ivig during the last months. screening for neoplasia was unremarkable in both patients (negative anti-neuronal antibodies, in particular anti-hu, anti-yo and anti-ri antibodies; normal computerized tomography scan of the chest and abdomen). in addition to routine studies, we tested synchronicity to spontaneous discharges in different motor units in simultaneous recordings made with two needle electrodes in the first dorsal interosseus muscle. time-locked fasciculations in these double recordings would represent abnormal ectopic activity initiated in a nerve trunk with ephaptic stimulation of a nearby axon. in patient , this research protocol was applied once, years after regular ivig treatment. patient was investigated before and year after ivig. both patients improved after ivig, mirrored by a striking decrease in the amount of spontaneous activity on emg. moreover, our technique did not detect synchronous spontaneous activity (time-locked fasciculations) on the second assessment, although this was predominant before treatment in patient . in nmt, abnormal discharges originate both in distal axonal branches and in more proximal segments. it appears that ivig is more effective in blocking antibody activity in proximal axonal segments, perhaps related to factors such as blood-nerve barrier, temperature or differing ion channel distributions. celiac disease (cd) is a chronic, multisystem and immune-mediated disorder characterized by small-bowel sensitivity to dietary gluten in genetically predisposed individuals. neurological manifestations may occur in about % of patients, including peripheral nerve involvement. recent growing evidence strongly suggests that peripheral neuropathy in cd may be autoimmune and associated with anti-ganglioside antibodies. a -year-old woman presented with slowly progressive weakness of her right hand and fingers extensors. medical and family history were unremarkable. on examination, muscle strength (medical research council-mrc) was scored for right wrist and finger extensors, for right abductor pollicis longus and for right ankle dorsiflexion. right triceps brachii and brachioradialis reflexes were weak, but normal elsewhere. the remainder of the neurological examination was normal. neuroaxis magnetic resonance was unremarkable, specifically with no gadolinium-enhancing lesions. motor and sensory nerve conduction studies were normal. no conduction block or abnormal temporal dispersion was found. needle electromyography showed severe neurogenic changes with abnormal spontaneous activity in right radial-innervated muscles, and chronic neurogenic changes in homolateral tibialis anterior, peroneus longus and extensor digitorum brevis. ganglioside antibody testing was positive to igg anti-gm antibody, but negative to anti-gm and other anti-ganglioside antibodies. additional blood tests were unremarkable, in particular cryoglobulin testing was negative. intravenous immunoglobulin improved weakness, as right extensors of the wrist and fingers scored (mrc). a monthly treatment was initiated, which after months was changed to every weeks to preserve function. a diagnosis of mama was established. later, cd was diagnosed in her daughter due to chronic diarrhoea. our patient underwent anti-tissue transglutaminase antibodies determination and small-bowel biopsy after years disease' duration, and a diagnosis of cd was made. gluten-free diet was started, but her neurological picture did not change after six months. in our case the presence of igg anti-gm antibody may support a causal link between mama and biopsy-confirmed cd, and the lack of response to gluten-free diet may be explained by chronic axonal injury induced by memory t-cells. this case broadens our knowledge about neurological manifestations in cd, raising a probable association with purely axonal multifocal motor neuropathies and anti-ganglioside antibodies. Õunpuu s , , pogemiller k , acsadi g , pierz k , , . center for motion analysis, connecticut children's medical center, farmington, ct, usa; school of medicine, university of connecticut, farmington, ct, usa; division of neurology, connecticut children's medical center, farmington, ct, usa; division of orthopaedics, connecticut children's medical center, farmington, ct, usa. orthopaedic surgical intervention of the foot and ankle is performed in persons with charcot-marie-tooth (cmt) for improving foot pain, ankle instability, orthosis fitting/comfort issues and shoe wear. the impact of these surgeries on ankle function during gait is not known. therefore, the goal of this study was to measure gait changes in ankle function following surgical intervention by means of computerized motion analysis. fourteen patients with cmt ( ± years pre and ± years post-operative) with bilateral orthopaedic surgery were included. all patients had a plantar fascia release plus some combination of other soft-tissue (posterior tibialis and tendo-achilles lengthenings; extensor hallucis longus, peroneus longus and anterior tibialis transfers) and bony procedures (metatarsal and cuboid osteotomies). all patients completed two gait analyses (pre and on average . years post-surgery) during barefoot walking using a standardized d motion analysis protocol. the changes in ankle kinematics and kinetics and temporal-spatial parameters were analyzed in reference to a control group of patients with cmt without intervening surgeries with . years between gait analyses as well as normal reference data. the surgical group showed a significant increase in height between the pre and post-operative analyses and no changes in walking velocity (pre: ± , post: ± , normal: ± cm/sec). similarly, the ankle kinematics and kinetics showed no changes as a result of surgery. however, there was a trend for increased peak ankle dorsiflexion (pre: ± , post: ± , normal: ± degrees). as in the surgical group, the control group showed an increase in height but no simultaneous increase in walking velocity (test : ± , test : ± cm/sec). the control group also showed no changes in ankle kinematics and kinetics suggesting that the impact of the disease is, for the most part, not noticeable over . years. the results show that surgical intervention that primarily addresses foot alignment does not negatively impact ankle kinematics and kinetics during gait. the comparison with the control group supports this finding. however, there are some other findings such as the increase in peak dorsiflexion during stance in some patients that will need to be examined further in larger cohorts. Õunpuu s , , pogemiller k , pierz k , , , acsadi g , . center for motion analysis, connecticut children's medical center, farmington, ct, usa; school of medicine, university of connecticut, farmington, ct, usa; division of orthopaedics, connecticut children's medical center, farmington, ct, usa; division of neurology, connecticut children's medical center, farmington, ct, usa. ankle-foot-orthoses (afos) are commonly prescribed for patients with charcot-marie-tooth (cmt) to improve gait and reduce ankle instability and falling. there are no studies that examine bracing outcomes using objective evaluations. the purpose of this study was to examine the impact of various afo designs on gait parameters in children and adolescents with cmt. we predicted that the afos would improve excessive and delayed peak dorsiflexion in stance and equinus in swing. fifteen patients (mean age ± years) were analyzed barefoot and with their prescribed afos by means of a standardized motion analysis protocol. a full clinical examination was also completed including strength and passive range of motion measures. the afos included solid, hinged and posterior leaf spring (tapered, less supportive) designs. sagittal plane ankle kinematics and kinetics and temporal-spatial parameters were analyzed in comparison to normal controls. walking velocity improved from ± to ± cm/sec demonstrating the functional benefits of afos. ankle plantar flexion angle at initial contact improved from − ± to − ± degrees demonstrating improvement in drop foot in swing and at initial contact reducing the risk of tripping. however, the degree of peak ankle dorsiflexion in stance remained the same and excessive at ± degrees barefoot and ± degrees with afos suggesting that in many patients the afo design was not sufficiently supportive. peak ankle plantar flexor moment showed improvement from . ± . to . ± . nm/kg highlighting the improved base of support provided by the afos that compensate for ankle plantar flexor weakness. however, peak power generation was reduced from . ± . to . ± . w/kg indicating that some of the available ankle strength was impeded with the afos. the results of this study suggest that orthoses can provide improved gait outcomes which will improve overall function. however, there are individual differences in patient impairment (strength, range of motion and bony deformity) and associated gait presentation that need to be accounted for in afo design. afos do not always function as intended due to the complex interaction between patient impairment, orthosis stiffness and orthosis design. motion analysis can assist in identifying the specific afo needs for an individual with cmt. painous c , lópez-pérez ma , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, barcelona, spain; hospital san pedro, logroño, la rioja, spain; centro para la investigación biomédica en red en enfermedades raras (ciberer), madrid, spain. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune neuropathy with a heterogeneous clinical spectrum. specific autoantibodies define different clinical phenotypes. cidp with nf antibodies constitutes a specific cidp subset in which a high incidence of postural and intention tremor has been described and that responds poorly to immunoglobulins. we report a patient with an anti-nf positive cidp that presented head, voice and limb tremor that improved with immunotherapy. a -year-old man with unremarkable medical history, presented at the age of with progressive distal weakness and paraesthesia. numbness, gait ataxia and action tremor involving voice, head and limbs appeared sequentially. the emg showed features of acquired demyelination fulfilling cidp diagnostic criteria. a first course of intravenous immunoglobulins was ineffective. the weakness, ataxia and tremor worsened significantly, needing two walking aids first and becoming wheelchair bound later on. he received six plasmapheresis cycles that were also ineffective and oral corticosteroids ( mg/kg) were started with mild improvement. after corticosteroid tapering the patient developed a severe relapse and was referred to our centre. five plasma exchange cycles followed by rituximab ( mg/m , doses) were added to the corticosteroids. three months later the weakness, ataxia and tremor, including the head and voice tremor, started to improve significantly. six months later the patient presented a significant improvement and was able to walk unaided. the anti-nf antibody titres fell from / pre-rituximab to be undetectable. limb tremor is known to occur in patients with inflammatory neuropathies and, specifically in anti-nf + cidp patients but, to our knowledge, this is the first report of a cidp patient presenting with head and voice tremor ever reported. the improvement of tremor with immunotherapy strongly suggest that anti-nf autoantibodies are involved in its pathogenesis, expanding the phenotype of anti-nf specific clinical features. palaima p , peeters k , l. pelayo-negro a , garcía a , gallardo e , garcía-barredo r , de vriendt e , infante j , berciano j , jordanova a . vib and university of antwerp, center for molecular neurology, molecular neurogenomics group, antwerp, belgium; university hospital "marqués de valdecilla", santander, spain. charcot-marie-tooth disease type g is an autosomal dominant and slowly progressing inherited neuropathy which was first described over years ago. it has been attributed to a single spanish family consisting of individuals with affected members spanning four generation. initially, the genetic defect was linked to a . mb region in q -q . . however, extensive sequence and structural variant analyses using whole genome sequences (wgs) of three affected individuals did not reveal any known or novel genetic causes within the region. over the last decade, serial clinical re-evaluation of the entire pedigree was performed leading to changes to the affection status of seven individuals redefining the disease-linked region to chr q . -q . (z max = . at theta = ). additional family members were then submitted for whole exome sequencing. this has led to the identification of a novel missense variant in the e ubiquitin-protein ligase lrsam (p.cys tyr) that was previously not covered by wgs. the variants co-segregated with disease and were absent from controls. other mutations that are known to disrupt the ring domain of lrsam have been previously reported to cause both autosomal dominant and recessive cmt type p (cmt p). unlike prior reports, we demonstrated that the mutation does not influence overall protein levels of lrsam , nor of its ubiquitylation target tsg in patient derived lymphoblasts. transcriptomics analysis identified significant upregulation of another e ubiquitin-protein ligase (nedd l) and of a key regulator of axonal degeneration (tnfrsf ). notably, magnetic resonance imaging of lower-limb musculature systematically showed fatty atrophy in both clinical and subclinical mutation carriers emphasizing its use for the identification of mildly affected members. our findings demonstrate that the isolated genetic entity cmt g is caused by a missense mutation in lrsam and should be reclassified as cmt p. moreover, we reveal novel molecular players associated with lrsam dysfunction, and highlight pathways and therapeutic targets shared with amyotrophic lateral sclerosis and alzheimer disease. damage to peripheral nerves is a prerequisite for neuropathic pain. however, it remains unclear why some neuropathy patients have pain, but others with identical nerve conduction, qst and ienfd do not. we propose to examine the distribution of trpv and its co-localization with cgrp and sp in epidermal nerve fibers in patients with and without neuropathic pain. we aimed to define the expression pattern of trpv in normal healthy subjects first, and study the co-localization of trpv with cgrp and sp in normal controls and patients with painful neuropathy. skin biopsies from neuropathy patients and normal subjects were utilized. anti-trpv antibody generated in our university with support from ninds-funded dept. of neuroscience monoclonal core (ns ) was used. combined immunohistochemistry were performed to identify co-expression of trpv with cgrp, sp and pgp . . the distribution of trpv in controls revealed a proximal to distal gradient similar to that observed for ienf labeled by pgp . . trpv staining was more intense in nerve terminals in the epidermis. combined immunostaining revealed that % of pgp . labeled fibers in the epidermis were trpv +, while % of cgrp+ fibers trpv +. patients with pain had a higher density of trpv + fibers compared to that of patients with numbness. a greater proportion of cgrp+ fibers ( %) in the painful patients were trpv +. expression of trpv in controls exhibits a distal to proximal gradient. trpv expression and co-localization with cgrp were altered in neuropathic pain patients, suggesting that this receptor plays an important role in pathological states. activation of the nop-receptor (nop-r) by its endogenous ligand nociceptin/orphanin fq or non-peptide agonists modulates nociception and analgesia in neuropathic pain models. the wide distribution of nop-r and its endogenous ligand represents an attractive treatment target. since pain is the most prominent feature in pc, we defined expression of the nop-r in plantar skin biopsies and assessed whether alterations exist in pc-affected vs pc-unaffected and anatomically matched control skin. skin biopsies from k a pc and control subjects were immunohistochemically stained for nop-r. combined immunostaining for nop-r with pgp . , neurofilament h (nfh) and cgrp was used to define nop receptor expression in the epidermis and upper dermis. robust nop-r was detected in epidermal keratinocytes and a subset of pgp . + fibers in both epidermis and dermis. staining was inhibited through pre-incubation with a nop-r blocking peptide and western blot analysis using homogenized human skin tissue demonstrated a band at ∼ kd consistent with nop-r molecular weight. nop-r expression occurred in dermal nfh+ a beta-fibers in all groups though no cgrp+ fibers co-expressed nop-r. pc-affected skin had slightly lower nop-r expression than in pc-unaffected skin and a similar pattern in anatomically matched locations from healthy control subjects was observed. nop-r is expressed in human plantar skin epidermal keratinocytes as well as a subset of epidermal and dermal nerve fibers. these fibers are pgp . +, cgrp-and many co-express nfh. nop receptor is a viable pharmaceutical analgesic target in pc patients irrespective of its slight down-regulation as compared to pc-unaffected skin. this work was supported by grünenthal gmbh. pareyson d , stojkovic t , leonard-louis s , reilly mm , laurà m , parman y , battaloglu e , tazir m , bellatache m , bonello-palot n , sacconi s , guimarães-costa r , attarian s , latour p , megarbane a , schenone a , ursino g , sabatelli m , luigetti m , santoro l , manganelli f , quattrone a , valentino p , murakami t , scherer ss , dankwa l , shy me , bacon cj , herrmann dn , pisciotta c , previtali s , bolino a . milan, italy; paris, france; london, united kingdom; istanbul, turkey; algiers, algeria; marseille, france; nice, france; lyon, france; genoa, italy; rome, italy; naples, italy; catanzaro, italy; kurashiki, japan; philadelphia, usa; iowa city, usa; rochester, usa. cmt b and b are characterized by recessive inheritance, early onset, severe course, slowed nerve conduction, myelin outfoldings, association with loss-of-function mutations in myotubularin-related protein- and − (mtmr , mtmr /sbf ), respectively, involved in the metabolism of ptdins p and ptsins( , )p phosphoinositides, key regulators of membrane trafficking. in a multicentre retrospective study to better characterise cmt b, we collected a minimal dataset of information including cmtes/cmtns on cmt b patients, cmt b ( unrelated families) and cmt b ( families). cmt b patients were younger and with earlier onset than cmt b : last visit was performed at a mean age of years (sd . ; range - ) for cmt b and years ( . ; - ) for cmt b ; disease onset occurred at a mean age of . years ( . ; - ) in cmt b as compared to . years ( . ; - ) in cmt b ; delay in motor milestones occurred in / cmt b and / cmt b subjects. eleven cmt b patients became chair-bound, whereas all cmt b subjects but one are still ambulant, although with afos for patients and requiring unilateral support in two cases. both disease types are characterised by vocal cord involvement ( / cmt b and / cmt b ); respiratory involvement was seen almost exclusively in cmt b patients (n= , four requiring non-invasive ventilation and one tracheostomy, as compared to one cmt b patient on niv at age ); two cmt b subjects and an affected cmt b relative not included in the present study died of respiratory complications. glaucoma (n= ) and buphthalmos (n= ) occurred only in cmt b . cmtes/ cmtns scores were higher in cmt b patients in spite of their younger age, indicating more severe disease: cmt b = cmtes mean . (n= ; sd . ; range - / ), cmtns mean . (n= ; sd . ; range - / ,; cmt b = cmtes mean (n= ; sd . ; range - / ), cmtns mean (n= ; sd . ; range - / ). our data confirm that cmt b is more severe than cmt b . interestingly, mtmr interacts with mtmr . mtmr is a catalytically active phosphatase, whereas mtmr is a catalytically inactive protein, known to increase mtmr enzymatic activity. thus, in cmt b nerves a residual enzymatic activity of mtmr may result in a less severe clinical phenotype as compared to cmt b . the charcot-marie-tooth disease (cmt) national registry is fully operative (https:www.registronmd.it) with collection of clinical/genetic information (minimal dataset) and reporting of clinical scales; cmt patients have registered thus far and have chosen one of the reference centers; information collected during the ad hoc visit have been entered in the registry for of them. registered patients have the chance to participate to a study that requires filling online self-reported questionnaires related to five important issues: disease course and complications during pregnancy; use, efficacy and tolerability of orthotics and assistive devices; outcome of surgery for skeletal deformities; safety of anesthesia; occurrence of sleep disorders (including evaluation of fatigue, anxiety and depression). by february , patients and relatives/friends (as healthy controls) have filled the questionnaires. we are performing a first explorative analysis of results, but data collection on all questionnaires will be prolonged until novemebr , , to obtain a larger sample. pregnancy: / cmt women had at least one pregnancy; complications ranging from mild to severe occurred in / pregnancies vs / in controls. cmt worsened in pregnancies ( patients) with no recovery in instances. prenatal diagnosis was performed in / pregnancies. satisfaction related to surgical procedures for foot deformities, assessed with vas (score - ), was . ± . (n = ). repeat surgery was required in / instances. sleep: the epworth sleepiness scale questionnaire revealed abnormalities of sleep in / cmt patients ( %) and in / controls ( %). the vast majority of cmt subjects ( / ; mean . ± . ), but also of controls ( / ; . ± . ) were not good sleepers according to the pittsburgh sleep quality index (psqi) (range - , good sleep). fatigue: scores of modified-fatigue impact scale (range - , no fatigue) were higher for cmt (mean ± . ) than controls (mean . ± . ). hospital scale for anxiety and depression: / cmt subjects had mild-to-severe anxiety and / mild-to-severe depression as compared to / and / controls, respectively. data analysis on orthotics and anesthesia is ongoing. in conclusion, the first data analyses confirm that there are problems related to all the five domains explored, that will need to be specifically addressed in patients' care. supported by telethon-uildm grant gup . charcot-marie-tooth disease (cmt) is the most frequent motor and sensory peripheral neuropathy and is known to have the uniform demyelination. the traditional definition of conduction block is the reduction of negative peak of compound muscle action potential (cmap) of proximal segment relative to adjacent distal segment more than %. in this study, we tried to find the frequency of conduction block in cmt a patients and to investigate the differences of the clinical manifestation. we enrolled unrelated cmt a patients ( males and females) with pmp duplication and undertook nerve conduction studies from to . stimulation sites were wrist, elbow and axilla for median nerve study. eleven patients ( . % of all enrolled patients) had ncs features suggestive of conduction block. compared to cmt a patients without conduction block, functional disability scale was significantly higher in cmt a patients with conduction block (p < . ). however, onset age and disease duration were not different between cmt a patients with and without conduction block. in addition, conduction block was more frequently observed in distal segments than proximal segments. we suggest that the frequency of conduction block in cmt a patients is not low, and there is some heterogeneity of demyelination in cmt a patients. also, in cmt a patients, the conduction block might have relationship with clinical disability. park jg , park ms . yeungnam university college of medicine, daegu, korea. the subacute combined degeneration (scd) is diagnosed by a characteristic clinical manifestation, spinal mri, and decreased serum vitamin b levels. we report a case of scd with elevated homocysteine and methlymalonic acid level in the situation of spurious elevation of vitamin b concentration. an years old man presented with progressive gait disturbances and sensory disturbances. about a year ago, he felt both hands and feet paresthesia and gait disturbances. four months before the visit, nerve conduction tests were performed elsewhere due to symptoms. polyneuropathy was found. the laboratory test showed megaloblastic anemia. however, serum vitamin b concentration was increased to pg / ml (normal: - pg / ml). his symptoms progressed gradually. at presented, neurological examination showed spasticity of the lower limb without weakness, and vibration and position sensation were decreased in both lower limbs, and showed ataxic gait. spinal cord mri showed a lesion with a long t high signal intensity from c to t in the posterior column of the spinal cord. further laboratory test were added, then while folate was normal level but homocysteine at . umol/l (normal: - umol/l), and methylmalonic acid at . um / l (normal: - . um / l) were increased. finally, scd caused by vitamin b deficiency was diagnosed. in addition, gastric endoscopy was performed to find the cause of vitamin b deficiency and chronic atrophic gastritis was found. after cobalamine treatment for months, hemoglobin level was improved and his symptoms were all improving with a little gait disturbances. false-elevation of vitamin b level could lead to delayed diagnosis and cause irreversible changes in the nervous systems. one of the most commonly used methods to measure vitamin b concentrations is competitive-binding assay, which may result in normal levels even with vitamin b deficiency due to the effect of anti-intrinsic factor antibodies. when vitamin b deficiency is suspected, even if the vitamin b concentration is normal, additional tests of homocysteine and methylmalonic acid should be considered. parman y , durmus h , deymeer f , oflazer-serdaroglu p , battaloglu e . istanbul university, istanbul faculty of medicine, department of neurology, istanbul, turkey; bogazici university, istanbul, turkey. hereditary motor and sensory neuropathies, also called cmt neuropathies, are the most common group among the hereditary neuropathies. cmt subtypes are grouped by axonal, demyelinating or intermediate phenotype, or by autosomal-dominant (ad), autosomal-recessive (ar) or x-chromosomal inheritance. cmt- is considered axonal and characterized by distal muscle wasting, mild sensory loss and normal or near-normal nerve conduction velocities. mutations in more than genes are known to be able to cause autosomal dominant (ad) or recessive (ar) cmt- to date. the genetic heterogeneity in charcot-marie-tooth (cmt) is a challenge for genetic diagnostics. clinical clues and frequencies of mutations in cmt genes from large cohorts may help to develop strategies for efficient genetic testing. here, we present the clinical, electrophysiological and genetic features of patients from turkey, diagnosed as genetically confirmed cmt- in the department of neurology, istanbul faculty of medicine our clinic between and . genetic testing was performed for gdap by dna sequencing and samples from patients were exome sequenced (wes). twenty-one male and ten female patients from unrelated families were investigated. segregation was ar in eight, ad in six families and two were isolated cases. intraand interfamilial variability in the age of onset with a range of - (mean . ± . years) and disease progression rate were striking. slowly progressive weakness and atrophy of distal muscles in the feet and/or hands were the most common presenting symptoms. mfn was found in four unrelated ad kinship and in unrelated ar kinships and was most common gene mutated among whole cohort. gdap was the most commonly mutated gene among ar families ( / ). wes revealed further mutations in aars, nefl, kif b and hspb , however, the causative mutation could not be identified in known cmt genes in about % of patients. our data indicates the marked intra-and inter-familiar phenotype variability in cmt- as previously described in literature. many more genes causing arcmt- remain to be discovered. pasnoor m , veerapaneni k , murphy r , statland jm , kimple d , hamasaki a , glenn md , herbelin l , barohn rj , jawdat o , dimachkie mm . department of neurology, neuromuscular division, the university of kansas medical center, kansas city, ks, usa. electrodiagnostic (edx) studies involving electromyography/nerve conduction study (emg/ncs) are useful for diagnosis of neuromuscular disorders. although these are safe procedures, emg and ncs often evoke anticipated pain and anxiety. there are no published research studies to support the notion that providing detailed information to patients prior to these procedures would reduce procedural pain and ameliorate anxiety levels afterwards. the objective of this study was to perform a prospective survey to assess anticipated pain and anxiety in patients presenting for edx studies, design a detailed patient education form, and assess the change in perceived pain and anxiety pre and post procedure in the standard of care (soc) education versus the printed detailed education instrument group. after completing a brief pain/anxiety questionnaire, patients were randomly assigned to either soc verbal education group or to read the detailed education form. another brief questionnaire was completed post procedure. seventy-eight patients were enrolled at the time of abstract submission, in intervention and in soc groups. mean age of patients enrolled was ± years. pain was anticipated by % patients as a visual analog scale (vas) score mean of . ± . with no significant difference between both groups (p= . ). post-procedural pain was reported in % of patients with mean pain score of . ± . and no significant difference between the two groups (p= . ). anxiety pre-procedure was reported by % patients with a mean likert-like score of . ± . score in all cases (p= . between both groups). post-procedural anxiety frequency was reduced to % with mean level of . ± . (p= . between both groups). most patients reported pain with both emg and ncs ( %). we found that edx testing evokes moderate pain in most and in some cases moderate anxiety level. the detailed education intervention did not attenuate the frequency or severity of pain. post-procedural anxiety was expectedly reduced in severity and frequency in both groups with a suggestion that anxiety severity is lesser in the detailed education group but numbers were small. further studies with more detailed questionnaire and perhaps web-based education in larger population may be useful to reduce pain/anxiety in patients presenting for edx studies. pasnoor m , roach c , barohn rj , statland j , jawdat o , dick a , glenn m , dimachkie mm . department of neurology, neuromuscular division, the university of kansas medical center, kansas city, ks, usa. diagnostic criteria for chronic inflammatory demyelinating polyneuropathy (cidp) have been designed for use in clinical trials. there is limited data on the comparative diagnostic utility of these criteria in the clinic setting and it is unknown if some of these criteria are better for predicting treatment response. the objective of this study is to compare the sensitivity of various diagnostic criteria and review treatment response. after obtaining local irb approval, we performed a retrospective chart review of cidp patients seen at the university of kansas medical center between and . we abstracted the clinical, electrodiagnostic, and treatment information. we determined the frequency of patients fulfilling each of the following criteria: efns , aan, saperstein and incat. we defined treatment response based on treating physician's impression of change, patient-reported functional improvement or one point grade change in the mrc grade. fifty-three cidp patients charts were reviewed, were excluded due to missing data. mean age was . , and ( %) were female. elevated csf protein was seen in / ( %). twenty-eight ( %) fulfilled efns definite criteria, ( . %) efns probable, ( . %) aan, ( . %) incat, and ( . %) the saperstein criteria. treatment response in patients who fulfilled efns definite/probable criteria included / to ivig, / to iv or oral corticosteroids, / to mycophenolate mofetil; among patients who fulfilled aan criteria, these were respectively / , / and / ; among those meeting incat criteria / , / and / ; and for saperstein criteria it was / , / and / . half to two-thirds of patients responded to plex based on different criteria. while all cidp criteria can similarly predict ivig treatment response, the efns criteria are most sensitive for the clinical diagnosis of cidp. pellegatta m , canevazzi p , forese mg , podini p , quattrini a , taveggia c . division of neuroscience and inspe, axo-glia interaction unit, san raffaele scientific institute, milan, italy; division of neuroscience and inspe, experimental neuropathology unit, san raffaele scientific institute, milan, italy. axonal nrg type iii is an essential instructive signal for pns myelination, since its expression determines whether axons are myelinated and also the thickness of the myelin sheath. previous studies have shown that secretases' cleavage controls nrg type iii activity at post-transcriptional level. specifically, the beta-secretase bace- cleavage of nrg type iii promotes myelination, whereas the alpha-secretase tace inhibits pns myelination by inactivating nrg type iii. after a damage, the axon located distally to the site of injury degenerates and loses the myelin sheath, following a process termed as wallerian degeneration. unlike the cns, axons in the pns can regenerate and regrow. several studies have shown that regeneration is favored by the trans-differentiation of schwann cells, which start to create a permissive environment for axonal regrowth. in addition, it has been shown that nrg regulates the early stages of the dedifferentiation process and is essential for creating a permissive environment to regeneration. similarly, the beta secretase bace- promotes pns regeneration and remyelination. in the present study, we analyzed the role of the alpha-secretase tace during wallerian degeneration. our data indicate that tace is upregulated in sciatic nerves after injury, distally to the site of damage. since tace is expressed in schwann cells and axons, we analyzed the role of both glial and neuronal tace during degeneration and regeneration processes. thus, we performed crush injury in sciatic nerves of two different transgenic lines carrying a conditional deletion of tace either in schwann cells (p -cre//tace flox/flox ) or in neurons (chat-cre//tace flox/flox ). the analyses of the different phases of the wallerian degeneration in these animals suggest a role of glial tace during the early stage of the process. in fact, we observed an increased number of myelinated axons only in p -cre//tace flox/flox mice, while we did not detect substantial differences in mutant chatcre//tace flox/flox mice. these results suggest that even during regeneration tace has an inhibitory role, as deletion of tace in schwann cell likely induces the activation of a neuro-protective program that we are currently investigating. péréon y , nizon m , küry s , besnard t , quinquis d , boisseau p , magot a , mussini jp , mayrargue e , barbarot s , bézieau s , isidor b . reference centre for neuromuscular disorders; dept. of genetics; dept. of paediatric surgery; dept. of dermatology, university hospital, nantes, france. the hereditary sensory and autonomic neuropathies (hsan) constitute a clinically and genetically heterogeneous group. hsan are associated with sensory dysfunction, altered pain and temperature perception with varying degrees of autonomic dysfunction, and abnormal small fibers neurodevelopment. more than ten genes have been described so far in the hsan group. original classification encompassed four entities, but additional subgroups continue to be described. hereditary sensory neuropathies (hsan) type ii are characterized by autosomal recessive inheritance, onset at birth and self-mutilating behavior. until now, one homozygous frameshift variant, c.[ _ del], p.(lys phefs* ), was reported in arl ip (adprribosylation-like factor -interacting protein ) in a consanguineous family. patients presented with spastic paraplegia, diffuse sensory and motor polyneuropathy and acromutilation. the disorder was classified as autosomal recessive spastic paraplegia spg . here, we described a new patient with congenital insensitivity to pain, sensory neuropathy, self-mutilation, and spastic paraplegia. whole exome sequencing showed a homozygous frameshift variant c. [ _ del], p.(lys phefs* ) in arl ip . the protein harbours reticulon-like short hairpin transmembrane domains and has a role in endoplasmic reticulum shaping. the variant causes an additional c-terminus hydrophobic domain which could alter its function. arl ip interacts with atlastin- responsible for spg a and hsan type d. this report highlights the role of arl ip in pathophysiology of insensitivity to pain and spastic paraplegia. péréon y , nguyen a-l , leclair-visonneau l , fayet g , nguyen j-m , magot a . laboratoire d'explorations fonctionnelles, hôtel-dieu, nantes, france; département de statistiques médicales, hôtel-dieu, nantes, france. carpal tunnel syndrome (cts) is responsible for sensory and/or motor symptoms that may be increased by wrist flexion or extension (phalen sign). previous studies have shown that short duration (e.g. min or less) flexion or extension was not responsible for significant changes of median nerve conduction parameters. the objective is the study was to determine how prolonged extension (reverse phalen's test) affects median and ulnar nerve conduction parameters in normal subjects. after providing informed consent, normal subjects ( females, age rank - years) underwent motor and sensory testing of both median and ulnar nerves from the dominant side. edx testing was performed using standard techniques with wrist in neutral position, then during passive wrist extension ( ∘ ) for minutes, with sequential recording of both motor action potential (cmap) and sensory action potential (snap) latencies and amplitudes every minutes after extension onset. ulnar nerve conduction parameters remained unchanged in all the subjects. regarding median nerve, groups of subjects could be individualized: subjects with no changes; subjects with only significant decrease of snap amplitude (− %); subjects with both snap amplitude and velocity decrease; subjects with both cmap and snap significant changes. when present, changes did not appear before min, with snap decrease being the earliest observed abnormality. prolonged wrist extension was responsible for median nerve edx parameter modifications in / normal subjects. sensory fibers were firstly affected, corresponding to the chronology clinically encountered in cts. based upon these results, the same protocol will be used in patients presenting with mild clinical cts, in order to try and identify an additional edx technique being useful in the diagnosis of cts. one could also wonder whether the subjects with the most important changes would be more prone to present cts in the future. perez-siles g , , drew a , , ellis m , kidambi m , takata r i , speck-martins c e , hagerman k a , siskind c e , day j w , ginzberg m , nicholson g , , , kennerson m l , , . northcott neuroscience laboratory, anzac research institute, sydney, australia; sydney medical school, university of sydney, sydney, australia; molecular medicine laboratory, concord repatriation general hospital, sydney, australia; sarah network rehabilitation hospitals, brasilia, df, brazil; department of neurology, stanford health care, stanford, ca, usa; pediatric neuromuscular unit, wolfson medical center, holon, israel. mutations in the atp a gene cause of x-linked hereditary distal motor neuropathy (dhmnx). to date, missense mutations (t i and p s) in this copper transporting atpase are the only confirmed mutations causing dhmnx in two independent families. next generation sequencing (ngs), including whole exome and whole genome sequencing, is increasing the rapid detection of variants in genes known to cause disease, however the absence and size of additional families, make it challenging to determine the pathogenic or benign status of variants identified. we have recently identified new variants in atp a in patients with progressive peripheral neuropathy, suggesting further genetic heterogeneity of dhmnx. two of these new variants, pe v and pm v, are located at highly conserved amino acids within domains of atp a (a-domain and p-domain, respectively) that are critical for the catalytic cycle of the copper transporter. we have also identified a third variant (c. - a>g) located bases upstream exon that is predicted to abolish a conserved branch-site, a consensus intronic sequence necessary for the processing of immature rna during splicing. our recent investigations using both patient fibroblasts and an atp a conditional knock in mouse model for dhmnx expressing atp a t i , the orthologue of the human atp a t i mutation, showed reduced atp a protein levels and defective retrograde trafficking of atp a, as pathological hallmarks of dhmnx atp a causative mutations. to elucidate if the newly identified pe v, pm v and c. - a>g atp a variants are pathogenic, we have systematically assessed patient fibroblasts harbouring these mutations for altered parameters previously found in the pathogenic t i atp a mutation. we predict that functional characterisation of atp a using patient fibroblasts harbouring newly identified variants will provide the evidence to ascertain whether these variants are disease causing. establishing a systematic functional readout for atp a variants will improve accuracy of genetic counselling and patient management of dhmnx in those cases where genetic evidence is limited. this study represents a complementary and necessary approach to the use of ngs, for validating the pathogenic status of variants identified and for expanding the genetic heterogeneity of dhmnx. month of birth (mob) have been defined as a risk factor for more than diseases. for instance, susceptibility for multiple sclerosis (ms) seems to be associated with being born in may, while lower risk of ms is observed in those born in november. there are no studies that assessed potential association between mob and a risk for guillain-barré syndrome (gbs). we sought to determine if the risk of gbs is associated with the mob. study included gbs patients diagnosed in serbia, montenegro and republic of srpska in the period from january , until december , . mob of patients and of general population were compared. patients with gbs had tendency to be born in october ( % increase compared to general population, p= . ) and were less likely to be born in june ( % decrease, p= . ). when consider specific seasons, gbs patients were more likely to be born in winter months ( % increase, p< . ). no associations were found between month/season of birth and disease severity. results of this pilot study showed that gbs patients are more likely to be born in cold months, and less likely to be born in june. this might be explained by higher exposure to different pathogens during pregnancy in cold months. in accordance with this, it is well known that early exposure to infective agents reduces risk of allergic and autoimmune diseases. further studies are needed to test our findings in different cohorts and in regions with different climate. these results may shed new light on the disease pathogenesis. majority of patients with guillain-barré syndrome (gbs) tend to have a successful recovery, but in number of them significant long-term consequences may negatively affect their quality of life. aim of this research was to analyze the outcome of the disease one year after the acute episode of gbs. among patients diagnosed with gbs in seven tertiary centers in serbia, montenegro and republic of srpska during , subjects were retested after one year ( % males, mean age ± years). functional disability of patients was estimated based on the gbs disability scale (gds). severe form of the disease (gds score - ) was registered in % of patients at admission, % at nadir, and % on discharge. after one year follow-up period, % of patients had no symptoms of gbs, % had mild symptoms, % was able to walk but not to run, % needed unilateral support during walking, while % was wheelchair bound or bedridden. lethal outcome was registered in three patients during acute phase of gbs and in four more during one-year follow-up. paresthesias were present in % of gbs patients, musculoskeletal pain in %, and fatigue in % one year after acute phase of the disease. factors associated with the worse functional outcome (gds grade above ) after one year were: age above , preceding respiratory infection, and worse gds on discharge. one year after the onset of the disease, significant number of our gbs patients had neurological impairments including sensory symptoms, pain, fatigue and muscle weakness which may significantly affect patients' everyday functioning. phetteplace je , saade d , bacon c , shy me . university of iowa hospitals and clinics, iowa city, ia, usa. charcot-marie-tooth (cmt) disease, also known as hereditary motor and sensory neuropathy, is a heritable peripheral neuropathy caused by genetic mutations in many different genes. cmt type a is the most common form of cmt. individuals affected with cmt a commonly have distal muscle atrophy, foot deformities, abnormal gait, and reduced nerve conduction velocities caused by demyelination of the peripheral nerves. cmt a is known to be caused by a duplication of the pmp gene at p . . the proposed disease mechanism of cmt a is believed to be increased gene dosage of the pmp gene, although the exact function of pmp is not known. there have been previous reports in the literature of individuals who carry a homozygous duplication of pmp , therefore having four copies of the gene. however, many of these reports have focused on adult patients who symptoms may not vary significantly from individuals who harbor only a duplication of pmp . a month old male presented to the university of iowa hospital due to a family history of cmt a and concern for delay in independent ambulation. he was known to have both a maternal and paternal family history of cmt a, although the families were believed to be unrelated. on exam he was unable to walk independently, but could take a few steps when his hands were held. per report he began crawling and scooting at months of age. he was not found to have any structural abnormalities or atrophy in his upper or lower extremities. electrodiagnostic testing revealed a demyelinating neuropathy. only one nerve was tested, as the patient was unable to tolerate additional shocks. the peroneal nerve was found to have a nerve conduction velocity of m/s. based on the clinical presentation, electrodiagnostic examination and family history deletion and duplication testing for pmp was performed. this testing revealed a homozygous duplication of pmp , which has been previously reported to cause cmt a. this case supports the gene dosage disease mechanism, as our patient appears to be more severely affected than a typical infant or toddler affected with cmt a. piscosquito g , saveri p , provitera v , stancanelli a , ciano c , magri s , taroni mpz mutations are associated with the demyelinating early-onset charcot-marie-tooth (cmt) type b and with the axonal later onset cmt i/cmt j. paresthesias/dysesthesias and pain have been already reported in some cmt types and also in cmt i/j. we report patients ( from families, sporadic cases,) carrying the mpz c. dela mutation (p.asp thrfster ). this is predicted to be a loss-of-function mutation, leading to p haploinsufficiency. most of the subjects have genetic ancestry from puglia region (southern italy). two patients carried the mutation in homozygosity, showing a severe phenotype: onset at birth, severe scoliosis, proximal weakness, distal limb plegia, no autonomous walking, cmt examination score (cmtes) - / . all the other heterozygous patients had mild adult-onset (mean age , range - ) neuropathy, walked without aids or ankle-foot orthoses, had cmtes < / . five showed mild foot deformities, / had foot and / hand weakness (mrc never less than / ), / sensory loss in lower limbs (ll). paresthesias/dysesthesias (mostly tingling and burning in type) localized in hands and ll were reported by / subjects, neuropathic pain by / . in the homozygous patients, all cmaps and saps were absent. in all other subjects, motor conduction velocities (cv) were > m/s (range - m/s) and sensory cv > m/s (range - m/s). cmaps as well as saps were normal or moderately reduced in amplitude, indicating only slight axonal loss. we confirm that heterozygosity is associated with very mild cmt, whereas homozygosity causes a very severe neuropathy. our data suggest that paresthesias and neuropathic pain are very common and should be considered as part of the phenotype. although the clinical picture suggests a small fiber neuropathy, no such evidence is provided in the current literature. in the attempt to further investigate this field, we found in a -year-old male patient of our series that all thresholds were increased at quantitative sensory testing, and several abnormalities of small nerve fibers were present in skin biopsy. apparently, p aneuplody causes neuropathy and small fiber involvement through a mechanism different from other mpz missense mutations. poitras t , chandrasekhar a , singh v , martinez j , zochodne dw . neuroscience mental health institute, and division of neurology, department of medicine, university of alberta, edmonton, alberta, canada. a microarray mrna analysis of dorsal root ganglia examined in long-term diabetic mice yielded an unexpected upregulation of a unique set of proteins known as major urinary proteins (mups & ). originally, this family of barrel shaped proteins was described as including mediators of pheromone secretion in the urine of rodents, apparently unconnected to other functions. we identified its unanticipated expression in sensory neurons of adult mice and rats and explored its potential impact on the properties of these neurons. analysis of rat and mouse sensory neurons showed widespread and pan-neuronal cytosolic mup expression, not only in the cell body, but also in distal sensory projections located in the dermis of the footpad. to assess the role that mup might offer in the growth behaviour of adult neurons, we examined sirna-induced knockdown of mup on pre-injured dissociated drg primary cultures. mup knockdown in both species showed a significant increase in neurite outgrowth following mup sirna treatment when compared to a scrambled sequence control. mice treated with streptozotocin (stz) model features of human type diabetic polyneuropathy, including decreases in multi-fiber motor and sensory measurements, and changes in thermal sensation in the extremities. to explore the potential role of mup upregulation specifically in diabetes, we examined the impact of non-viral intranasal injection of mup sirna on indices of neuropathy in chronic type diabetic mice. following treatment, mice treated with mup sirna showed improvement in both motor and sensory nerve conduction velocities compared to scrambled controls. taken together, this data suggests that mup plays a growth-suppressive role in both diabetic and non-diabetic neurons and may also contribute to the electrophysiological and behavioral abnormalities associated with diabetic polyneuropathy. the repurposing of mups in sensory neurons identifies an interesting role for a supposed pheromone protein. [supported by cihr, cda and adi] enhancement of their outgrowth properties (singh et al, ) . here we asked whether similar activation, using low dose capsaicin, might similarly ramp up the regenerative activity of sensory neurons. using rat adult primary sensory neurons, we identified a dose dependent relationship between capsaicin exposure and their outgrowth with enhancement at lower doses and expected neurotoxicity at higher doses. doses that enhanced outgrowth were nonetheless associated with rises in intraneuronal calcium. we next tested whether a similar impact of trpv activation in vivo might exist. we used a high sciatic crush injury mouse model to assess the impact of capsaicin at low ( um) or high ( mm) doses applied directly at the site of axon outgrowth after injury. by days following the single application of capsaicin, low doses of capsaicin showed more rapid recovery of thermal, but not mechanical sensation, whereas high dose capsaicin showed no significant difference compared to vehicle treated mice. in parallel with the neurotoxicity observed in vitro, the high dose capsaicin resulted in a decrease in sensory nerve conduction velocity, while the low dose showed no significant difference when compared to the vehicle treated animals. there was no impact on motor axon recovery. taken together, subtoxic trpv activation with intraneuronal calcium mobilization, as applied to regenerating sensory axons, enhances their outgrowth after injury, and improves behavioural recovery similar to that following extracellular electrical stimulation. [supported by cihr] hereditary transthyretin (hattr) amyloidosis is a rapidly progressive, life-threatening disease caused by ttr gene variants, resulting in ttr misfolding and amyloid deposition in multiple organs, including peripheral nerves. hattr amyloid polyneuropathy has prominent small fiber involvement characterized by neuropathic pain and dysautonomia. measurement of intraepidermal and sweat gland nerve fiber density (ienfd, sgnfd) in skin biopsies has been useful in identifying small fiber abnormalities in hattr. we report the effect of patisiran, an investigational rnai therapeutic for the treatment of hattr, over a -month period on ienfd, sgnfd and dermal amyloid content from the phase open-label extension (ole) study. skin biopsies (distal leg, distal thigh) were obtained every months for up to years within the phase ole study (patisiran . mg/kg iv, q w, nct ) and analyzed in a blinded manner by a central laboratory. all analyses are exploratory; nominal p-values are reported without adjustment for multiple testing. twenty-seven patients enrolled; preliminary data indicated patisiran was generally well tolerated; resulted in sustained mean serum ttr lowering of ∼ % for > months and a mean . -point decrease in mnis+ (n= ). twenty-four patients underwent serial skin punch biopsies. at baseline, mean ienfd was . and . fibers/mm, while mean sgnfd was . and . m/mm for distal thigh and distal leg, respectively. amyloid was detected in ∼ % of skin biopsies, mean baseline dermal amyloid content . % and . % for distal thigh and distal leg, respectively, which inversely correlated with ienfd and sgnfd. overall, ienfd was stable throughout the -year treatment. sgnfd increased over time relative to baseline, reaching statistical significance at , , and months for distal thigh (p< . , all time points) and at months for distal leg (p= . ). dermal amyloid content decreased over time and reached statistical significance at , and months for distal thigh (p< . , all time points) and at , , , and months for distal leg (p< . , all time points). in summary, improvements in sgnfd and dermal amyloid content observed over a -year period suggest that these parameters have the potential to serve as biomarkers of response to patisiran treatment. polydefkis m , neuhaus s , doherty l , ebenezer gj . department of neurology, johns hopkins university, baltimore, md, usa. misdiagnosis and delayed identification of an etiology for sensory neuropathies hampers adequate management and delays initiation of therapy. we present several cases that illustrate variable presentations of amyloid neuropathy (an) and an complicated by amyloid arthropathy. a -year-old former college athlete developed painless difficulty walking. he was unable to keep up with family members during routine outings and subsequently was unable to navigate uneven ground during a hike, something he had previously excelled at. balance decreased, making bicycling difficult. neurological evaluation -months after symptom onset led to a diagnosis of peripheral neuropathy. a reversible neuropathy panel was normal though a c was elevated ( . %). over several months, his strength/sensation deteriorated despite diet improvement, losing lbs and a c improvement ( . %). upon referral, emg revealed a sensorimotor neuropathy with markedly reduced/absent sural, peroneal and tibial motor amplitudes. there was mild orthostasis. nis was . skin biopsies revealed severe ienfd, sgnfd reductions and positive congo red staining, confirmed with birefringence. a sural nerve biopsy confirmed amyloid deposits. genetic testing revealed the ttr-fap variant leu his. there was no history or suspicion for a family history of amyloid. a -year-old male with a history of controlled diabetes x years developed abrupt onset of rls months after a lymphoma diagnosis. treatment with rituximab led to radiographic resolution of the lymphoma though the patient developed progressive peripheral neuropathy confirmed by ncv/emg. new-onset early satiety, mild orthostasis and ed emerged. skin biopsies demonstrated a lichenoid pattern of amyloid deposition consistent with aa-amyloid. a -year old woman with leu his ttr-fap since developed progressive leg weakness, reduced ability to walk and low back pain. emg demonstrated progression of her sensorimotor neuropathy and superimposed multilevel radiculopathy. spine mri demonstrated severe lumbar stenosis requiring surgical decompression. postoperatively, she regained strength, walking more easily. congo red staining of vertebral bone demonstrated widespread positive staining with birefringence consistent with amyloid arthropathy. these cases illustrate to wide sprectrum of amyloid neuropathy presentation, an example of amyloid arthropathy-induced spinal stenosis and the utility of skin biopsy to diagnose and manage amyloid neuropathy. the differentiation between hereditary neuropathy and chronic inflammatory demyelinating polyneuropathy (cidp) in children is especially significant because of completely different treatment possibilities and prognosis in these conditions. the aim of the study was to compare electrophysiological abnormalities in a group of children and young adults with demyelinating neuropathy of chronic or subacute onset. retrospective analysis of clinical and nerve conduction study (ncs) data included children and young adults with charcot-marie-tooth neuropathy x type (cmtx ), children with charcot-marie-tooth neuropathy type a (cmt a) and children with cidp. in our study / cmtx had both axonal and demyelinating changes in ncs study. the aan and efns electrophysiological cidp criteria were fulfilled in / cmtx , / cmt a and / cidp patients. additionally patients with cmtx are classified with efns criteria as "probable/possible cidp". a distal compound muscle action potential (dcmap) was > ms in all cmt a and / cidp patients but none with cmtx . abnormal median/normal sural snap (amns) parameter was observed in // cidp and / ctmx patients but not any cmt a patient while abnormal sural/normal median snap (asnm) parameter was found in / patients with cidp. a difference between conduction velocities (cv) of two corresponding nerves > m/s was seen in / cmtx and / cidp patients but no one with ctm a. one patient with cmtx had especially conspicuous difference between nerve conduction in lower limbs, with axonal neuropathy and demyelinating features, and upper limbs with no changes. one -years-old patient with genetically confirmed cmtx had no abnormalities in ncs. electrophysiological data of cmtx , cmt a and cidp indicate diffuse demyelination, however in cmtx axonal changes coexist, seen in / patients, and asymmetrical abnormalities between corresponding upper and lower limb were observed in / patients. our study has showed that duration of dcmap is useful not only in diagnosing an inflammatory neuropathy but also in differentiating cmtx from cidp. however presence of the not homogenous abnormalities in ncs in patients with cmtx may mimic inflammatory neuropathy. prada v , mori l , francini l , accogli s , ursino g , gemelli c , schizzi s , grandis m , bellone e , mandich p , schenone a . department of neurosciences, rehabilitation, ophthalmology, genetics and maternal and child health, university of genoa, genoa, italy. the overwork weakness (ow) problem in cmt disease has been debated for long time. it has been reported that the non-dominant hand (ndh) of patients with cmt disease is stronger than the dominant hand (dh) as a result of ow and some authors verified this hypothesis using mrc on different muscles (van pomeren, ). more recently, piscosquito et al. ( ) found that the ow phenomenon does not exist using a dynamometer and the hole peg test, a dexterity test. we recruited cmt patients and healthy controls. we evaluated the strength of the -pinch and of the hand-grip with a dynamometer, the opposition ability with the thumb opposition test (tot) and applied an instrumental testing of hand function using the sensor engineered glove test (segt), which previously demonstrated its sensitiveness to measure severity of hands dysfunction in cmt patients. tot was significantly higher in the ndh compared to dh (ndh= , ± , ; dh= , ± , , p< . ) in cmt patients, instead there was no difference in dh and ndh in healthy controls. in the evaluation of -pinch and of the hand-grip, ndh performed slightly but not significantly better than the dh ( -pinch: dh= , ± , n; ndh= , ± , n; ± , n; ndh= , ± , n, p:ns) . in normal controls we confirmed the % rule (noguchi & demura, ). finally, segt results were similar between the ndh and dh in cmt patients but in normal controls there was a better performance in the dh. in conclusion, this study supports the existence of the overwork weakness in cmt, evident in every measurements. dexterity and overall ability of the hands impaired in the dh, probably because of compensating movements, compared with the healthy controls in the weaker hand. finally, our results support the importance of avoiding supramaximal exercises and educating the cmt patients to prevent incorrect movements, which may overload the hand muscles. prasad k , ohnmar o , teng a , cheng yj , umapathi t . national neuroscience institute, singapore; yangon general hospital, yangon, myanmar; yong loo lin school of medicine, national university of singapore, singapore. antecedent infections that have been linked to guillain-barré syndrome (gbs) include campylobacter jejuni, haemophilus influenza, epstein-barr virus, cytomegalovirus, mycoplasma pneumoniae and influenza a virus. in south-east asia, the burden of mosquito-borne flavivirus infections is considerable. even in a small country like singapore, with a population of only . million and in spite of excellent sanitation and public health measures, dengue virus (denv) infections can exceed , a year. both symptomatic and asymptomatic denv infections are associated with gbs. zika virus (zikv) infections were detected in singapore in late august . doctors working in south-east asia anecdotally report that gbs cases increase after rainy season when mosquitos breed. we are currently planning studies to estimate the hitherto unknown burden of gbs associated with denv, zikv and other mosquito-borne viruses in this region. as a preliminary step, we reviewed the gbs cases seen at our institution in for evidence of denv and zikv. three out of the tested cases were positive for denv serology. one of these was also positive for denv pcr. all these patients had symptomatic dengue. two had acute inflammatory demyelinating polyneuropathy (aidp) and acute motor axonal neuropathy (aman) subtype of gbs. none the patients tested were positive for zikv pcr in serum and urine. one patient, with miller fisher syndrome (mfs), had serological evidence of recent zikv. he did not have clinical zikv symptoms. in mfs cases, the initial detectable zikv igg was later proven to be due to cross reactivity with previous denv infection. in cases ( amsan, aidp, mfs) the initial zikv igm was raised. we await analysis of convalescent sera to decide if they indeed had zikv or the results were related to previous and co-infection with denv. our preliminary findings indicate that flavivirus infections may account for at least some of the gbs cases in singapore. the lack of symptoms in some cases and the interactions between zikv and denv antibodies make accurate diagnoses challenging. prior r , , benoy v , , vanden berghe p , van den bosch l , . ku leuven-university of leuven, department of neurosciences, experimental neurology and leuven research institute for neuroscience and disease (lind), leuven, belgium; vib-center for brain & disease research, laboratory of neurobiology, leuven, belgium; laboratory for enteric neuroscience, translational research center for gastrointestinal disorders, k.u. leuven, leuven, belgium. charcot-marie-tooth disease (cmt) is the most common inherited neurodegenerative disorder of the peripheral nervous system. it is divided into two main subtypes, a demyelinating subtype (cmt type ) and an axonal form (cmt type ). cmt is a length-dependent disease, in that it effects the longest neurons in the body, thus the muscles in the peripheral regions are affected first and foremost. moreover, the majority of cmt patients share a classical phenotype with shared pathological hallmarks, such as distal muscular atrophy, reduction in nerve conductions, etc. but also molecular pathological hallmarks, like the breakdown in the transport of organelles and vesicles in neurons in a process called axonal transport. currently, there is no cure or effective treatment available to cmt patients. histone deacetylase (hdac ) has been shown to be a key regulator in axonal transport. moreover, inhibiting hdac has been demonstrated to stabilize microtubules, which act as molecular tracks for motor proteins and facilitates axonal transport of cargos. our labs current focus is on cmt type a, the main cause of cmt, which is caused by a duplication of a segment of chromosome p . containing the gene encoding peripheral myelin protein (pmp ). pmp is mainly expressed by schwann cells, the cells that myelinate neurons in the peripheral nervous system. in the work presented, the commercial mouse schwann cell-line, sw cells, was transfected to overexpress pmp using a lentivirus vector system to mimic that of cmt a patient schwann cells. the overexpression of pmp was confirmed using immunofluorescence and western blot techniques. these transfected sw schwann cells were then co-cultured with primary mouse dorsal root ganglion neurons (drgs) isolated from adult mice. these co-cultures where then analysed after weeks in vitro for axonal transport. the investigated groups were: a co-culture of drgs + sw cells, a co-culture of drgs + sw cells transfected with pmp , and a co-culture of drgs + a sw cell-line transfected with green fluorescent protein using a lentivirus vector system, and a monoculture of drgs. furthermore, this work demonstrates that the overexpression of pmp in schwann cells can impair axonal transport in co-cultured drgs in comparison to the other co-culture groups. moreover, these axonal transport defects were able to be rescued by the treatment of a hdac inhibitor, tubstatin a. to conclude, selective hdac inhibitors have been shown to be a beneficial treatment for a number of cmt subtypes in preclinical studies in our lab and offer as a viable treatment for a currently, untreatable debilitating disease. provitera v , caporaso g , stancanelli a , piscosquito g , di caprio g , saltalamacchia am , santoro l , nolano m . "maugeri" clinical and scientific institutes irccs, institute of telese terme (bn), italy; department of neurosciences, reproductive and odontostomatological sciences, university 'federico ii' of naples, naples, italy. the observation of rate and patterns of cutaneous nerves regrowth after mechanical or pharmacological distal axonotmesis, has proven to be an excellent tool to study human nerve regeneration in normal and pathological conditions. most of the observations, however, are referred to small fibers, possibly due to the availability of excellent models of reversible distal cutaneous small fiber axonopathy. no such model is available to study the regeneration of myelinated fibers in human. we studied regrowth of cutaneous large fibers in fingertip of patients with carpal tunnel syndrome after surgical decompression. we recruited patients (m/f / ; age . ± . years) with carpal tunnel syndrome candidate to surgery. patients underwent clinical and electrophysiological evaluation, quantitative evaluation of discriminative threshold at rd fingertip. in the same site, patients also underwent mm punch skin biopsy. skin sections were stained by immunohistochemical techniques and cutaneous innervation was analyzed by confocal microscopy. meissner corpuscles and their myelinated afferences were assessed following previously published procedures. twelve months after surgery, patients repeated functional evaluation and underwent a second skin biopsy two mm apart from the first one. mean density of mcs/mm was . ± . at time and . ± . at follow-up, mean density of myelinated endings was . ± . at time and . ± . at follow-up. however, not in all patients regeneration occurred. based on the variation of mc density between time and follow-up we were able to identify patients in which active regeneration had occurred. in this subgroup mean density of mcs/mm was . ± . at time and . ± . at follow-up (p< . ) with a mean delta of + . . in the same group, mean density of myelinated endings was . ± . at time and . ± . at follow-up (p< . ) with a mean delta of + . . we describe morphological patterns associated to nerve regrowth in the biopsies of patients in which nerve regeneration occurs. we propose an in vivo model to study regeneration of large myelinated fiber endings in human skin. in addition to the count of nerve endings, the identification of patterns associated to nerve regeneration can increase the diagnostic yield of skin biopsy. the most common type of charcot-marie-tooth disease is caused by a duplication of pmp , leading to dysmyelination, axonal loss and progressive muscle weakness (cmt a). currently, no approved treatment for cmt a patients is available. among others, preclinical therapeutic approaches aim to correct the pmp overexpression in order to ameliorate axonal loss and muscle weakness. we previously reported that the novel polytherapeutic drug pxt , a low-dose combination of baclofen, naltrexone and sorbitol, improved dysmyelination and axonal loss after long-term application in adult pmp transgenic rats, a known animal model of cmt a. interestingly, after short-term application in young cmt a rats we observed a long-lasting prevention of muscle weakness as well but without obvious effects on dysmyelination and axonal loss. improved distal motor latencies in the electrophysiological recordings and a shift in the axonal diameter distribution raised the hypothesis that therapy may improve the neuromuscular endplate pathology as another therapeutic target for cmt a. here, we report a preclinical trial in adult cmt a rats treated from postnatal week for consecutive weeks as the most effective regimen in order to facilitate a deeper understanding of the mode-of-action of pxt . long-term therapy effects were confirmed by an ameliorated behavioral phenotype, improved distal motor latencies and also nerve conduction velocity in the electrophysiological recordings. histological analysis revealed an increased number of neuromuscular endplates, which were lowered to wildtype level after pxt treatment. further investigations include detailed analysis of peripheral nerve myelin thickness, internodal and nodal length, terminal axonal sprouting at the neuromuscular endplate and muscle fiber subtyping in treated cmt a rats. charcot-marie-tooth disease (cmt) is a rare hereditary neuropathy for which novel treatments are being developed and urgently needed. only few clinical trials and natural history studies have been performed. evaluation of intervention efficacy is hampered by slow progression and lack of sensitive outcome measures. the interindividual disease variability is high and prognostic disease measures are not established for cmt patients. previously, we have identified skin-derived disease and progression biomarkers in a large european and us-based cmt a cohort. within the german charcot-marie-tooth disease network (cmt-net, www.cmt-net.de) we aim to establish easier accessible biomarkers in blood of cmt a patients. blood analysis is less invasive and can be repeatedly performed during clinical trials and routine patient care. we have started clinically assessing approximately young, adolescent and adult cmt a patients at clinical centres (münster, münchen, göttingen) including a large set of clinical outcome measures (cmtnsv , onls, mwt, mwt, walk , hpt, sf- , fss, psqi, ess, bdi-ii). we will sample blood and skin tissue once a year at , and months for an observational period of years. tissue samples will be analyzed on the transcriptional, translational and epigenetic level. we envision that the diagnostic and progression biomarkers may be used to measure therapeutic effects within clinical trials and later in clinical routine monitoring. this is the first trial testing "circulating" biomarkers from blood in a prospective observational trial in cmt a patients. igm peripheral neuropathy is a slowly progressive and heterogeneous disease with symptoms ranging from mild foot numbness and minor imbalance to severe neuropathic pain and sensory and motor dysfunction. international consensus regarding assessment and treatment of patients with igm peripheral neuropathy is lacking. this might be caused by the repeated use of suboptimal outcome measures, the small trial sizes with low numbers of treated patients, the indolent disease course needing a longer follow-up period to capture relevant changes, or the possibility that administered treatments were not aggressive enough. the imagine study is an international, multi-centre, prospective, observational cohort study, which will result in a unique collection of a large number of prospectively collected and highly standardized clinical data, and a biobank from a large population of well-defined patients with igm peripheral neuropathy. the main objective is to describe in detail the variation in clinical subtypes, clinical disease course, past and current practice of treatment, antibody titres, and clinical picture at the various levels of assessing outcome. patients being at least years, and fulfilling the international criteria for igm peripheral neuropathy are eligible. exclusion is primarily based on concomitant diseases influencing peripheral nerve function. several study parameters measuring weakness, sensation, activity and participation, ataxia, pain, and quality of life are of interest. in february an enmc kick of meeting is organized, bringing together an igm peripheral neuropathy study group. the meeting will focus on the development of a core set of outcome measures to be used in future studies in igm peripheral neuropathy. the imagine study started in the netherlands in september , and to date patients were included. the imagine study starts in the united kingdom, italy, and france in the beginning of . centres recruiting at least patients with igm peripheral neuropathy are eligible to participate in the study. during the pns global recommendations from the enmc meeting, as well as the first results of the imagine study, will be presented. puget s , paolantonacci p , burlot l lfb biomédicaments, les ulis, france; lfb biotechnologies, les ulis, france. in dysimmune neuropathies patients (mmn, cidp, gbs), ivig have become the gold standard due to their efficacy and tolerability. acute renal impairment, a rare but serious adverse event, can be induced by all ivig. its incidence is not determined precisely for each ivig. it mostly occurs in patients at risk, with pre-existing conditions (prior renal insufficiency, diabetes, mellitus, arterial hypertension, elderly > years old, dehydratation, hypervolemia, sepsis, paraproteinemia or concomitant use of other nephrotoxic drugs) and treated by immunomodulatory doses. as the number of these patients increases over the years, the choice of ivig in association with the precautionary measures will be decisive to reduce or avoid the occurrence of this adverse event. from s to s, the nephrotoxicity of ivig has been explained by tubular toxicity related to osmotic nephrosis. sucrose, a stabilizer of some ivig, has been implicated as one of the causes of this. even if several new ivig without sucrose are used, renal impairment cases have been described and studies have highlighted that sucrose is not the only ivig stabilizer associated with tubular damage. however, in a recent study, renal impairment cases ( / ) have occurred in cidp elderly patients treated by free-sucrose ivig. the occurrence of renal failure seemed to be low (only / ) for patients treated with sucrose-ivig in association with precautionary measures (hydratation, adaptation of dosage according body mass index, rate infusion). otherwise, since s, cases of renal impairment secondary to hemolysis have been increased with some ivig but would be rare for full ethanolic fractionated products as they have been identified to generate very low occurrence of hemolysis. the occurrence of renal impairment related to ivig seems to be multifactorial, need to find out all causes (mechanisms of different stabilizers, hemolysis, and concentration of ivig). registries would be helpful for physicians to define the incidence, relative risk of ivig-related renal impairment and the right ivig in association with precautionary measures in these patients at risk. the published ultrasound (us) studies on chronic inflammatory demyelinating polyradiculoneuropathy (cidp) report diffusely increased cross-sectional area (csa) of nerves. these data have not been correlated with histological patterns. to date, no further information about us ultrastructural nerve modification has been provided because of limited frequency range ( - mhz) of the us probe. the aim of this preliminary study is to evaluate the correlation between histological findings from sural nerve biopsies and us patterns found with uhfus ( mhz) from sural nerve at the ankle. four patients with cidp underwent uhfus nerve evaluation of clinically affected sural nerve before undergoing a sural nerve biopsy. us findings included: cross-sectional area of the nerve, connective tissue depth and changes in echogenicity of fascicles. those data were then compared to the histological findings obtained in transversal and longitudinal sections. in all patients, uhfus nerve changes were as following: csa was increased; connective tissue was thickened and in two of the four patients hyperechogenicity of fascicles was observed. also, thanks to the ultra-high-resolution of the probe, a direct correlation of the histological and us images was possible, so as a direct measurement of internal microstructure of the nerve. uhfus may be of adjunctive diagnostic value in cidp assessment. more detailed images of sural nerve can be obtained compared with high-frequency us, allowing a better evaluation of the internal structure of the nerve. the increased csa observed in all our subjects seems to relate to an enlargement of connective tissue, as confirmed by the histological study. in particular, we have observed a hyperechogenicity of fascicles in most severe patients; in those cases, histology confirmed an increase of endoneurial depth. we speculate that those findings may explain a preliminary involvement of connective tissue in the pathogenesis of the cidp; our findings of nerve enlargement may be tool to monitor disease activity in cidp, and better understand disease pathogenesis. further studies are needed to confirm these findings and additional data are being processed and will be presented during the meeting. puma a , cambieri c , panicucci e , desnuelle c , raffaelli c , sacconi s . high-resolution ultrasound (hfus, mhz) of peripheral nerves is a valuable non-invasive, painless complement to neurophysiology, especially in the workup of cidp. nevertheless, the current spatial resolution of echographic images doesn't allow a detailed study of the nerves. ultra-high frequency ultrasound (uhfus, mhz) is a new tool with a - times higher spatial resolution than traditional hfus. the aim of this preliminary study is to evaluate sensory and motor nerves structural characteristics found with uhfus in patients with definite cidp. seven patients with cidp underwent uhfus nerve evaluation of median, ulnar, peroneal and sural nerves, bilaterally. us findings included: cross-sectional area of the nerve, connective tissue depth, nerve vascularization and changes in echogenicity of fascicles. patients also underwent electroneurography (eng) and plexus mri. in all patients, uhfus nerve changes were as following: csa was increased; connective tissue was thickened. two of the seven patients presented an epineural hypervascularization, observed at the doppler evaluation; none of them was treated by iv immunoglobulines. echographic changes were present even in the absence of mri abnormalities (root hypertrophy). eng characteristics correlated with the us patterns. uhfus may be of adjunctive diagnostic value in cidp assessment. more detailed images of nerves can be obtained compared with high-frequency us, allowing a better evaluation of the internal structure of the nerve. the increased csa observed in all our subjects seems to relate to an enlargement of connective tissue. in particular, uhfus allows a more detailed study of nerves, demonstrating that the structural abnormalities hit connective tissue, while -conversely to previous studies -fascicles anatomy seems to be spared. we did not show nerve vascularization except in non-treated patients. we speculate that those findings may explain a preliminary involvement of connective tissue in the pathogenesis of the cidp; our findings of nerve enlargement may be tool to monitor disease activity in cidp, and better understand disease pathogenesis. further studies are needed to confirm these findings and additional data are being processed. purcell l , , wojciechowski e , , gibbons p , , jamil k , , menezes, m , , burns j , . sydney children's hospitals network (randwick and westmead), new south wales, australia; university of sydney, new south wales, australia; universiti kebangsaan, kuala lumpur, malaysia. - % of children with cmt are reported to have hip dysplasia. we aimed to investigate the relationship between radiological hip dysplasia indicators and walking pattern in children with cmt. thirty children ( female; . ± . yrs; ± . cm; ± . kg cmt a, cmtx , cmtx , cmt f, cmt a) underwent d gait analysis ( dga), and had an anterior-posterior pelvis radiograph within months of assessment. radiographs were reviewed by two orthopaedic surgeons, and the reliability of measures of dominant limb hip health via radiograph was assessed. of the measures, measures had an intraclass correlation coefficient > . between raters, and the more experienced surgeon's measures were used for further analysis. the measures of acetabular index (ai), centre edge angle (cea), neck shaft angle (nsa), medial joint space, head width, lateral uncoverage, migration percentage and triradiate were used to investigate correlations with kinematic and kinetic dga variables of the pelvis and dominant limb hip, knee and ankle in planes, temporal spatial parameters and gait profile score. dga data were captured with an -camera vicon nexus motion capture system using the lower body plug-in-gait model. gait data were compared to typically developing children ( female; . ± . yrs, ± . cm, ± . kg). five of affected children had a migration percentage > ∘ , representing % of our sample. maximum hip abductor moment in terminal stance was significantly lower than normative reference values, and was moderately correlated with of the radiographic measures (ai r=− . , p= . ; nsa r=− . , p= . and medial joint space r=− . , p= . ). walking speed (normalised to leg length) was correlated with medial joint space (r= . , p= . ) head width (r=− . , p= . ) and triradiate (r=− . , p= . the ganglionopathies are a unique group of disorders with a varied etiology which includes autoimmune disorders, paraneoplastic syndromes and toxin induced causes. it has been observed that despite extensive investigations the cause of a sensory neuronopathy is often idiopathic in approximately - % cases. electrodiagnostic criteria to diagnose a possible ganglionopathy requires at least one sensory action potential absent or three sensory action potentials < % of the lower limit of normal in the upper limbs plus less than two nerves with abnormal motor nerve conduction study in the lower limbs. ulnar sensory-motor amplitude ratio values (usmar) lower than . is useful in differentiating ganglionopathy from axonal length-dependent neuropathy. we performed a retrospective analysis of patients who were either admitted or evaluated in the outpatient department in department of neurology, nizam's institute of medical sciences, with a possible ganglionopathy as per the diagnostic criteria proposed by camdessanchéet al. consecutive patients who sought treatment during a year period with a clinical pattern of sensory neuronopathy were analysed retrospectively. we reviewed the electrodiagnostic studies of patients and calculated the ulnar sensory-motor amplitude ratio values in comparison with patients with a clinical and electrophysiological diagnosis of diabetic axonal sensorimotor neuropathy. the clinical profile of a ganglionopathy from the indian subcontinent comprised of a prominent sensory ataxia as the initial presenting clinical manifestation with a predilection towards involvement of large-fiber sensory modalities. electrophysiologically a pattern of absent snaps was characteristically encountered with a significant value of usmar less than . , enabling differentiation from axonal length-dependent neuropathy. rahman im , jahan it , nahar s , khalid mm , jahan i , hayat s , , , islam z . guillain-barré syndrome (gbs) is a post-infectious autoimmune polyradiculoneuropathy where immune response is triggered by molecular mimicry between glycolipid antigens expressed by an infective agent such as campylobacter jejuni (c. jejuni) and human peripheral nerve gangliosides. cd molecules are mhc-like structures specialized in capturing and presenting a variety of glycolipids to antigen-specific t lymphocytes. the objective of this study was to investigate the possible role of coding region polymorphisms of cd genes in the pathogenesis of gbs in bangladeshi population. single nucleotide polymorphisms (snps) of exon of cd a (* /* ) and cd e (* /* ) in well defined gbs patients and healthy controls were studied to delineate their effect in developing gbs. we genotyped exon of both cd a (cys/tryp) and cd e (gln/arg) genes through pcr-rflp. to validate these findings, direct sequencing of pcr product was performed for at least samples for each position. we found no differences in genotypes and allele frequencies of both genes in gbs patients compared to controls. however, compared to control individuals with cd a* /cd e* haplotype were . times more likely to develop gbs, whereas individuals with cd a* /cd e* haplotype had a reduced relative risk by . times. a positive association of cd a* /cd e* haplotype was observed only in axonal form of gbs ( . % vs. . %, p = < . ). haplotype cd a* /cd e* was prevalent among anti-gm antibody positive gbs patient compared to anti-gm antibody negative patients ( . % vs. . %) though it was not statistically significant. snps in cd a and cd e were not associated with antecedent c. jejuni infection, disease severity and disease outcome at months of follow up. in conclusion, cd polymorphisms are not a susceptibility or disease causing factor in gbs. conversely, increasing knowledge of this field may offer new dimension for the research to elucidate better answer for disease pathogenesis and also contribute to conduct high power meta-analysis. extensive genetic testing was performed on a unique patient, who received national media attention due to her severe cmt phenotype. the index patient was never able to walk independently, had difficulty breathing and swallowing and demonstrated aspiration by a barium swallow study. nerve conduction velocity testing revealed velocities of m/s in her upper extremity. she required continuous positive airway pressure (cpap) for an upper respiratory infection and developed bilateral vocal cord paralysis. we used the combined brief assessment of motor function (bamf) scale to evaluate her disability. her fine motor scale was a / , her upper extremity gross motor scale was a / and her lower extremity gross motor scale was / . unfortunately, she died at age in her sleep, presumably from respiratory arrest. the proband's father was asymptomatic, however, his neurological exam showed pes cavus bilaterally and mild atrophy of the first dorsal interossei muscles in his hands. his nerve conduction velocities were slowed to m/s in the upper extremities and his cmt neuropathy score (v ) was / . we performed whole exome and subsequently whole genome sequencing in the trio. comprehensive structural variant analysis for copy number variations, large deletions, and recombinations was completed by a combination of software tools. known cmt genes were excluded as the underlying cause and the only viable candidate gene remaining was sh tc . we showed expression of sh tc in peripheral nerve and schwann cells. sh tc is a paralogue to sh tc , which causes the recessively inherited dysmyelinating form cmt c. sanger sequencing confirmed the variants in our family, p.v m (c. g>a, chr : ) and p. a p (c. g>c, chr : ) , and showed the segregation of the heterozygous variations. we hypothesize that the heterozygous paternal allele had a minimal effect and let to a very mild or subclinical form of peripheral neuropathy. of interest, the recessive sh tc gene has also been shown to cause mild aberrant forms of peripheral nerve degeneration in carriers of heterozygous disease alleles. in summary, this paper will present genetic and molecular evidence from an extensive n= study proposing a novel cmt gene. charcot-marie-tooth (cmt) is an important cause of morbidity worldwide. it is a heterogeneous disease manifesting as progressive weakness, wasting and loss of feeling in a length-dependent pattern. there are an increasing number of cmt-related genes in the literature. more recently, recessive mutations in the hint gene have been reported as causative of predominant motor axonal neuropathy associated with neuromyotonic discharges on emg. one of the characteristics of autosomal recessive cmt is it varying frequency in different populations and ethnic groups. we sought to evaluate the frequency of mutations in the hint gene in a brazilian cohort with axonal hereditary neuropathy. all patients included in this study born within south east area of brazil. the group consists of consecutive patients with axonal neuropathy screened for recessive or sporadic axonal neuropathy in the neurogenetics laboratory of clinical hospital of ribeirão preto. direct sequencing of the coding region of hint gene was done. among patients screened, were suspected of having recessive axonal neuropathy without neuromyothonic discharges, and two have axonal neuropathy associated with neuromyothonic discharges on emg. we did not find any disease causing mutations among our patients. some previously reported studies reported a high frequency of mutations in the hint gene among recessive axonal neuropathies in some european countries. our results demonstrated that frequencies of mutation underlying genetic hereditary neuropathies are different between different ethnic groups and have implications for the organization of services management of cmt, for genetic counseling, and for gene flow in different world populations. funded by cnpq, fapesp, faepa, pronas (ministry of health). rodriguez-menendez v , ballarini e , malacrida a , , ceresa c , semperboni s , , meregalli c , cavaletti g , nicolini g . school of medicine and surgery, experimental neurology unit, university of milano-bicocca, monza, italy; phd program in neuroscience, university of milano-bicocca, monza, italy. bortezomib (btz) is a proteasome inhibitor widely used for multiple myeloma treatment. btz induced peripheral neuropathy (bipn) is the most frequent adverse effect. bipn in humans, is a dose dependent painful sensory neuropathy characterized by nerve axonopathy and a tendency to recover in the follow-up period after btz withdrawal. bipn affects principally dorsal root ganglia (drg) and different rodent models have shown alterations in sensory neurons, small unmyelinated axons, large myelinated axons, axonal mitochondria and schwann cells. in this work, we evaluated the effects of btz in vitro on drg neurons isolated from adult mice. our interest focused on dystonin, a protein able to interact with all the three components of cytoskeleton (microtubules, microfilaments and intermediate filaments) and able to bind map b (a mt-associated protein) through which can influence golgi apparatus morphology. there are different neuronal isoforms of dystonin and in particular dystonin-a is able to modulate tubulin acetylation and stability through interaction with map b. it is noteworthy to underline that map b expression in the mature nervous system is restricted to sensory neurons. western blot analysis demonstrated that a treatment with nm btz induces a statistically significant decrease in tubulin acetylation. this result does not go along with our previous study where we observed a tubulin polymerization increase after btz treatment. therefore, we have decided to focus our interest on soma organelle organization that is well known to be dependent from cytoskeleton structure. immunofluorescence images showed an altered distribution of acetylated tubulin in soma cytoplasm after hour treatment. moreover, confocal analysis of cis golgi (gm ) demonstrated that in btz treated neurons the normal golgi structure is lost showing a spot-like non perinuclear labeling distribution. additionally, dystonin distribution seemed comparable to that of cis golgi apparatus suggesting that btz could induce a change in dystonin localization which in turn affects golgi organization, probably through map b.these data suggest that the cytoskeleton alterations, induced by btz, could probably cause wrong maturation and trafficking alteration of golgi vesicles consequently impairing the correct sensorial function. romão tt , aleixo bfl , wedemann d , herculano fgn , prado h , cal h , pupe c , bittar c , nascimento ojm . universidade federal fluminense (uff), rio de janeiro, brazil. acute motor and sensory axonal neuropathy (amsan) is a clinical variant of guillain-barré syndrome (gbs) which has rarely been reported in association with human immunodeficiency virus (hiv) and neurosyphilis. we describe a case of a -year-old woman who started with muscle weakness in the left leg followed by weakness in the right leg and in upper limbs in one month. motor symptoms were followed by urinary and fecal incontinence. no preceding symptoms, as diarrhea or fever were referred. neurological examination showed global areflexia with impairment of vibratory and tactile modalities, as well as loss of proprioception. pinprick sensation was moderately compromised. no impairment of higher mental functions was observed. other causes of neuropathies were ruled out. cerebrospinal fluid (csf) examination showed cell/mm , increased protein level, normal glucose level and negative vdrl test. blood analysis showed hiv and positive serology tests, vdrl : and positive t. pallidum hemagglutination assay. igm cytomegalovirus antibody was negative, cd was in normal range and hiv load was undetectable. cranial and spinal cord magnetic resonance imaging revealed extensive involvement of the posterior spinal cord tracts which supports the diagnosis of neurosyphilis. nerve conduction studies showed a significant reduction in compound motor action potentials amplitudes, particularly at lower limbs, and absent sural nerve responses. electromyography revealed positive waves and fibrillation at lower limbs. treatment with methylprednisolone, human intravenous immunoglobulin and ceftriaxone has been settled, resulting in improvement of upper limbs paresis and sphincters dysfunction. antiretroviral therapy was initiated. this amsan case is associated with central nervous system involvement, encompassing an encephalomyeloradiculopathyrelated to hiv/t. pallidum co-infection. c.jejuni is the main etiological agent associated with amsan, but there are some reports of hiv/cmv co-infected patients who presented this gbs variant. facing a patient with amsan, mainly when cns involvement is present, it is of crucial importance to undergo investigation of an ongoing infectious process, such as hiv and syphilis, considering the impact of early treatment on prognosis. roodbol j , , yiu e , doets a , , de wit mcy , monges ms , van nes s , catsman-berrevoets ce , van den berg b , , jacobs bc , and on behalf of the igos-kids consortium. only a few prospective cohort studies so far have investigated the clinical presentation and course of guillain-barré syndrome (gbs) in children using age-adjusted outcome scores. in we started a study based on the igos protocol but adjusted for children with gbs (igos-kids). (pediatric)neurologists from argentina, australia and the netherlands joined forces to determine the clinical and biological determinants of disease progression and recovery in gbs in children from different geographical areas. for each age category specific pain scales are used including the comfort score for children < years old, pain faces revised age - years old and numerical rating scale in children ≥ years old and outcome measures. a new strength score was developed: gbs kids score. this score will be used and validated in addition to the mrc-sum score. the onls, r-ods and fss are also be used in igos-kids. age-dependent quality of life questionnaires were added to the igos-kids protocol, namely the pedsql and the pedsql multidimensional fatigue scale. these scales are available for children ≥ years old. an activity score validated for neuromuscular disorders for children ≥ years old was also added. currently there are children included in igos-kids, eight children from australia and five from the netherlands. additional clinicians and researchers interested in gbs in children are most welcome to participate in igos-kids. prognostic models have been developed to predict the highly variable clinical course of guillain-barré syndrome (gbs) in adults. the clinical course of gbs is equally variable in children, but the current prognostic models have not been validated in children. in this study, we aimed to identify in children with gbs the characteristics at hospital admission that predict the clinical severity and outcome. the study was conducted in two patient cohorts from europe: one (largely) german cohort of children and one dutch cohort of children. clinical information was obtained regarding preceding infection, first symptoms, neurological deficits at admission and nadir, results of additional investigations (cerebrospinal fluid and nerve conduction studies). clinical severity, course and outcome was defined by the gbs disability score, especially the ability to walk unaided, at a follow-up of month, months, months and months after onset of symptoms. univariate and multivariate regression analyses were performed initially on the two separate cohorts. combined the cohorts consisted of children (age median years, range - years) including boys ( %). the median duration between onset of symptoms and hospital admission was days (range ). pain at admission was remarkably frequent and present in ( %) children. in the combined cohorts, children ( . %) developed respiratory failure and one child died. multivariate regression analysis showed that in both cohorts strong predictors of respiratory failure available at hospital admission were cranial nerve involvement, a higher gbs disability score and a shorter period in days between onset of symptoms and admission. this information was used to develop and validate a prognostic model for children with gbs that will be presented at the conference. in conclusion, based on the analysis of two independent cohorts of patients the predictors of respiratory failure and clinical recovery were identified and validated. similar factors were identified for adult patients, although the prognosis in children in general is better than in adults. this information will be used to develop a simple prognostic model for current clinical practice to predict the chance of respiratory failure and outcome in children with gbs. the negative trials of vitamin c in cmt a have highlighted the lack of sensitive outcome measures in charcot-marie-tooth disease (cmt). neurofilaments are abundant neuronal cytoskeletal proteins and their concentration in blood is likely to reflect axonal breakdown. we therefore examined plasma neurofilament light chain (nfl) concentration as a potential biomarker in cmt. blood samples were collected from patients with cmt and age matched healthy controls over a -year period. disease severity was measured using the weighted cmt examination and neuropathy scores. plasma nfl concentration was determined using an in house developed simoa assay based on the nfl antibodies from the nf-l light elisa kit (umandiagnostics). plasma nfl concentrations were significantly higher in cmt patients (median: . pg/ml; iqr: . - . ) than in controls (median: . pg/ml; iqr . - . , p< . ) and correlated with disease severity as estimated by the weighted cmt examination (n= , r= . , p< . ) and neuropathy scores (n= , r= . , p= . ) . concentrations were also significantly higher when subdividing cmt patients by genetic subtype; cmt a (n= , p< . ), sptlc (n= , p< . ) and gjb (n= , p= . ) or into demyelinating, cmt (n= , p< . ) or axonal, cmt (n= , p< . ) forms compared to healthy controls. there was no significant difference in the plasma nfl concentration after year in patients with cmt (n= , mean difference − . pg/ml, % confidence interval − . - . ) or healthy volunteers (n= , mean difference − . pg/ml, % confidence interval . - . ) which is unsurprising as cmt is a slowly progressive disease in which the rates of axonal degeneration are likely to be constant and elevated. in summary, we have shown that plasma nfl levels are significantly raised in patients with cmt and that they correlate with disease severity. this is of relevance not only for the field of cmt but for peripheral neuropathies in general, and suggests that plasma nfl holds promise as a biomarker of peripheral neuropathy in both routine practice and clinical trials. neurolymphomatosis (infiltration of the peripheral nervous system by lymphoma cells) is a rare and usually devastating condition belonging to the spectrum of lymphoma-associated neuropathies. cerebrospinal fluid examination with cytologic examination, flow cytometry and clonality testing by pcr may show malignant cells especially when nerve root involvement is prominent. however, nerve biopsy remains a useful tool to confirm the presence of malignant cells invading the nerve. neuropathological features are important and pcr-based immunoglobulin or t-cell receptor clonality testing on nerve fragments may add notable value for the diagnosis of neurolymphomatosis, although this has not been systematically investigated. we retrospectively studied clinicopathological data and clonality results of nerve biopsy samples in patients with nl from centres, performed between and . among patients with nl, we found % of b-cell lymphoma and one t-cell lymphoma. nl was the first manifestation in , % of patients. the main clinical pattern was symmetrical progressive sensorimotor polyneuropathy in % of patients and pain was a prominent feature in %. clonality testing showed a monoclonal rearrangement in ( %) cases, oligoclonal rearrangement and no amplification in ( %). the main histological pattern was perivascular infiltration of predominant b-cells in the epineurium, without signs of vasculitis. the extent of axon loss was highly variable between patients. various chemotherapeutic regimens were used and the median overall survival was months. only one case showed a monoclonal pattern among control nerve samples from patients with other types of neuropathy including vasculitis and cidp. overall, we found that clonality testing on nerve biopsy specimens may provide decisive information on the presence of neurolymphomatosis, with a sensitivity of . %, a specificity of . %, a positive predictive value of . % and a negative predictive value of . %. we confirm the utility of nerve biopsy for the diagnosis of nl and show the great yield of pcr-based clonality testing to assess the malignant feature of peripheral nervous system lymphoid infiltrates. despite an accurate diagnosis, neurolymphomatosis still remains a devastating disease with an overall poor prognosis. in the last years, cumulative data have shown that patients with amyotrophic lateral sclerosis (als) and mouse models of the disease present loss of small epidermal and dermal nerve fibers and sensory dysfunctions, in addition to the classical motor symptoms. our objective is to characterize this small fiber neuropathy and to clarify if axonal loss involves all sort of fibers equally, or if there is some specificity. for this purpose, we performed an immunohistochemical characterization of total intraepidermal nerve endings (protein gene product; pgp . ), peptidergic (calcitonin gene-related peptide; cgrp) and nonpeptidergical nerve epidermal endings (isolectin b ; ib ) of the sod g a mouse at different stages: presymptomatic stage ( weeks), disease onset ( weeks) and in symptomatic stage ( weeks). the sympathetic sweat gland innervation was immunolabeled for vasoactive intestinal peptide (vip) from very early stage ( weeks) to disease onset ( weeks). the results showed a marked reduction of the intraepidermal nerve fibers already in the presymptomatic stage compared to the wildtype littermates (p< . ). this axonal loss affected more markedly the nonpeptidergic axons from the disease onset stage ( % axonal loss, p< . ), whereas no significant differences were found in the cgrp positive fibers ( . % axonal loss). a reduction of the sympathetic innervation of the sweat glands was also found from the disease onset stage ( % axonal loss, p< . ). in summary, we have found that nonpeptidergic and sympathetic innervation of the skin are predominantly affected in the sod g a mouse model. these findings suggest that this specificity could be used as an accessible biomarker for the disease. hereditary and inflammatory neuropathies are peripheral nerve disorders of different pathophysiology whose identification is crucial for therapeutic approach. diagnosis of cmt is easy when there is a family history, disease course is slowly progressive, neurophysiological findings are homogeneously abnormal. cases of cmt patients with inflammatory-like phenotypes leading to a misdiagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (cidp) have been described. the presence of "red flag signs", such as slowly progressive decline, mild sensory symptoms, minimal electrophysiological progression and no response to therapy should prompt re-evaluation of the diagnosis of cidp. the introduction of nerve neuroimaging has contributed to the diagnostic work-up. we describe cmt patients ( men, women, mean age . ± . yrs, mean neuropathy duration . ± . yrs) with genetically confirmed cmt, who were initially diagnosed with cidp. neurophysiology showed demyelinating features in patients, the remaining patients had axonal features. csf analysis showed albumin-cytological dissociation in patients, oligoclonal igg bands were present in patient. nerve ultrasound in patients with demyelinating neuropathy showed diffuse increased cross-sectional area. mr-neurography in one patient confirmed diffuse nerve hypertrophy and increased signal intensity, supporting the hypothesis of a overlap syndrome. all patients were treated with immunomodulatory therapies. among patients with demyelinating features, patients underwent ivig for months, without benefit; the remaining were treated with steroids, showing temporary improvement. the patients with axonal neuropathy complaining of a progressive history, underwent ex adjuvantibus plasma exchange and ivig, without benefit. given the lack of benefit from therapies, screening for hereditary neuropathies was performed. among patients with demyelinating neuropathy, had cmt a, cmt b, cmt d. among the patients with axonal neuropathy, one was diagnosed with cmt k. in patients an overlap syndrome was present. several clinical and laboratory features (csf protein elevation), might contribute to the misdiagnosis of cidp in cmt patients. refractoriness to immune-modulatory treatment should rise the suspicion of a hereditary neuropathy. an overlap cmt/cidp syndrome may be considered, in front of acute/subacute deterioration and/or proximal muscles involvement. two patient with an overlap syndrome showed benefit after either steroids or ivig. ruiz m , tripodi sm , campagnolo m , zara g , salvalaggio a , ruggero s , toffanin e , anglani m , briani c . neurology, department of neuroscience, university of padua, padua, italy; neuroradiology, padua hospital, padua, italy. facial diplegia is a rare regional subtype of guillain-barrè syndrome characterized by rapidly progressive bilateral facial palsy in absence of other cranial neuropathies, ataxia or limb weakness. the diagnosis is based on history, clinical examination, laboratory data. the outcome of this variant appears to be better than that of classical gbs. although about % of gbs patients clinically exhibit facial nerve involvement, there are only few reports that demonstrate mri gadolinium facial nerves enhancement. a -year-old previously healthy man sought neurological advice for the acute onset of right perioral paresthesias and hyposthenia at the extensor hallucis longus bilaterally. in the previous month, during a trip to china and japan, he had suffered from gastroenteritis. at neurological examination he presented with bilateral extensor hallucis longus hyposthenia. ankle jerk reflexes were absent. one week later, he developed bilateral facial palsy. neurophysiology disclosed absence of activity in the facial nerves innervated muscles bilaterally and signs of mild partial denervation at right l metamer. csf analysis showed increased protein levels ( mg%) and . leucocytes/ l. serological tests for infectious agents were negative and serum levels of angiotensin converting enzyme were normal. antibodies to monosialoganglioside gm of igg isotype were positive ( / ). t brain mri showed, after gadolinium administration, marked bilateral enhancement of the facial nerves in their extra-and intra-canalicular segments. t lumbosacral mri scan ruled out the presence of disc diseases as well as signal modifications within conus medullaris and cauda equine nerve roots. the patient was treated with iv immunoglobulins ( . g/kg/die for days) with benefit on facial weakness. at follow-up examination months later, the patient presented a further improvement of the facial diplegia and neurophysiology disclosed a partial recovery of activity in the facial nerves, with persistent axonopatic damage. bilateral extensor hallucis longus hyposthenia persisted. in conclusion, we report on a regional subtype of guillain-barrè syndrome with the curious association of facial diplegia and bilateral extensor hallucis longus hyposthenia, and igg anti-ganglioside antibodies, that are not commonly described in facial diplegia. moreover, we provide t brain mri evidence of facial nerve involvement. rumora ae , tabbey ma , lograsso g , dolkowski j , haidar j , lentz si , feldman el . department of neurology, university of michigan, ann arbor, usa; department of internal medicine, division on metabolism, endocrinology and diabetes, university of michigan, ann arbor, usa. diabetic neuropathy (dn) is the most common complication of diabetes affecting up to % of diabetic patients. the pathogenesis of dn in type diabetes is directly related to the metabolic syndrome associated with the western diet composed of elevated levels of long chain saturated fatty acids (sfas) and low levels of medium chain sfas. long chain sfas are associated with insulin resistance and dyslipidemia while medium chain sfas have been associated with decreased lipid accumulation and improved mitochondrial function. since dn is primarily a disorder of the sensory dorsal root ganglion (drg) neurons, we sought to evaluate the impact of sfa hydrocarbon chain length on mitochondrial trafficking mechanisms that are critical for distributing atp throughout drg axon to provide energy for synaptic transmission. we hypothesize that sfas with longer hydrocarbon chains will impair mitochondrial trafficking whereas medium length sfas will not impact mitochondrial movement along the drg axon. in this study, we examined the impact of sfa hydrocarbon chain length on mitochondrial trafficking, directionality and velocity in primary mouse drg neurons. drg neurons were treated with increasing concentrations of long chain sfas, stearate and palmitate, or medium chain sfa, laurate, ranging from . to micromolar for twenty-four hours. drg neurons treated with long-chain sfas, palmitate and stearate, showed a significant decrease in the percentage of motile mitochondria whereas medium chain sfa, laurate, does not alter mitochondrial motility. we next assessed whether motile mitochondria in drg neurons treated with palmitate or stearate exhibited altered directionality or velocity of mitochondrial trafficking. palmitate and stearate treatments resulted in a trending decrease in the number of mitochondria trafficking in both anterograde and retrograde directions. furthermore, . to micromolar palmitate and stearate induced a significant decrease in mitochondrial velocity. laurate treatment, on the other hand, retained directionality and velocity of mitochondrial trafficking. these results suggest that hydrocarbon chain length of saturated fatty acids plays an important role in regulating mitochondrial trafficking mechanisms in dorsal root ganglion neurons. impaired mitochondrial trafficking in drg neurons exposed to elevated levels of long-chain sfas may play a critical role in the progression of dn. dyslipidemia is a critical factor that contributes to the development of diabetic neuropathy (dn). the progressive nerve damage associated with dn correlates with the dyslipidemic state characterized by elevated circulating levels of harmful saturated fatty acids (fas) and low levels of beneficial unsaturated fas. in dn, primary sensory dorsal root ganglion (drg) neurons exhibit energy dyshomeostasis and mitochondrial dysfunction, however, little is known about the differential impact of saturated and unsaturated fas on mitochondrial mechanisms in drg neurons. mitochondrial trafficking is an essential mechanism for transporting mitochondria throughout drg axons to provide cellular atp for neuronal function and neurotransmission. since mitochondrial trafficking is regulated by metabolic flux, we sought to determine whether saturated fa, palmitate, and unsaturated fa, oleate, have differential effects on mitochondrial trafficking in drg neurons. we evaluated mitochondrial trafficking patterns and the mitochondrial membrane potential (mmp) in primary drg neurons treated with physiologically relevant concentrations of palmitate, oleate, and combinations of both fas. primary drg neurons treated with . to micromolar palmitate induced a significant and dose-dependent reduction in the percentage of motile mitochondria. these palmitate treatments also induced a dose-dependent reduction in mitochondrial velocity but did not impact the directionality of mitochondrial movement. alternatively, . to micromolar oleate treatments did not impair the percent of motile mitochondria, the direction of mitochondrial movement, or mitochondrial velocity. since palmitate and oleate have differential effects on mitochondrial motility, we next assessed whether oleate could counter the inhibitory effect of palmitate on mitochondrial trafficking. surprisingly, oleate/palmitate mixtures at ratios of : or : prevented palmitate-induced impairment of mitochondrial trafficking. we assessed the impact of palmitate and oleate on mmp by staining palmitate and oleate-treated drg neurons with tetramethylrhodamine methyl ester. drg neurons treated with micromolar palmitate exhibited an increase in the percentage of depolarized mitochondria while mitochondria in oleate-treated drg neurons retained mmp. interestingly, drg neurons treated with : oleate/palmitate mixtures also maintained polarized mitochondria. these results suggest that saturated and unsaturated fas have a distinct impact on mitochondrial trafficking mechanisms in drg neurons and that equimolar ratios of oleate/palmitate can prevent impairment of mitochondrial trafficking. of g alpha- in m r overexpressed sensory neurons significantly reversed the m r-induced reduction in relative levels of total neurite outgrowth (mean±sd: . ± . (control) vs . ± . (g alpha- knockdown), p< . , n= neurons). further, treatment of m r overexpressing neurons with mt or pirenzepine sequestered g alpha- / proteins at the m r and significantly reversed the impaired cytoskeleton-mediated reduction in total neurite outgrowth (mean±sd: . ± . (control), ± (+ nm mt ), ± . (+ micromolar pirenzepine); n= , and neurons, respectively; for treated groups p value= . vs control by one-way anova). mt and pirenzepine also significantly restored mitochondrial trafficking and abundance of mitochondria in the distal neurites. our findings suggest a novel mechanism in which modulation of m r influences tubulin polymerization, mitochondrial distribution in nerve terminals and controls axonal outgrowth and regeneration. funded by cihr and nih. muscarinic receptors are a group of five g-protein coupled receptors (gpcrs) that are targeted by drugs for the treatment of several human pathophysiological conditions. we have recently shown that selective (pirenzepine) and specific (muscarinic toxin : mt ) antagonists of the muscarinic acetylcholine type receptor (m r) induced elevated neurite outgrowth and protected from small and large fiber neuropathy in adult sensory neurons in various animal models (calcutt et al., ) . one of the major cellular effectors activated by gpcrs is extracellular signal-regulated kinase (erk). the erk signaling cascade regulates a variety of cellular processes including growth and proliferation. both g protein and beta-arrestin mediated signaling pathways can lead to erk activation by phosphorylation through different kinases. activated erk in turn can phosphorylate about cellular substrates, thereby mediating diverse functions. in this study, we have analyzed beta-arrestin recruitment, as part of the receptor internalization process induced by agonist/antagonist binding. in addition, we studied phosphorylation of erk by mt and pirenzepine using isoelectric focusing with phospho-erk specific antibodies and a variety of cell-based assays including beta-arrestin and g protein (gnas/gnasl/gnaq/gna /gna /gna ) knockout cell lines. our study revealed that beta-arrestin is recruited to the m r upon mt and pirenzepine treatment. treatment with nm mt and micromolar pirenzepine significantly increased the dual phosphorylation of tyr and tyr residues of erk / in primary rat sensory neurons (p< . ) in comparison to muscarinic agonist carbachol ( micromolar) and general antagonist atropine ( micromolar). we have identified multiple distinct phosphorylation events on the m r by isoelectric focusing that are specific to mt and pirenzepine induction. further, we have shown that mt and pirenzepine-mediated erk phosphorylation is dependent on both g protein and beta-arrestin recruitment to m r. finally, we reveal that increased erk phosphorylation by mt and pirenzepine significantly (p= . and . , respectively) increased phosphorylation of camp responsive element binding protein (creb) at ser . these results show for the first time that antagonists of the m r can activate the erk signaling pathway and possibly drive phenotypic change in adult sensory neurons. funded by cihr # mop- . charcot-marie-tooth disease (cmt) is representative of inherited neuropathies affecting an estimated in people. the immense advances in gene discovery gained from next-generation sequencing (ngs) projects have revealed the extent of cmt's genetic heterogeneity, with over loci already identified. this knowledge is rapidly translated into clinical comprehensive gene testing panels, often containing over genes. such a large genomic space will invariantly yield variants of uncertain clinical significance (vus) in nearly any person tested. this rise of the number of vus creates major challenges for genetic counseling. in addition, less individual mutations in already known genes are being published as the academic merit is decreasing, and most such testing now happens in clinical laboratories. we propose to capture more of this data in the cmt field to gain a more complete collection of alleles in cmt genes, ideally in conjunction with detailed phenotypic data. this represents a rational approach to eventually reduce the number of vus. thus, we have created a unique, community-driven variant database for cmt researchers and clinicians. the inherited neuropathy variant browser provides simple, user-friendly access to currently reported cmt variation, including patient-level genotypic and phenotypic information. we have also designed an interactive rating system of genetic variation to assist the community with interpretation of vus. for the initial release, we have collected genetic variation, along with genotypic and phenotypic data when available, from published literature, clinical lab reports, and our in-house database. we highly encourage new submissions of not only observed pathogenic variation, but also variation of unknown significance. the goal is to provide a platform for the cmt community to store, share, and discuss genetic data in order to resolve variation of uncertain significance as a joint-effort. with active participation, we aim to provide the community with a more complete mutational spectrum in cmt genes to assist allelic interpretation and patient diagnosis. neurofilaments are strictly neuron-specific intermediate filaments crucial for maintaining axonal architecture. pathogenic mutations in nefl, which encodes the light chain of neurofilament, cause dominantly and recessively inherited charcot-marie-tooth neuropathy (cmt). the nefl-associated neuropathy can be either axonal (type e) or demyelinating (type f). most pathogenic nefl variants are dominantly inherited missense mutations, which are thought to cause disease by inducing nefl aggregation, leading to the disruption of axonal transport. however, investigation of disease mechanisms caused by the mutations has been complicated by the neuronal specificity of nefl. we identified a homozygous nefl variant c. c>t predicting a nonsense change p.arg * in a patient with early-onset cmt. to elucidate the disease mechanism, we used pluripotent stem cells, reprogrammed from patient's skin fibroblasts, to differentiate patient-specific spinal motor neurons. the motor neurons revealed a near complete loss of nefl mrna, and absence of nefl protein. our results establish that nefl is not essential for the development of human nervous system but its absence causes progressive axonal neuropathy. we currently profile the transcriptomic alterations of the motor neurons lacking nefl using single cell rna sequencing to identify compensatory pathways. friedreich's ataxia (frda) is the most common autosomic recessive ataxia, due to a trinucleotide expansion within the frataxin gene. within the wide phenotype, frda patients present also with an axonal neuropathy whose pathological mechanisms are not completely known. eight patients ( women, mean age . yrs, range - ) with genetically confirmed frda underwent neurophysiological and nerve ultrasound evaluation at four limbs bilaterally. echogenicity and cross-sectional area (csa) of median, ulnar, radial, peroneal, tibial, and sural nerves were recorded. mr neurography and diffusion tensor imaging (dti) analysis were performed in one patient; fractional anisotropy (fa), radial (rd) and axial (ad) diffusivity of median, radial and ulnar nerve were calculated at proximal, intermediate and distal sites. all patients presented with sensory axonal neuropathy. seven patients ( %) presented with increased csa of median and ulnar nerves at arm and axilla. mean median nerve csa at mid-upper arm was . mm (normal values < mm ), mean ulnar nerve csa at mid-upper arm was . mm (normal values < mm ). mean median nerve csa at axilla was . mm (normal values < mm ), mean ulnar nerve csa at axilla was . mm (normal values < mm ). mr neurography (performed in one patient) confirmed diffuse swelling and signal hyperintensity of median and ulnar nerves at the arm and dti analysis showed abnormal values of fa, ad and rd along the whole course of evaluated nerves thus suggesting a wide alteration of nerves structure. frda patients presented with an axonal neuropathy characterized, at ultrasound, by a nerve enlargement strictly limited to mid-upper limbs in all patients, findings that cannot be solely explained by a dying-back axonopathy, as suggested by several authors. neither a dorsal root ganglia neural loss could explain by itself our findings, because a diffuse csa reduction would have been expected. on the whole, these findings represent a peculiar feature in frda, but its pathophysiologic meaning remains unclear. inherited peripheral neuropathies are an important health concern for which there is currently no disease-modifying therapy. dogs are affected by a variety of peripheral neuropathies that are breed-specific, indicating a strong genetic component. the use of in-bred populations, such as pure-bred dogs, is advantageous for genetic dissection of disease. acquired peripheral neuropathy (apn) is an inherited late-onset generalized polyneuropathy with high prevalence in labrador retrievers. the most prominent features of apn, laryngeal paralysis and pelvic limb weakness, are associated with the longest peripheral motor nerves in the dog. the pathologic features of apn are similar to human peripheral neuropathy. our aim is to understand the genetic and pathologic features of apn for development of this condition as a naturally occurring large animal model for human disease. we performed a genome-wide association study (gwas) and short-read high-depth whole genome sequencing (wgs) to investigate the genetic underpinning of apn in the labrador. our gwas data indicates that apn is an autosomal dominant disease. the initial analysis from the wgs study resulted in a potential causal variant with an autosomal dominant pattern; this variant is associated with an axonal gene. the neuropathologic progression and histologic features of apn are poorly defined. using genetic markers from our gwas study, we are able to confidently identify labradors with pre-clinical apn, from which nerve biopsies can be obtained. preliminary analysis of biopsies from labradors suggest apn is an axonopathy. further histologic data from preclinical and symptomatic dogs is being obtained to further define the pathogenesis in this model. mutations in morc lead to an axonal form of neuropathy (cmt z). to date, nine families have been published with mutations in the morc gene, showing that this gene is frequently involved in cmt. while the recent genetic data clearly established the causative role of morc in cmt z, its phenotypic consequences in patients and role in neuronal biology remains to be clarified. therefore, we aim to look for altered genetic and biochemical pathways with a transcriptomic approach in order to investigate the role of morc in hereditary peripheral neuropathy. we have performed transcriptomic analysis using a human gene expression microarray (v x k, agilent technologies), in three hek- t cell lines: control, morc knock-down (kd) and the overexpression of the most common morc mutation, the p.r w (np_ ) (kr). differential gene expression assessment was carried out using limma moderated t-statistics. standard analysis techniques perform one test for each gene. thus, for each gene, a t-test statistic is reported together with its corresponding p-value. in this analysis we have used the conventional multiple testing p-value correction procedures proposed by benjamini hochberg to derive adjusted p-values. the preliminary results reveals that kd shows up-regulated genes involved in transmission of nerve impulse and cilium metabolism, suggesting that morc might act at this level in the peripheral nervous system. otherwise, kr shows a major alteration of main axonal metabolic pathways, including the overexpression of genes related to the generation of neuronal action potential, transport through the axon and its targeting in synapse formation. kr also shows a marked alteration of gene expression related to organization, assembly and cilium movement and with the axonemal dynein complex assembly. this study provides an important step towards understanding the pathomechanism underlying to morc p.r w and its role in cmt z. aifm encodes a mitochondria associated apoptosis inducing factor. mutations in aifm lead to a wide spectrum of neurodegenerative disorders: cowchock syndrome; a combined oxidative phosphorylation deficiency (coxpd ) with severe encephalomyopathy; x-linked deafness with peripheral sensory neuropathy; spondyloepimetaphyseal dysplasia (semd) with mental retardation; and an infantile motor neuron disease. previous studies showed severe defects in mitochondrial metabolism, related to redox function, mitochondrial fragmentation, and respiratory deficiencies. in addition, some mutations impair the protein expression of aifm and cause an increase in caspase-independent apoptosis. by targeted next-generation sequencing, we detected the aifm c. c>t (p.phe ser), in a year-boy. this mutation was confirmed in his year-old affected brother. electromyography and nerve conduction velocities studies revealed an axonal polyneuropathy with exclusive involvement of motor fibers, with an early childhood-onset. both children currently show normal cognitive and cranial nerves functions. the in silico structural modeling of human aifm showed that the mutation of a phenylalanine to serine at position disrupts the hydrophobic interaction between phe and pro , and consequently, it destabilizes an alpha-loop domain. cartoon of protein superposition between two different human aifm structures suggest that a lack of constraints in this region could affect the interaction between - helix and the - -hairpin regions, a very important stage for the functional activity of the aifm protein. patient-derived fibroblasts were used to investigate the pathological effect of the p.phe ser mutation: fibroblasts from patients show a similar mrna but different protein expression of aifm compared to healthy control fibroblasts. however, they have an aberrant morphology, from fibroblastic to polygonal shape, and they are larger than control fibroblast; mitochondria from mutant fibroblasts are markedly fragmented compared to controls; the viability of the patient's fibroblasts is lower, but it does not correlate with an increase in apoptosis. instead, it seems to be caused by an increase in the expression of genes activating the senescent program, like p and p . our study confirms that variable effect of different mutations on the protein function may contribute to the clinical variability observed in aifm patients. funds: isciii (pi / ); fundació per amor a l' art. sango k , takaku s , niimi n , yako h . diabetic neuropathy project, tokyo metropolitan institute of medical science, tokyo, japan. coculture models of neurons and schwann cells have been utilized for the study of myelination and demyelination in the peripheral nervous system; in most of the previous studies, however, these cells were obtained from the primary culture with embryonic or neonatal animals. because it is recognized that some biological properties of both neurons and schwann cells change with maturation and aging, culture systems of adult animal cells appear to mimic peripheral nerve degeneration and regeneration better than those of immature animal cells. we have established spontaneously immortalized schwann cell lines from long-term cultures of adult fischer rat peripheral nerves. one of these cell lines, designated ifrs , has been shown to retain distinct schwann cell phenotypes, such as spindle-shaped morphology with expression of glial cell markers, synthesis and secretion of neurotrophic factors and cytokines, and fundamental ability to myelinate neurites in cocultures with adult rat dorsal root ganglion neurons and nerve growth factor-primed pc cells. our current investigation focuses on the establishment of the coculture system of ifrs cells and nsc- motor neuron-like cells. nsc- cells were seeded at a low density ( x /ml) and maintained for a week in serum-containing medium supplemented with non-essential amino acids and brain-derived neurotrophic factor (bdnf, ng/ml). after overnight exposure to mitomycin c (mmc, micro g/ml), nsc- cells were cocultured with ifrs cells ( x /ml) and maintained in serum-containing medium supplemented with bdnf ( ng/ml), ciliary neurotrophic factor (cntf, ng/ml) and coenzyme q ( micro m). under this culture condition, overgrowth of nsc- cells was prevented and gradual movement of ifrs cells toward the neurites emerging from nsc- cell bodies was observed. double-immunofluorescence staining carried out at day of the coculture showed myelin protein zero-immunoreactive ifrs cells surrounding the beta iii tubulin-immunoreactive neurites. this coculture system can be a beneficial tool to study the pathogenesis of motor neuron diseases (e.g. amyotrophic lateral sclerosis, charcot-marie-tooth diseases and immune-mediated demyelinating neuropathies) and novel therapeutic approaches against them. santos pp , torezani gs , maciero l , pagliarini lfd , romão tt , abunahman ms , ferreira is , bittar c , pupe c , nascimento ojm . universidade federal fluminense (uff), rio de janeiro, brazil. zika virus (zikv) is a flavivirus related to dengue, yellow fever and west nile viruses, and has been recently associated to the occurrence of neurological complications in children and adults. previous studies have linked zikv to the development of guillain-barré syndrome (gbs), myelitis, meningoencephalitis and ophthalmological manifestations in adults. guillain-barré syndrome (gbs) encompasses a spectrum of post-infectious neuropathies characterized by different distributions of weakness and sensory impairment. serum anti-ganglioside antibodies are often found and are related to different clinical patterns. recently we have encountered patients in rio de janeiro, brazil, with distal edema in lower limbs, acute weakness, pain and sensory disturbances during the acute stage of an acute febrile exanthematous illness. the symptoms persist for up to days, with complete resolution afterwards, without specific therapy. blood concentrations of muscle enzymes show normal values, and electromyography and nerve conduction studies (emg/ncs) are usually unremarkable. of note, all cases have had positive rt-pcr for zika virus, indicating an illness that occurred during the viremic phase of this arbovirus infection, and complete recovery within the expected timeframe for the resolution of the systemic viremia. there is a subset of patients who developed acute weakness very early after the initial viral symptoms, with clinical, laboratorial and electrophysiological findings that substantially differ from gbs. we describe three cases with similar features suggestive of an acute infective polyneuritis (aipn). one might hypothesize that zikv might lead to a direct neurotropic injury, significant enough to cause weakness and sensory complaints, but not severe enough to cause permanent damage, resolving in conjunction with decreasing blood viremia. we consider that these patients differ from classical gbs because their illness begins during the acute febrile stage of an infective illness and the clinical course is more rapid leading to complete resolution in a few days. savransky a , mozzoni j , massaro sanchez mp , reisin r , monges ms . department of neurology, hospital de pediatria j.p. garrahan, buenos aires, argentine; department of physical therapy, hospital de pediatria j. p garrahan, buenos aires, argentine; department of neurology, britain hospital, buenos aires, argentine. our objective is to describe a series of children with guillain barré syndrome (gbs) included in the igos protocol. as part of the igos multicenter protocol, pediatric patients meeting gbs diagnostic criteria, who consulted within the first weeks of symptom onset and had parental consent to participate in the study were included. patients were also offered to participate in the extended -year protocol. all patients were evaluated according to the igos protocol. patients were recruited between october and june . twenty-four patients, eight girls, participated. ages ranged from months to years (mean . years). all patients agreed to participate in the extended protocol, which was completed by of them. eight patients completed the -year follow-up and are still under evaluation. five patients were lost to follow-up.twenty-two had the classic variant of gbs and two miller fisher. radicular pain in the back or lower limbs was reported by %. twenty-three patients underwent lumbar puncture and albumin-cytological dissociation was found in . in all cases, csf was stored for proteomic studies. all patients underwent emg showing aidp in , and amsan and aman in each. one patient with aidp developed to cidp. in patients full-spine mri was performed and cauda equina enhancement was found in every case. three patients required ucip, two with invasive and one non-invasive ventilation. all patients were treated with gammaglobulin, with a second dose at weeks in cases with a poor response. all patients who followed the protocol were evaluated with the gbs disability score. median score was at baseline and between and at the -year assessment. we describe a series of children with gbs as a part of an international protocol including patients of different ages. pain was a frequent and early symptom and could be determined despite the young age of our patients. most patients fully recovered. we were invited to participate in igos kids to better assess this age group. saysavath k , somchit v , t. umapathi . mittaphab hospital, vientiane, lao pdr; setthathilath hospital vientiane; national neuroscience institute, singapore, singapore. laos people democratic republic is a country of . million people in south east asia. largely agricultural, its capital and metropolis is vientiane. adult neurological services are concentrated at mittaphab hospital, vientiane serviced by three neurologists. on the average about six cases of guillain-barré syndrome (gbs) are seen per year. it is believed that most patients do not seek medical attention. cases appear to cluster during the rainy season. most patients present late, often at the second week of illness when recovery is unapparent after seeking treatment from traditional medicine doctors and at district hospitals. a substantial number of patients seek treatment at hospitals across the border, in neighboring provinces of thailand. common antecedent symptoms are viral prodrome and diarrhea. miller fisher syndrome appears to be rare, possibly because of the mild deficits that do not prompt patients to seek medical attention. diagnosis is made largely from clinical features and from spinal fluid analysis. nerve conduction studies are not available. patients are often treated with steroids by internists. intravenous immunoglobulin and plasma exchange are not available. common complications include pneumonia, autonomic dysfunction (fluctuating blood pressure), pressure sores and depression. patients who develop respiratory failure are nursed at a twelve-bedded intensive care units. plans are afoot to set up a prospective gbs database, systematically study antecedent infections, including of flaviviruses, and develop low-volume plasma exchange as a feasible therapeutic modality. a gene therapy approach for treating cmt c neuropathy schiza n , markoullis k , richter j , tryfonos c , kagiava a , sargiannidou i , christodoulou c , kleopa ka , . neuroscience laboratory; department of molecular virology; neurology clinics, cyprus institute of neurology and genetics and cyprus school of molecular medicine, nicosia, cyprus. charcot-marie-tooth type c (cmt c) is the most frequent form among recessively inherited demyelinating neuropathies and results from mutations in the sh tc /kiaa gene. sh tc mutations cause loss of function of the sh tc protein suggesting that gene replacement therapy may be useful for treating cmt c. sh tc −/− mice develop all major aspects of the human pathology including early onset progressive peripheral neuropathy with hypo-and demyelination along with decreased motor and sensory nerve conduction velocities, offering a relevant model for testing treatments for cmt c. in order to develop a gene replacement strategy for cmt c, we generated a novel lentiviral vector, lv-mpz-sh tc .myc, to drive expression of the human sh tc cdna under the control of the myelin protein zero (mpz/ p ) promoter specifically in myelinating schwann cells. a c-terminus myc tag was included to facilitate expression analysis. a control vector (mock) was also produced in which the sh tc cdna was replaced by the egfp reporter gene. we first confirmed expression of hsh tc in hela cells transfected with the pcdna -cmv-sh tc .myc vector. immunofluorescence analysis confirmed a strong expression of sh tc specifically at the plasma membrane with additional localization in a dotted pattern intracellularly. for in vivo gene delivery we used both intraneural and intrathecal injections of the lv-mpz-sh tc .myc vector in -week to -month old sh tc −/− mice. expression of virally delivered hsh tc was assessed weeks after injection. immunofluorescence analysis showed hsh tc immunoreactivity in perinuclear schwann cell cytoplasm in sciatic nerve teased fibers of sh tc −/− mice following both intraneural and intrathecal delivery, while lumbar intrathecal gene delivery resulted additionally in expression of hsh tc in the lumbar roots. real time pcr analysis confirmed hsh tc mrna expression in both lumbar roots and sciatic nerves. thus, we have developed a novel lentiviral vector for schwann cell targeted gene delivery to treat cmt c and for testing possible therapeutic effects in the mouse model of the disease. hereditary neuropathies are a group of disorders which are characterised by the systemic impairment of peripheral nerves. more than neuropathies are associated with causative gene defects [ ]. charcot marie tooth (cmt a) neuropathy is the most frequent hereditary neuropathy, triggered by a mutation in the peripheral myelin protein gene (pmp ). cmt a leads to a primary loss of myelin sheath and afterwards to a degeneration of axons [ ] . symptoms appear with the degeneration of axons, whereas demyelination is thought to be largely asymptomatic. for that reason, we investigate in the mechanisms of axonal degeneration. for our analysis, we used purified axoplasma without detectable myelin proteins of the sciatic nerves of weeks old pmp -c mice. in this early stage of the axonal degeneration sarm was significantly increased. the nad + concentration in the axoplasma was dramatically reduced. this correlates to the previous finding that sarm promotes axonal degeneration by cleavage of nad + . additional studies showed an increase of the nad + -dependent axonal protective factor sirt in pmp -c sciatic nerves. nmnat- , known as the active component of the wallerian degeneration slow gene, was unaffected in axoplasma of pmp -c sciatic nerves. summarised, these results indicate that the pathway of sarm , nad + and sirt may play a critical role in axonal degeneration in neuropathy. we report two unrelated czech patients with cmt b , both sporadic cases in the family. patient is a year old man with congenital glaucoma after ophthalmological surgeries. his early motor development was normal. at the age of years parents noted gait problems, first neurological examination was at the age of years, when emg showed diffuse motor and sensory neuropathy with severely decreased ncv ( - m/s). he developed foot deformities (pes cavovarus) and underwent corrective orthopedic surgeries at the age of years. after several dna tests for demyelinating cmt the sfb gene was sanger sequenced and a novel missense mutation p.ile asn was found in homozygous state in the patient and in heterozygous state in both parents. the patient was later tested also by ngs of a panel of all genes to be causal for hereditary neuropathies and no other potentially causal variants were detected. patient is years old man, with normal early motor development. at the age of years parents noted gait problems with distal leg weakness which progressed into distal leg plegia at the age of years. hand weakness was noticed since the age of years. he has severe atrophies of distal muscles of all extremities, is self ambulant. at the age of , emg showed unrecordable responses from nerves of lower limbs and ncv was measurable only on ulnar nerve and was m/s. at the age of years increased intraocular pressure was diagnosed and he uses anti glaucoma eye drops. after many single gene tests for demyelinating cmt, we used ngs of a panel of genes known to be causal for hereditary neuropathies and two novel heterozygous, probably pathogenic variants affecting invariant splice sites were detected: c. - a>g and c. - _ del, both confirmed by sanger sequencing. the first variant is also in the father, but the second is probably de-novo (not detected in parents, despite correct parentity). sekiguchi y , kikuchi si , konno si , sekiguchi m . department of orthopaedic surgery, fukushima medical university school of medicine, fukushima, japan. carpal tunnel syndrome (cts) is the most common entrapment neuropathy. ultrasonography can be used to detect anatomic changes in cts. more recently, it has been shown that doppler ultrasonography can detect increased intraneural blood flow in cts. the purpose of this study was to determine the most suitable finding of pre-and postoperation in cts by ultrasonography. a total of wrists of patients with nerve conduction study (ncs) proven cts were evaluated with ultrasonography. we measured the median nerve's cross-sectional area (csa) and intraneural blood flow of median nerve by ultrasonography. the correlation between these ultrasonographic measurements, ncs severity and duration of clinical cts symptoms was analyzed. the csa (mean, mm ) was no significantly reduction after successful carpal tunnel release. morphologic median nerve changes may persist for a longer period regardless of successful surgery and clinical improvement. however, intraneural blood flow is increasing after successful carpal tunnel release. we conclude that doppler ultrasonography results strongly correlate with post operated cts improvement. hence doppler ultrasonography is a useful method for functional improvement of pre-and post cts operation. senger jl , chan km , olson jl , webber ca . university of alberta, edmonton, canada. the beneficial effects of a preinjury crush conditioning lesion (cl) on peripheral nerve regeneration is well-documented in animal models. no human studies have been attempted to date, given the ethical dilemma of deliberately injuring an intact nerve, and the difficulty in predicting the timing of a nerve injury. recent studies demonstrate that hour of electrical stimulation (es) produces effects similar to cl in neuronal cultures. this, coupled with a surgical environment favoring nerve transfers, in which an intact nerve is deliberately cut to reinnervated a denervated muscle, means that es may be clinically translatable to enhance regeneration. this study hypothesizes that es prior to nerve injury will enhance nerve regeneration. twelve sprague-dawley rats were divided into four groups based on conditioning-type to the mid-common peroneal (cp) nerve: es ( ), crush ( ), sham-es ( ), and naïve ( ). one week following conditioning, they underwent a cut/coaptation of the cp nerve at the sciatic trifurcation. post-cut day , nerves and dorsal root ganglia (drgs) were collected. axonal counts of nerves stained with nf- revealed similar regeneration between es and crush ( . vs. . mm, p= . ) that was superior to sham-stimulation ( . mm) or no-conditioning ( . mm, p< . ). a greater number of axons at the distal tip were present in animals that received either type of conditioning compared to the unconditioned cohorts. drgs were stained with neuronal injury marker growth associated factor- (gap- ), and satellite cell glial cells with glial fibrillary acidic protein (gfap). significant increase in gap- expression at three days was observed in es and crush cohorts compared to sham or naïve (p< . ) cohorts. the satellite glial cells of es and crush conditioning showed a significant increase in gfap expression ( . % and . % respectively) compared to sham ( . %) and naïve ( . %) drgs. by demonstrating similar improvements in axon regeneration, this proof of principle project suggests that es conditioning may produce regenerative outcomes comparable to the classical crush injury model. in turn, this suggests that es may be a promising method for delivering conditioning lesions in clinical trials for conditioning nerves prior to surgical intervention. we report a unique case of newly developed waldenstrom's macroglobulinemia (wm) in a patient with chronic inflammatory demyelinating polyneuropathy (cidp) with antibodies against myelin-associated glycoprotein (mag) and sulfatide who was undergoing treatment with intravenous immunoglobulines (iv-ig). subsequent rituximab infusions did not have a positive impact. patients with wm can develop demyelinating and axonal polyneuropathies and few patients have anti-mag and/or anti-sulfatide antibodies. anti-mag antibodies ( % of wm) are associated with sensorimotor axon loss and demyelination and anti-sulfatide ( % of wm) with sensory axonal loss. rarely, both antibodies can be present, with a more severe clinical phenotype. cidp associated with anti-mag and anti-sulfatide antibodies can represent independent entities, not associated with wm. there are no reports to date of patients with cidp associated with anti-mag and anti-sulfatide antibodies that developed wm during immunomodulatory therapy with iv-ig. in addition, subsequent rituximab infusions after the iv-ig were stopped have not been proven beneficial, as has been previously reported for anti-mag cidp patients. seventy-six year old right-handed gentleman presented with persistent numbness in his left foot, three months following artificial disc placement in his lumbar spine. gradually he developed sensory ataxia. no radicular signs were present on exam or impingement on serial spine mri's. ncs/emg studies were consistent with a cidp variant with severely prolonged distal motor latencies. serum anti-mag and anti-sulfatide antibodies were elevated. chronic therapy with iv-ig was able to partially stabilize the symptoms; however, six years later he newly developed wm. subsequent infusions with rituximab, after iv-ig was stopped, did not improve the clinical picture or the ncs/emg findings. wm can newly develop in an autoimmune setting, such as cidp associated with anti-mag and anti-sulfatide antibodies. in this particular case, there was an ongoing immunomodulatory therapy for our cidp patient, as he had monthly iv-ig infusions. this may reflect a possible induction of pathological b cell clone proliferation during the iv-ig treatment. subsequent rituximab infusions, after the iv-ig was stopped, did not improve the symptoms or the demyelination features on ncs/emg. he continues to be symptomatic despite efforts. charcot-marie-tooth disease (cmt) is an inherited neuropathy without known cure (prevalance: : ). duplication of the gene encoding the peripheral myelin protein of kda (pmp ) underlies the most common subtype cmt a. severely affected cmt patients suffer from sensory and motor symptoms with wheelchair-boundness. the clinical phenotype is highly variable and is determined by the amount of axonal loss, but the molecular mechanisms of the disturbed neuron-glia interaction are poorly understood. risk factors have not been investigated. therefore, cmt-net, a german network funded by the german ministry of education and research (bmbf, bonn, germany) includes interdisciplinary expertise from molecular biology, neurology, neuropathology and human genetics in order to identify genetic and non-genetic risk factors of disease severity of cmt by: (i) examining the mechanisms of the disturbed axon-glia-interaction and neuronal vulnerability, (ii) identification of genetic modifiers and (iii) novel therapeutic targets, (iv) validating outcome measures in children and adults, (v) establishing a biobank and (vi) exploring the disease burden via an internationally harmonised patient registry. cmt-net includes three service structures cmt-net will focus on cmt a, but also includes rarer subforms. we will provide the scientific basis for the development of translational approaches to therapy in patients. our approach bridges cutting edge molecular screening techniques, transgenic animal models of altered axon-glia interactions (fly, chick, mouse, rat), state-of-the-art genomic technologies and human patient trials in order to understand and treat the disease aggravation in cmt. sezer g , tekol y , sezer z , . erciyes university, betül ziya eren genome and stem cell centre, kayseri, turkey; erciyes university, school of medicine, pharmacology department, kayseri, turkey; erciyes university, good clinical practice and research centre, kayseri, turkey. analgesic effects of antidepressant drugs are well known for a long time, however, their systemic side effects limit their usage as an analgesics. venlafaxine is an antidepressant drug that has different structure. our purpose was to investigate whether systemic analgesic effect has been proved drug, venlafaxine, has local peripheral antinociceptive action. we applied vanlafaxine ( μl , , , μg) to male, sprague-dawley rats' paws by intraplantar injection and also by intraperitoneal route ( , , , mg/kg) in formalin test, a model for acute and tonic pain. we also pretreated another groups of rats with mg/kg naloxone (opioid receptor antagonist), mg/kg cpt (adenosine a receptor antagonist) or saline (ip.) before μg/paw venlafaxine injection. to check the effect is local or not, we determined the blood levels of venlafaxine in at different time points after both the local and systemic applications by gc-ms method. datas were expressed as number of flinches and total time for biting/licking of the injected paw over phase ( - min) or phase ( - min) and analyzed using the student's t-test. venlafaxine induced antinociception at , and μg concentrations by the local peripheral application and at , mg/kg doses by the systemic application in formalin test and the effects were comparable. pretreatment with naloxone diminished the effect of venlafaxine in the both phases, however, it was not statistically significant. pretreatment with cpt decreased venlafaxine induced antinociception only in phase . neither naloxone nor cpt changed formalin induced nociceptive behaviors alone. this is the first that determines the peripheral antinociceptive actions of venlafaxine in rat formalin test. with roles of opioid and adenosine a receptors in this action. our results suggest that venlafaxine has local peripheral antinociceptive effect and such an activity may led to trials for to use this drug as a cream-gel formulation for analgesia in clinics in the future. topical application might permit the attainment of higher and more efficacious concentrations in the region of the sensory nerve terminal, with limited systemic side effects. shah a , hoffman em , klein cj , staff np . mayo clinic, rochester, usa. cipn is a common dose-limiting complication for patients with cancer. the long-term disease burden of cipn is compounded by increasing cancer survivorship, yet there are minimal data on long term outcomes following onset of cipn, especially in population-based studies. we utilized the rochester epidemiology project to examine incidence and disease burden of cipn among individuals of olmsted county, minnesota with neurotoxic chemotherapy exposure between and . clinical records were queried for the presence of neuropathic signs, symptoms and icd- diagnostic codes as well as for patient provided information on impairment with activities of daily living and use of pain medications. a total of individuals with incident exposure to neurotoxic chemotherapy agents between and were identified. based on aan criteria for identifying peripheral neuropathy, ( . %) of these individuals were determined to have cipn, while ( . %) controls did not. the median time from incident exposure to reported symptom onset was days (iqr . - ). patients with cipn received a neuropathy icd- diagnosis in merely cases ( . %). median survival following incident chemotherapy exposure among all cases and controls was . years with a significantly longer mean survival in cases with cipn as compared to that of controls ( . years vs. . years, p< . ). in addition to acute effects in cipn, individuals surviving greater than years following exposure to neurotoxic chemotherapy continue to self-report increased symptoms of numbness (or . , % ci . - . ) and pain (or . , % ci . . . ) of the extremities. through utilization of patient provided information, our study was able to collect data on long-term impairment associated with previous history of exposure to neurotoxic chemotherapy. our results are consistent with previous reports of the high incidence of cipn in the first two years following incident exposure. additionally, our results provide evidence of high incidence of cipn independent of individual chemotherapeutic agent used. additionally, our results indicate icd- -cm diagnostic code attribution may dramatically underestimate the magnitude of cipn. increased survival following exposure to neurotoxic chemotherapy and its long-term disease burden necessitates further study of among survivors. the utility of quantitative muscle ultrasound as a marker of disease severity in charcot-marie-tooth (cmt) disease subtypes was investigated. muscle ultrasound was prospectively performed on individual muscles from cmt patients ( cmt a, cmtx , cmt a) and compared to muscles from age and gender-matched controls. muscle ultrasound recorded echogenicity and thickness in representative muscles including first dorsal interosseus (fdi) and tibialis anterior (ta charcot-marie-tooth (cmt) disease is the most frequent inherited peripheral neuropathy, and there is currently no available cure. the most common subtype of cmt, cmt a, is completely associated with duplication of the pmp gene, which encodes peripheral myelin protein of schwann cells. previous studies of cmt a mainly relied on rodent models, and it is not yet clear how pmp overexpression leads to the phenotype in patients. based on induced pluripotent stem cell (ipsc) technology, we herein developed a brand new in vitro cell model of cmt a, called cmt a-hipscs, in the hopes of simulating the developmental progress of the disease and gaining new insights into its pathogenesis. here, we efficiently derived ncscs from cmt a-ipscs and assessed the potential of the isolated cmt a-neural crest stem cells (ncscs) to differentiate into peripheral neurons and schwann cells using defined media. we found that, unlike normal control ncscs, cmt a-ncscs rarely generated schwann cells. instead, cmt a-ncscs produced numerous endoneurial fibroblasts in the schwann cell differentiation system. we further established a pmp -overexpressing ipsc model, and obtained similar results when pmp -ncscs were subjected to schwann cell differentiation. these results suggest that the development of schwann cells in cmt a patients is interrupted by the duplication of pmp . with the exception of the demyelination-remyelination process, developmental disabilities of schwann cells should be considered as an underlying cause of cmt a. shimoi t , yamada t . international university of health and welfare, tochigi, japan, cmt japan, tokyo, japan. charcot-marie-tooth (cmt) disease is the most common hereditary motor and sensory neuropathy. our preliminary report suggests that a certain cmt patient has the recruitment disorder of motor units during muscle fatigue and this disorder may be a factor of "super fatigability" in motor neuropathy patients. if the "super fatigability" occurs, we would expect patients with this characteristic to become slower in recovery from muscle fatigue than patients without this characteristic. in order to verify this hypothesis, we measured characteristic of recovery from muscle fatigue in charcot-marie-tooth patients with electromyographic study. twenty three participants were asked to maintain their % of maximal voluntary isometric contraction (mvc) of elbow flexor until exhaustion as the fatigue exercise. in addition, the participants asked to perform s of their % of mvc at , , , , , , , , , s after the fatigue exercise as recovery tasks. the surface emg (semg) signals of biceps brachii muscle were determined during the exercise and tasks. muscle force, median power frequency (mdpf) and the root mean square of semg amplitude (rms) were used as objective parameters of muscle fatigue. borg scale was used as a subjective parameter of muscle fatigue. six of twenty three participants showed significant decrease of rms during the fatigue exercise. in consideration of this result, we compared alteration of mdpf in recovery task between six participants with decrease of rms (abnormal group) and seventeen participants with increase of rms (normal group). as the result, the abnormal group had at least s as the recovery time from muscle fatigue in contrast with s of normal group. the recovery time from muscle fatigue in subjective parameter was shorter than the time in objective parameters in each group. our data support the "super fatigability" hypothesis. and that hypothesis may induce "hidden muscle fatigue". comparison between complex regional pain syndrome type and based on electrophysiologic, imaging and clinical findings shin jy , moon jy , sung jj . seoul national university hospital, seoul, republic of korea. complex regional pain syndrome (crps) is a constant regional neuropathic pain characterized by various kinds of motor, sensory, and autonomic changes. conventionally, the crps is divided into type i and ii according to the absence and presence of nerve injury. but the pathogenesis of crps is not fully understood yet. and there is still no systematic comparative study between crps type i and ii. we compared between crps type i and ii using multimodal approaches including electrophysiologic, imaging, and clinical findings. the patients ( type i and type ii) diagnosed with crps using the international association for the study of pain (iasp) diagnostic criteria were included. type i and ii were divided by electromyography and nerve conduction study. we obtained clinical information such as continuing pain, allodynia, hyperalgesia, edema, temperature, skin color, sweating, trophic change from patients. all patients were evaluated by bone scan, thermography, quantitative sudomotor axon reflex test (qsart), quantitative somatosensory test (qst). the ratio of qsart and temperature threshold abnormality in type ii was higher compared to type i ( . % vs . %, . % vs . % respectively, p = . and . ), among clinical symptoms, sweating change significantly high in type ii compared to type i ( . % vs . %, p = . ). other electrophysiologic and imaging, clinical findings were not significantly different in both type. in this study, we identified that crps type i and ii are distinguished not only by the nerve injury but also by the sudomotor function, and qsart can serve as a good technique to differentiate between crps type i and ii. it is estimated that there are two distinct pathogenesis in crps. our results may be helpful to diagnose crps correctly and understand the pathogenesis of crps. sjogren syndrome (ss) is an autoimmune inflammatory disorder of exocrine glands resulting in xerophthalmia and xerostomia. ross syndrome is a rare entity characterized by tonic pupil, hyporeflexia, and segmental anhidrosis. we present a -year-old hispanic woman with debilitating sensory and autonomic neuropathies, and persistently elevated anti-ss-a and anti-ss-b antibodies, without the classic sicca complex. she initially developed diarrhea and an ear infection, then felt toe and later leg numbness, which eventually spread to her cheeks and tongue, over few months. four years later, her thumbs and index fingers started tingling. also, she developed orthostatic lightheadedness, tachycardia, segmental hypohidrosis of the right abdomen, and hyperhidrosis of the remaining trunk, intermittent erythema, chronic diarrhea, and a -pound weight loss. her exam demonstrated orthostatic hypotension, bilateral tonic pupils and light-near dissociation, sectoral palsy of the right iris sphincter, stocking-distribution diminished sensation to all modalities, pseudoathetosis, areflexia, dysmetria, intention tremor, romberg, and sensory gait ataxia. mri of neuraxis demonstrated t -weighted hyperintensity in the dorsal spinal cord from c to the lower thoracic level, with mild atrophy. electrodiagnostic testing was consistent with moderate-to-severe, length-dependent, asymmetric, sensory polyganglionopathies. csf showed oligoclonal bands. serology showed elevated antinuclear antibody ( : , reference < : ), ss-a ( , reference < u), ss-b ( , reference < u), and rheumatoid factor ( , reference < iu/ml) titers. the remaining workup was negative for infection (syphilis, hiv, htlv, hepatitis, lyme disease), paraneoplastic syndrome (anti-hu and ganglionic nicotinic acetylcholine receptor antibodies), pyridoxine intoxication, malignancy (chest/abdomen/pelvis ct, breast ultrasound, axillary lymph node flow cytometry, colon and esophagus biopsy), celiac disease, vitamin deficiency, autoimmune disease (anti-aquaporin and anti-gq b antibodies), and adrenoleukodystrophy. the patient received ivig and steroid with some gait improvement and currently takes mycophenolate. midodrine and fludrocortisone resolved her dizziness. this case highlights an important, treatable sensory ganglionopathy and systemic autonomic neuropathy due to sjogren syndrome, and illustrates the overlapping clinical triad of ross syndrome, which may guide future management. siles am , , assylbekova d , , diaz-manera j , , rojas-garcia r , , cortes e , , gallardo e , , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, univeristat autònoma de barcelona, barcelona, spain; centro para la investigación biomédica en red en enfermedades raras (ciberer), madrid, spain. inflammatory neuropathies are a heterogeneous group of peripheral nerve diseases that respond to immune-therapies. chronic inflammatory polyradiculoneuropathy (cidp) and multifocal motor neuropathy (mmn) are two chronic inflammatory neuropathies responding to intravenous immunoglobulins (ivig). b lymphocytes are involved in their pathogenesis. while widely used in clinical practice, ivig's mechanism(s) remain not completely understood. ivig are reported to lead to b-cell anergy and to increase regulatory t-cell function and frequency. regulatory b cells (bregs) are a rare subset of b lymphocytes that suppress immunopathology acting upon several target cells in the immune system. impaired breg yields have been described in a plethora of autoimmune conditions. the presence of regulatory b cells in inflammatory neuropathies and the effect of ivig therapy on their frequencies has not been studied. the aim of this study is to describe the frequencies of bregs in cidp and mmn and the effect of ivig on their frequencies. patients fulfilling diagnostic criteria for cidp or mmn and matching controls were included. pbmcs were obtained by gradient centrifugation before ivig infusion and one week after treatment. b-cells were isolated with negative selection magnetic beads, cultured and activated with the tlr agonist odn and anti-human igg+iga+igm. il- secretion capacity was assessed by flow-cytometry. twenty-eight patients were included of whom where cidp and mmn. of all patients included, received ivig and were suitable for pre and post ivig breg frequency comparisons. breg frequencies did not differ in patients (before ivig treatment) and controls (p= . , mann whitney test, two-tailed). however, the frequencies of bregs significantly increased one-week after treatment with ivig (p= . , wilcoxon matched pairs test, two-tailed). when stratifying by disease subtype, breg frequencies increased in cidp patients after ivig (p= . , wilcoxon matched pairs test, two-tailed) and in mmn (p= . , wilcoxon matched pairs test, two-tailed) although results did not reach statistical significance in mmn. this is the first study that studies the breg frequencies in cidp and the first study that addresses the effect of ivig on breg frequencies. our study provides the proof of principle that bregs could become a biomarker for response to ivig but this would need a larger and prospective study. siles am , , martínez-hernández e , diaz-manera j , , rojas-garcia r , , gallardo e , , illa i , , graus f , querol l , . paraneoplastic neuropathies (pn) are rare, immune-mediated disorders of the peripheral nerve with important prognostic implications. ectopical expression of neural antigens in the tumor leads to the development of onconeural antibodies. several autoantibodies associate to pn, including anti-hu, anti-caspr or anti-cv antibodies but a significant proportion of pn lack identifiable antigens. adhesion molecules that are autoantigens in other neuropathies, like contactin- , are present in several types of tumors. our study proposes a systematic screening of autoantibodies against neural cell-adhesion molecules and neural structures to detect novel antigenic reactivies in pn. thirty-five patients followed in our centre and at the neuroimmunology-multiple sclerosis unit at hospital clínic de barcelona, with pn fulfilling diagnostic criteria of possible (n= ; . %) and definite (n= ; . %) paraneoplastic disease were included. serum samples were obtained and tested by immunocytochemistry against contactin- (cntnt ), neurofascin (nf ) and the cntn /caspr complex. primary cultures of dorsal-root ganglia (drg) and rat schwann cells were incubated with patients' sera to detect antibodies targeting neural structures. ten individuals ( . %) presented with a tumor and a neuropathy involving both sensory and motor symptoms. the remaining patients ( . %) presented with a tumor and a classical sensory neuronopathy without anti-hu or any other onconeuronal antibody. among the latter, ( . %) patients were diagnosed with small-cell lung carcinoma. the rest of the individuals ( . %) associated diverse malignancies. we did not detect any sera reacting against cntn , nf or the cntn /caspr complex. in igg antibody screening experiments, patients ( . %) reacted against drg neurons, of them ( . %) reacting strongly, and patients ( . %) reacted mildly against rat schwann cells. in igm experiments, patients ( . %) reacted slightly against drg neurons and patients ( . %) against rat schwann cells, of them ( . %) featuring strong staining. experiments screening antibodies against motor neurons and immunoprecipitation assays are ongoing. overall, % of patients reacted strongly against either neurons or schwann cells. our study did not detect antibodies against the neural adhesion molecules cntn , nf and the cntn /caspr complex in patients with pn. however, a significant proportion of pn patients harbour antibodies targeting neural structures, which suggests that novel neoplasm-associated antigens remain to be discovered. simmons m , tao f , abreu l , zuchner s , li j . department of neurology, vanderbilt university school of medicine, nashville, tennessee, usa; hussman institute for human genomics, university of miami, miami, florida, usa. objective: despite of a shared genetic mutation of the trisomy of chromosome p (c p ), patients with charcot-marie-tooth type- a (cmt a) present with a high variability of their disease severities. the underlying cause for the variability is still unclear. in this study, we tested a hypothesis whether a second genetic mutation known to damage the nervous system is also present in cmt a patients with early onset and severe phenotypes. methods: from a cohort of patients with cmt a mutation (chromosome p duplication), we identified patients with an early onset (< or = years of age) of the disease. four of the eleven also had dna testing for a panel of known cmt-related genes and sequencing of mitochondrial dna in addition to the dna testing for c p duplication. results: besides the c p duplication, we identified three additional mutations in the four patients with early onset. the mutations were a missense mutation of arg his in mpz gene, an a g mutation in mitochondrial trna for glycin and a homozygous mutation of c p duplication. three of the four had symptomatic onset at birth. one showed symptoms at years of age. conduction velocities were severely reduced in all four patients (from to m/s). interpretation: traditional approaches to identify genetic modifiers, including snp association, assume that those modifiers are clustered in a small region of human genome and shared by the studied patients. however, our study suggests that genetic modifiers in cmt a may be highly diverse and scattered throughout the genome, which could make the conventional approach via the genetic variants association difficult. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr sjogren's syndrome(ss) is a systemic auto-immune disease that apart from exocrine glands may affect any organ. involvement of peripheral nervous system results in wide spectrum of neuropathic manifestations. the aim of our study was to evaluate the clinico-electrophysiological patterns and pathological characteristics of neuropathy in sjogren's syndrome (ss) patients presented to neuromuscular clinic in a tertiary hospital from south india. this is a retrospective study from the departments of neurology, rheumatology and pathology from nizam's institute of medical sciences. twenty one patients with diagnosis of ss and peripheral neuropathy between to were analysed. clinical records, conventional nerve conduction studies, lip and nerve biopsy reports were collected.in patients with ss associated neuropathy,male to female ratio was : . in ( . %) neuropathy was the initial manifestation,while in ( %)exocrinopathy preceded neuropathy. the patterns of neuropathy included mononeuropathy multiplex(mnm) in patients ( %),ganglionopathy in ( %),length dependant, trigeminal, autonomic neuropathy and cidp in ( %)and cranial neuropathy in ( %).eighteen( %) were seropositive..schirmer's test was positive in ( . %).nerve biopsy showed vasculitis in patients, demyelinating and axonopathy in patients each.we conclude that neuropathy is frequently the initial presentation of ss.mnm is the common pattern followed by ganglionopathy. pattern of neuropathy helps in arriving at the diagnosis of ss. confirmation of ss is not by mere serology. schirmer's test and lip biopsy are equally essential for the diagnosis especially in seronegative patients when clinical index of suspicion is high. siskind ce , tesi rocha c . stanford health care, palo alto, ca, usa; stanford university, stanford, ca, usa. here, we report the first case of a human found to have a homozygous, presumed disease causing variant in the arl ip gene, causing charcot marie tooth disease (cmt) with central nervous system findings. the proband was born at term with apgars of and at and minutes. he was found to have iugr and was hospitalized for days for weight gain. he developed respiratory distress during the admission and was intubated. due to inability to extubate, he was transferred to stanford children's hospital, where he remained for three months. noted during admission was hypotonia, areflexia, minimal voluntary movment, sinus tachycardia, and dysautonomia. brain mri found polymicrogyria and cerebral underdevelopment with generally normal-appearing brainstem, with moderate ventriculomegaly. ncs found length dependent polyneuropathy with axonal degeneration. follow up muscle and nerve biopsy found immature muscle and amyelinating neuropathy the patient had normal plasma amino acid, acylcarnitine, lactate, pyruvate, urine organic acid testing, opththalmology exam, newborn screen, and normal array cgh. genetics ordered whole exome sequencing through baylor genetics laboratory (houston, tx, usa), which found a homozygous nonsense variant in arl ip : c. c>t, p.r x. a second child had been identified by baylor with two variants in this gene. that child had hypotonia, respiratory distress and seizures, and a muscle biopsy consistent with sma. the parents of that child chose to withdraw care at months of age. our patient's parents continued with aggressive therapies, including tracheostomy and g-tube for feedings. he had several subsequent hospitalizations for respiratory distress, possible seizure activity, and buldging anterior fontenelle, but now, at two years of age, has made developmental progress and is living at home with his family. he is able to smile, reaching for toys and swatting objects. he has little voluntary movement, and no longer responds to light touch stimuli. overall, this is the first picture of a child affected with a severe amyelinating form of cmt that causes weakness, hypotonia, and possible seizures, with the main concerning feature being the severe respiratory distress that may be life threatening, but can be managed with extreme care. charcot marie tooth (cmt) disease is the most common inherited peripheral neuropathy. patients frequently ask whether pregnancy will affect their cmt, whether cmt will affect their pregnancy, the optimal delivery and whether they or their child will have a higher risk of complications during pregnancy or delivery. so far few studies address these questions. currently no guidelines exist for the management of pregnancy, delivery and postnatal care in cmt patients. the aim of the study is assess the impact of pregnancy on cmt and assess how cmt affects pregnancy, delivery and care of the new born baby. we designed a retrospective questionnaire with expert help from an obstetrician with a special interest in pregnancy in patients with medical conditions. the questionnaire is divided into four parts (prior, during, after pregnancy and delivery) and includes questions on impairment, falls, pain, fatigue and respiratory complications during those periods; type of delivery, possible complications, details of anaesthesia and difficulties looking after the baby in the first months postpartum. so far women ( pregnancies) with cmt and related disorders have answered the questionnaire. % of patients had cmt a, the remaining had various subtypes of cmt and related disorders. patients reported deterioration of cmt symptoms during pregnancy in % of pregnancies with resolution of symptoms after pregnancy in % of pregnancies. of symptoms questioned walking ( %), balance ( %), and hand function ( %) deteriorated the most. there was an increased use of orthoses and walking aids during pregnancy. the majority of women ( %) had natural delivery, % were assisted and % had caesarian sections which was similar to the uk population ( %). no complications with anaesthesia were reported. the survey is currently ongoing. we plan to survey consecutive patients. data acquired from this survey will provide valuable information on current practice and will inform future guidelines and standard of care in charcot marie tooth disease. multifocal motor neuropathy (mmn) is a slowly progressive disorder in adults, characterized by asymmetrical limb weakness, mainly affecting the arms. despite beneficial effect of immunoglobulins, weakness gradually progresses. a major determinant of muscle weakness is the degeneration of affected motor axons. treatments aiming to reduce loss of motor axons require objective tools to quantify such an effect. therefore, we applied the compound muscle action potential (cmap) scan, which is an electrophysiological method that, with increasing transcutaneous stimulus-currents, successively activates all motor units (mus) in a muscle. it captures the contribution of enlarged mus due to reinnervation by the presence of relative large discontinuities in the scan. the aim of the present study was to identify pathophysiological changes of mu-loss and reinnervation in mmn patients by means of the cmap-scan. recordings were obtained from mmn patients. cmap-scan recordings were performed in the median nerve at the wrist where motor responses were recorded from the thenar muscle. we determined the number of largest cmap-scan discontinuities by means of a novel marker, d , where a low number is indicative of mu-loss and enlarged mus. furthermore, we applied the recently developed method of professor hugh bostock for obtaining a mu-number estimate from the cmap-scan. the median peak cmap amplitude was . mv (range . - . mv) and median d was (range - ). in three mmn patients with a normal maximum cmap amplitude (> mv) a reduced d (< ) was found indicative of mu-loss and enlarged mus. furthermore, d and the estimate of mu number were significantly related (r = . , p < . , n = ). the findings suggest that the cmap-scan is a sensitive tool in detecting the underlying pathological changes of reinnervation and mu-loss in mmn, more so than standard maximum cmap amplitude. it is quick and easy to perform and has the potential to be useful for follow-up studies. smith ag , thurgood b , revere c , hauer p , aperghis a , singleton jr . university of utah, salt lake city, utah, usa. corneal confocal microscopy (ccm) directly and quantitatively assesses corneal innervation including nerve fiber length (nfl) and density (nfd). ccm has shown promise as a diagnostic test. we have previously demonstrated that ccm has a diagnostic performance for diabetic neuropathy (dpn) similar to skin biopsy with assessment of intraepidermal nerve fiber density (ienfd) and nerve conduction studies (ncs). the responsiveness of these surrogate measures to dpn progression and their relation to clinically meaningful outcomes has not been well explored. diabetic patients undergoing annual retinopathy examination were recruited. each underwent ccm, ienfd, ncs including sural sensory and peroneal motor responses, the utah early neuropathy score (uens), the norfolk quality of life -diabetic neuropathy (nqol-dn, a validated neuropathy specific qol scale), and a minute walk test ( mwt). with dpn based on symptoms ( %) or signs underwent repeat testing at months and at months. at baseline, nqol-dn correlated with sural sensory amplitude (ssa) (− . , p< . ), peroneal motor conduction velocity (pcv) (− . , p< . ) and ienfd (− . , p< . ). no ccm metric was related to qol. mwt distance correlated with ssa ( . , p< . ), nfl ( . , p< . ) and nfd ( . , p< . ). over months, there was a significant worsening in dpn signs assessed by the uens (increase . +/− . , p< . ). ssa declined . uv (p< . ) and ienfd . fibers/mm (p< . ). there was no change in any ccm metric, pcv or nqol-dn. these findings suggest measures of distal axonal integrity are most sensitive to neuropathy progression, with ienfd having the greatest responsiveness. in contrast, ccm was not responsive to dpn progression. both ncs and ienfd (but not ccm) were significantly correlated with neuropathy-specific qol, whereas ncs and ccm measures correlated with physical functioning. the responsiveness of ienfd and ssa, and their relationship to qol support their selection as endpoints in dpn clinical trials. chronic inflammatory demyelination polyradiculoneuropathy (cidp) affects in , people, and is marked by chronic autoimmune infiltration of peripheral nerves and destruction of the myelin sheath. with current therapies, only % of cidp patients achieve complete remission. to produce more effective, mechanism-based therapies, we study mice with a partial loss of function g w substitution in the autoimmune regulator (aire) gene on the non-obese diabetic (nod) background (nod.aire gw/+ ) that develop spontaneous autoimmune peripheral polyneuropathy (sapp) resembling cidp. autoimmunity can result from defective immunosuppression. the potent, immunosuppressive cytokine interleukin (il- ) is increased in the peripheral blood mononuclear cells (pbmcs) of active phase cidp patients relative to remission phase patients. further, pbmcs from cidp patients produce il- in response to the myelin protein p . despite these findings, whether il- is important for cidp pathogenesis is not known. thus, we sought to determine the role of il- in sapp. il- was highly upregulated in sciatic nerves of nod.aire gw/+ mice with sapp, suggesting it may play an immunosuppressive role in pathogenesis. however, genetic ablation of il- in nod.aire gw/+ mice lead to a paradoxical delay in disease development. age-matched il- -deficient nod.aire gw/+ mice exhibited no sciatic nerve infiltrate and no reduction in nerve conduction during electrodiagnostic studies. interestingly, the delay in sapp was specific, since the incidences of five other autoimmune manifestations in il- -deficient nod.aire gw/+ mice were unchanged relative to il- -sufficient nod.aire gw/+ controls. importantly, il- -deficient nod.aire gw/+ mice did not have colitis, which is consistent with previous studies of il- deficiency on the nod background. il- is known to perform effector functions in autoimmunity by promoting b cell secretion of immunoglobulins. however, genetic ablation of b cells did not affect neuropathy development in nod.aire gw/+ mice, suggesting b cells are dispensable for pathogenesis and unlikely to mediate the protective effect of il- deficiency. il- -deficient nod.aire gw/+ cd + t cells, which are sufficient to transfer sapp, exhibited increased activation, increased interferon gamma secretion, and preserved nerve-specific t cell activation. these data suggest t cell activation and priming are unperturbed and not the mechanism of protection. in summary, our data showed that il- was paradoxically an effector cytokine in sapp. long exercise test (let) has been used especially in myotonic syndromes and muscle channelopathies. marked decrement in compound muscle action potential (cmap) amplitude after prolonged exercise was previously reported in patients with paramyotonia congenita, hyperkalemic or hypokalemic periodic paralysis. we describe a patient with secondary hypokalemic paralysis who showed abnormal let results. a -year-old man presented with ascending flaccid paralysis which evolved in a hyperacute fashion. the patient became quadriplegic after two hours. initial laboratory evaluation revealed severe hypokalemia, with normal thyroid function. we performed electrodiagnostic studies including long exercise test as proposed by mcmanis et al. nerve conduction study was normal, but marked decrement in cmap amplitude (up to % decrease after minutes) was noted after prolonged exercise. despite oral and intravenous potassium replacement, serum potassium level was not corrected as expected. the unusual clinical course prompted for evaluation of secondary etiologies. abdomen computed tomography scan revealed a . x . cm-sized mass in the left adrenal gland. aldosterone to renin ratio was elevated, suggestive of primary hyperaldosteronism. genetic study for cacna s mutation turned negative. after receiving laparoscopic adrenalectomy, the patient experienced no further attacks, and also was able to stop his antihypertensive medication. let may show abnormal results in condition with reduced membrane excitability, even without true channelopathy. according to international criteria, the diagnosis small fiber neuropathy (sfn) is based on clinical symptoms in combination with a reduced intraepidermal nerve fiber density (ienfd) in skin biopsy and/or abnormal temperature threshold testing (ttt). the sensitivity of skin biopsy is moderate to good, although ienfd is normal in about % of patients with sfn complaints. furthermore, ttt is a widely available diagnostic tool, but lacks specificity. corneal confocal microscopy (ccm) has been described and is used in clinical practice as an objective, non-invasive diagnostic tool to detect small nerve fiber damage in patients with diabetes mellitus. this study examines the applicability of ccm in patients with sfn, and the value of ccm as an additional diagnostic tool in sfn. we will include healthy participants to compare the results with the recently published ccm normative values, and patients referred to the sfn center maastricht with the clinical picture of sfn. corneal nerve fiber density (cnfd), branch density (cnbd), fiber length (cnfl), and the tortuosity coefficient (cnft) will be determined in all participants. the results will be compared with the ienfd and ttt. preliminary results will be presented. southanalinh k , university of health sciences, vientiane capital, lao p.d.r. located in south east asia, lao pdr is a landlocked country with a population of about . million inhabitants. the health indicators are among the lowest in south east asia. the total health caregivers in consisted of , persons corresponding to a ratio of . health workers per inhabitants. the main network for health care service provision remains the public system. its health care facilities consist of four central teaching and referral hospitals; five regional hospitals, including one teaching hospital; provincial hospitals; district hospitals, and about health centers. only one in seven sick people receives modern health care treatment. most people rely on self-medication and/or reliance on self-healing. neurological care is a very new field. knowledge of common neurological disorders among both the lao population and medical staff is only beginning to be spread. there are three neurologists in the country. six neurology residents are currently being trained in a three-year program supported by the association pour la promotion des neuro-sciences au laos (association for the promotion of neuro-sciences in laos) and the asean neurological association. indeed, resources are scarce. in the peripheral nerve diseases domain for example, we have only one electromyography machine that was only temporary used when emg experts from france and from singapore came to teach residents. a significant mismatch between the provision of specialized neurologic services and the needs for them exists, especially in rural areas. also, health insurance is not available for the majority. as a consequence, patients have to bear the costs themselves, which constitutes a limit to the access of available healthcare facilities. neurologic training centers, laboratory facilities and equipments are limited. optimizing available human resources, integrating primary, secondary, and tertiary healthcare tiers and making medical treatment more affordable are need to improve neurologic care in the developing world. in certain low-income countries with limited human and financial resources, it may be difficult for governments to apply some of these recommendations on their own. in these circumstances, it is suggested that countries work with international agencies, nongovernmental organizations or other partners to put their plans into practice. spina e , topa a , iodice r , tozza s , dubbioso r , ruggiero l , santoro l , manganelli f . department of neuroscience, odontostomatological and reproductive sciences, university of naples "federico ii", naples, italy. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is a disabling disease and about % of patients may become persistently disabled over time. our aim was identify clinical prognostic factors of long-term disability in a large series of cidp patients. we collected data from cidp patients with definite diagnosis according efns/pns criteria and positive response to first-line therapies (immunoglobulin or corticosteroids) including sex, age of onset, phenotype, disease duration, course of disease (monophasic/relapsing-remitting or chronic progressive) and disability at the time of diagnosis assessed using the modified rankin scale (baseline mrs). all patients had clinical assessment of disability through mrs within the last months (last mrs). ordinal logistic regression model was applied to evaluate the relationship among the clinical parameters and last mrs, considered as ordinal outcome ( - ). anova test for repeated measures was applied to test the overall effects of different course on disability accumulation while t-test was performed to evaluate inter-group differences for parametric variables. we found a significant relationship between last mrs and the course of disease [p< . , z= . , or: . ]. disability accumulation was greater in patients with chronic progressive course than those with monophasic/relapsing-remitting course of disease [p= . ]. moreover, patients with progressive course were older [p= . ]. our data suggest that chronic progressive course of disease may be a major negative prognostic factor for long-term disability in cidp patients. to note that a chronic progressive course of disease is also associated with an older age from the beginning and a more pronounced worsening over the course of disease. sprenger a , lichtenstein t , henning t , lehmann hc . department of neurology, university hospital of cologne, cologne, germany; institute of diagnostic and interventional radiology, university hospital of cologne, cologne, germany; department of neurology, university hospital of cologne, cologne, germany. an unresolved problem in the treatment of inflammatory neuropathies is the lack of valid and reliable diagnostic biomarkers to evaluate axonal damage. we investigated if "diffusion tensor imaging" (dti) and mri t w multi echo dixon imaging are eligible methods to determine proximal nerve injury in chronic inflammatory demyelinating polyneuropathy (cidp). in this prospective observational cohort study the sciatic nerve of cidp patients and age matched healthy controls was investigated. all subjects underwent multimodal mri imaging to determine fractional anisotropy (fa) and muscle fat fraction of the biceps femoris and quadriceps femoris muscle. patients were evaluated by mri, clinical examination and nerve conduction studies at baseline and after six months. the mean fractional anisotropy (fa) value was significantly lower in the sciatic nerve from cidp patients compared to controls. fat fraction of the biceps femoris and quadriceps femoris muscle were significantly higher in cidp patients compared to controls. mri outcome parameters remained unchanged after six months. our study demonstrates the utility of mri imaging to differentiate between "healthy" and functional constricted proximal nerve segments. we postulate that dti and dixon mri might be eligible methods to assess proximal nerve damage in cidp. the presence of peripheral myelin protein (pmp ) has been known for decades, but its functional role was uncovered only recently. recent characterization of pmp -deficient mice revealed a role of pmp in the lipid homeostasis of myelinating schwann cells. in this study, we analyzed the functional impact of pmp on myelination. to decipher the role of pmp , experimental demyelination was performed in myelinating dorsal root ganglia cultures, and in vivo re-myelination was assessed after experimental peripheral nerve damage. we used the myelinating dorsal root ganglia (drg) model in pmp -deficient schwann cell cultures, combined with an established de-and remyelinating protocol in order to analyze myelination in vitro. we also performed experimental nerve crush in pmp -deficient mice. morphometric parameters were defined for the in-vitro experiments and functional parameters such as nerve conduction velocity and the clinical score were additionally measured for the in vivo experiments. structural analyses of the drg cultures revealed fibers expressing myelin basic protein (mbp) and pmp , as well as fibers positive for mbp alone. in contrast to our previous in vivo data, we were also able to detect myelin segments that stained positive for pmp , but were negative for mbp. pmp -deficient drg-cultures demonstrated slightly greater nodal lengths than the control cultures. this trend was significantly augmented after in vitro de-and remyelination, which also resulted in decreased internodal lengths only now, while conserving an intact myelin structure. concomitantly, in vivo nerve crush gives rise to a more severe phenotype in pmp -deficient mice than in wild-type controls. consistent with this, nerve conduction studies showed a delay in remyelination, and analysis of semi-thin sections demonstrated an altered fiber structure in the peripheral nerve biopsies. together, these data suggest that in addition to its role in glial cell lipid homeostasis, pmp also plays a role in remyelination of the injured peripheral nervous system. anti-mag neuropathy remains a difficult diagnosis to treat given its limited therapeutic options. of all interventions, rituximab has emerged as the most effective, although its effect has been with mixed results, especially in patients with advanced axonal loss. lenalidomide is another promising immune modulating therapy, whose effect has been well demonstrated in neuropathy associated with poems (polyneuropathy, endocrinopathy, organomegaly, m-spike protein, and skin changes) syndrome, a condition that has several striking parallels to anti-mag neuropathy. the use of lenalidomide has not been previously described in anti-mag neuropathy. herein, we describe a case of lenalidomide-responsive anti-mag neuropathy in a patient with advanced axonal loss. suichi t , misawa s , sato y , beppu m , sekiguchi y , shibuya k , watanabe k , amino h , kuwabara s . department of neurology, graduate school of medicine, chiba university, chiba, japan; clinical research center, chiba university hospital, chiba, japan. polyneuropathy, organomegaly, endocrinopathy, m-protein, and skin changes (poems) syndrome is a rare cause of demyelinating neuropathy associated with plasma cell dyscrasia and vegf overproduction. several diagnostic criteria for the disorder have been published, but sensitivity/specificity analyses, and their validation have never been performed. the aim of this study is to establish valid diagnostic criteria for poems syndrome. consecutive poems patients, seen at chiba university hospital since , were screened. of these, we have set a gold standard group of poems syndrome, based on treatment response and exclusion criteria during -year follow-up, and patients was diagnosed as having definite poems syndrome. we also collected patients with cidp (demyelinating neuropathy control) and with multiple myeloma, primary amyloidosis, or mgus (m-protein control). criteria for poems syndrome was defined as having two of the three major criteria (polyneuropathy, m-protein, and elevated serum vegf level) and at least two of the four minor criteria (extravascular volume overload, skin changes, sclerotic bone lesions, and thrombocytosis) which were determined by logistic regression analyses. according to the criteria the sensitivity was %, and the specificity was %. our results indicate that the proposed criteria have an excellent diagnostic accuracy, and are useful in clinical practice, presumably leading to early diagnosis and treatment. intraepidermal electrical stimulation (ies) is a new technique to that assesses the function of a-delta fibers in the epidermis. using this technique, we previously reported that the epidermal pain threshold was two-hold in asymptomatic diabetic patients than in normal subjects (muscle nerve : - , ). subsequently, we reported that the elevated pain threshold negatively correlated with intraepidermal nerve fiver density (jpns : s , ). empirically, it is known that lowering the skin temperature makes it less likely to feel pain. therefore, it is necessary to investigate whether the results of ies are affected by skin temperature. the aim of this study was to investigate the influence of a low skin temperature on pain threshold. we recruited subjects with a mean age of . years. for nociceptive stimulation, we used an ies method with a concentric micro-needle electrode that was developed specifically for the selective stimulation of cutaneous a-delta fibers. we placed the ies electrode onto the extensor digitorum brevis and began stimulation with intensity strong enough for the subject to feel a pricking sensation, then reduced the current in steps of . ma until no sensation was felt. we defined pain threshold as the minimum electrical intensity at which a subject felt a pricking sensation. firstly, we measured pain threshold at skin temperature above degrees celsius. then, we put an ice pack on the extensor digitorum brevis for min to lower the skin temperature, and measured pain threshold at skin temperatures below degrees. mean pain threshold values above degrees and below degrees of skin temperature were . and . ma (p< . ), respectively. our data indicated an elevated pain threshold in epidermis with a low skin temperature. one of most common methods for nociceptive stimulation is painful co laser stimulation. some co laser stimulation studies reported pain threshold increased with a low skin temperature. our result is similar to that of co laser stimulation. pain threshold using ies is very easy and non-invasive technique. it may be useful for the evaluation of small fiber neuropathy. svačina mkr , röth p , bobylev i , sprenger a , zhang g , sheikh ka , lehmann hc . department of neurology, university hospital cologne, cologne, germany; department of neurology, university of texas, houston, usa. intravenous immunoglobulins (ivig) are an effective treatment in guillain-barré-syndrome (gbs). in most patients, the optimal ivig dose and regime is unknown. in serum and ivig preparations, immunoglobulin (ig) g form igg dimers, which are assumed to consist of idiotypic/anti-idiotypic antibody pairs. however, data about kinetics of igg dimer formation in gbs are lacking. to study igg dimer formation, c bl/ mice were injected with ivig and anti-gd b antibody or pbs. blood sera were collected h, h and week post injection. a third cohort received an anti-gd a/gt b antibody and blood was collected h post injection. igg was extracted and subtyped into polymeric, dimeric and monomeric fractions using the Äkta fplc system. dialysed dimeric and monomeric igg fractions were examined for the presence of anti-ganglioside antibodies by anti-ganglioside antibody elisa. further, blood samples from gbs patients were collected before (pre-ivig) and after treatment with ivig (post ivig). serum samples were examined for igg dimers and monomers using the Äkta fplc system. in the mouse model, a maximum peak of igg dimer formation was observed h post injection. in gbs patients' samples, igg serum levels and igg dimer content was significantly higher after treatment with ivig. we demonstrate here the feasibility to assess igg dimer formation in an animal model and in gbs patients' samples after treatment with ivig. h after ivig treatment appears to be the optimal time point to assess igg dimer formation. further studies are warranted to determine the utility of igg dimer formation as surrogate marker for treatment response in gbs. svaren j , moran jj , wu x , gutmann l , shy m . university of wisconsin-madison, madison, wisconsin, usa; university of iowa, iowa city, iowa, usa. development of outcome measures for clinical trials in cmt a is a major challenge given the slowly progressive nature of the disease. outcome measures can be used to measure a) target engagement for a given therapy, as well as b) disease process and c) disease burden. several candidate therapies have been shown to reduce pmp levels in cmt a rodent models and thereby ameliorate the symptoms of pmp overexpression. measuring pmp mrna reduction in human trials has so far been limited to analysis of skin biopsies by qrt-pcr, which did not demonstrate clear elevations of pmp mrna during, nor a reduction following, ascorbic acid trials. the analysis of skin biopsies is hampered by variable amounts of schwann cells (sc) in skin biopsies, as well as the variable amount of pmp in sc as previously established by immuno-em in cmt a skin biopsies (katona et al., ) . therefore, it is important to develop optimal normalization criteria to address the variability inherent in skin biopsy analysis. ideally this will employ normalization to sc-specific genes that are not altered by cmt a status. to optimize normalization, we have performed rna-seq analysis of skin biopsies from patient and control skin biopsy samples. analysis of these data after normalization to read depth indicated that pmp levels were . fold higher in cmt a patient samples compared to control skin biopsies. however, there was significant variability in pmp levels particularly in cmt a samples, which may be due to variable amounts of schwann cells in cmt a skin. using a combination of sc-specific genes for normalization, we were able to reduce the apparent variability and optimize the differential levels between cmt a and control skin biopsy samples. we also identified other sc-specific genes that were apparently induced in cmt a skin biopsies relative to control. these studies provide a new framework for gene expression analysis in skin biopsies, enabling more precise evaluation of pmp levels in clinical trials for cmt a as a measure of target engagement. in addition, the normalization framework may also be applicable to other types of cmt. chronic inflammatory demyelinating polyneuropathy (cidp) is the most common chronic autoimmune neuropathy, with an estimated prevalence of between and per , people. it can cause temporary disability in the affected individuals and may eventually lead to permanent disability or death. cidp is commonly treated with intravenous immunoglobulin (ivig) therapy or corticosteroids. octagam ® % is licensed for cidp in france, while octagam ® % is licensed for cidp in germany and belgium. this analysis presents data from three open, multicenter, non-interventional, single-arm, non-controlled studies of a post-authorisation safety surveillance (pass) program for the subset of patients receiving octagam ® % or % for neurological indications, focusing on patients with cidp. briefly, data from in-and out-patients in austria, france, germany, and uk treated with octagam ® for neurological disorders were collected by physicians and analyzed to assess safety and tolerability of the treatment. of patients included in the three studies, patients ( . %) received octagam ® for neurological indications, of which patients ( . %; mean age . years [range - ]) had cidp. the mean dose of octagam ® per course was . g/kg bw for patients with cidp; for the other neurologic indications, the dose ranged from . (for multiple sclerosis) to . g/kg bw (for guillain-barré syndrome). premedication was not needed in . % of these patients. the development of clinical appearance since last observation (mean: every . months) was assessed for of the cidp patients by their treating physicians. the majority of observations ( . %) assessed the patients as stable and . % showed even an improved clinical appearance. only . % of the observation periods resulted in deteriorations. adverse drug reactions were rare: of the infusions received by patients with neurological disorders, . % of infusions were associated with an adr ( . % of infusions in cidp patients). overall, treatment with octagam ® was effective and well-tolerated in patients with cidp. these results are consistent with data for the overall patient population (including patients with primary and secondary immunodeficiencies, dermatological and other diseases). szepanowski f , szepanowski lp , kleinschnitz c , kieseier bc , stettner m . department of neurology, medical faculty, university duisburg-essen, essen, germany; department of neurology, medical faculty, heinrich-heine-university, düsseldorf, germany. lysophosphatidic acid (lpa) is a pleiotropic signaling lipid that acts as ligand for at least six specific g protein coupled receptors. schwann cells (sc) are known to mainly express the lpa receptor subtype. an emerging body of in vivo evidence has linked lpa with injury induced peripheral nerve demyelination as well as neuropathic pain. however, the molecular mechanism underlying its demyelinating effect has remained largely unclear. myelinated dorsal root ganglia (drg) cultures were treated either with lpa, lpa + am (lpa antagonist) or vehicle. we assessed myelin basic protein, tumor necrosis factor alpha (tnf-alpha) as well as the sc differentiation marker sox by immunocytochemistry. additionally, myelin was investigated by sudan black staining. to better understand the relevance of lpa signaling for demyelination in vivo, we performed sciatic nerve crush in c bl/ mice treated with am at mg/kg in order to study schwann cell expression of tnf-alpha, sox and sox , a marker for sc dedifferentiation, by immunohistochemistry. in drg cultures, lpa caused a significant reduction of myelin as demonstrated by both sudan black staining and immunocytochemical analysis of myelin basic protein. demyelination was paralleled by an upregulation of tnf-alpha as well as downregulation of sox . lpa mediated effects were found to be blocked by addition of the lpa receptor antagonist am . in c bl/ mice, am treatment prior to crush injury increased sox expression in scs in the distal nerve stump while reducing the number of cells expressing sox . these data indicate that lpa may be a critical factor to shift scs towards an injury-associated phenotype and contribute to the onset of wallerian degeneration. szepanowski lp , szepanowski f , kleinschnitz c , stettner m . department of neurology, university hospital essen, essen, germany. glyphosate-based formulations comprise the world's most commonly used herbicides. in non-resistant plants, glyphosate exerts toxic effects most likely via inhibition of aromatic amino acid synthesis by interfering with the shikimate pathway. while glyphosate is the active ingredient, herbicidal formulations contain several adjuvants, including polyethoxlated alkylamines (poeas). although glyphosate has long been considered safe for use in humans and animals, several studies have implicated glyphosate and/or the commonly used adjuvants in cytotoxicity, carcinogenicity and endocrine disruption. furthermore, glyphosate-based herbicide has been reported to mediate neurotoxicity in immature rat hippocampus involving glutamate excitotoxicity. however, it remains unclear whether glyphosate alone or in combination with its adjuvants may have detrimental effects on myelin integrity in the peripheral nervous system. myelinated dorsal root ganglia (drg) cultures were treated over the course of ten days with either pure glyphosate or a glyphosate-based herbicide at concentrations of . %, . % and . %. the concentration of the glyphosate-based herbicide was matched with regard to glyphosate content ( %). controls were treated with equal amounts of vehicle adjusted for the ph. subsequently, cultures were stained with sudan black and myelin content was assessed by determining the number of internodes per neurons. while glyphosate, regardless of its concentration, did not show any effect on myelin content, the glyphosate-based herbicide caused significant demyelination in a concentration-dependent manner. notably, at . %, drg cultures were completely devoid of myelin and appeared severely necrotic. these data raise the possibility that not glyphosate itself, but rather the adjuvants in glyphosate-based herbicide formulations may cause demyelination. the open question whether demyelination is a direct effect of the adjuvants or a consequence of increased cellular glyphosate uptake due to permeabilization warrants further investigation. tan cy , tan mp , yeoh ky , goh kj , shahrizaila n . department of medicine, university of malaya, kuala lumpur, malaysia. in guillain-barré syndrome (gbs), autonomic dysfunction is common and accounts for significant morbidity and mortality. there have been many studies investigating the electrodiagnosis of gbs but few have studied autonomic dysfunction in gbs. the current study comprehensively investigates quantitative autonomic function in patients with gbs and its variant. ten gbs patients were prospectively recruited and the results were compared to age-and gender-matched healthy controls. a series of autonomic function tests including computational (power spectrum analysis of heart rate variability (hrv) and baroreflex sensitivity (brs) at rest) and challenge tests (deep breathing, eyeball compression, active standing, valsalva manoeuvre, isometric exercise and ice-water hand immersion) were performed. parasympathetic function was represented by high frequency (hf) hrv, heart rate responses to deep breathing, eyeball compression, valsalva manoeuvre and active standing. sympathetic function was represented by low frequency (lf) hrv, blood pressure responses to active standing, sustained handgrip and ice-water hand immersion. in the frequency domain analysis of hrv, low frequency (lf: . ± . vs . ± . ; p= . ), high frequency (hf: . ± . vs . ± . ; p= . ) and total power spectral densities (psd: . ± . vs . ± . ; p= . ) were significantly reduced in patients compared to controls. the mean up slope ( . ± . vs . ± . ; p= . ), down slope ( . ± . vs . ± . ; p= . ) and total brs slope ( . ± . vs . ± . ; p= . ) were significantly lower in the gbs group. the diastolic rise in blood pressure upon ice-water hand immersion was significantly lower in gbs group compared to controls ( . ± . vs . ± . ; p= . ). our findings suggest that computation dependent tests (hrv and brs) were sensitive at detecting autonomic dysfunction and baroreceptor reflex insensitivity in gbs patients. in contrast, ice-water hand immersion was the only reliable challenge test making it useful as a bedside measure of autonomic function in gbs patients. sjögren's syndrome (ss) is an autoimmune disease that affects both east and west. nevertheless, we still have limited knowledge of how autoantibodies in ss affects the peripheral nervous system. in this study, we investigated the peripheral neuropathy in ss and sicca complex using the nerve excitability test, to elucidate how peripheral nerves are affected. we have enrolled a total of patients with ss or sicca complex. of these, two patients were excluded due to co-morbid carpal tunnel syndrome. each patient received clinical evaluation, examination for ssa/ssb antibodies titer, the nerve excitability test, conventional thermal quantitative sensory test, and conventional nerve conduction study. compared to normal control subjects, motor nerve excitability test of ss patients with positive ssa or ssb antibodies (n = ) were found to have increased rheobase (p< . ), increased relative refractory period (rrp) (p< . ), increased refractoriness at . ms (p< . ), increased accommodation toward depolarizing current in threshold electrotonus (te) (p< . ), and decreased superexcitability (p< . ). the sensory axonal study in seropositive ss also revealed increased rrp (p< . ), increased refractoriness at . ms (p< . ), and increased accommodation toward hyperpolarizing current in threshold electrotonus (te) (p< . ). meanwhile, in seronegative ss and sicca complex (n = ), we found no significant axonal properties changes. the present study revealed that peripheral nerves are affected differently in seropositive ss and in seronegative ss/sicca complex. in seropositive ss, motor axons tended to be depolarized, and both sensory and motor axons have increased refractoriness. the findings suggested that ssa and ssb antibodies might play a role in the inactivation of transient sodium channels. the effects of the antibodies on transient sodium channels might be the basis of peripheral neuropathies and even cardiac arrhythmias and heart block in ss. charcot-marie-tooth disease type a (cmt a), caused by the pmp duplication on chromosome p . , is the most common subtype of inherited peripheral neuropathies and affects in , individuals worldwide. while sharing the same genetic cause, cmt a patients often present great variability in their phenotypic presentation and disease severity. the cause of the phenotypic variability is largely unclear. in this study, we performed genome-wide association study (gwas) to identify novel genetic modifiers of various phenotypes in cmt a. dna samples from cmt a patients were genotyped on illumina omniexpress platform. after standard quality control, the dataset includes k markers in individuals ( individuals from european ancestry, and individuals from asian ancestry). we focused our analyses on the european population. logistic regression in plink was used to analyze the association between the clinical outcomes and patients' genotypes in an additive model. for cmt neuropathy score (cmtns), the analysis was performed using linear regression in plink, adjusting for patients' age. the analyses yielded several suggestive association signals. an association peak on chromosome was identified in difficulty with eating utensils (lead snp rs , chr : , p= . e- , odds ratio= . ). the peak is located within a non-coding gene linc . hearing loss showed an association peak on chromosome (lead snp rs , chr : , p= . e- , odds ratio= . ), located in an intergenic region near the megf gene. in foot plantar flexion, an association signal was identified in the dscam gene on chromosome (lead snp rs , chr : , p= . e- , odds ratio= . ). cmtns showed an association signal on chromosome (lead snp rs , chr : , p= . e- , beta= . ), located within an intergenic region close to dffb, c orf , and linc . while these suggestive signals require further validation, our study provides novel insights into the genetic architecture of cmt a. novel genetic modifiers may serve as potential targets for therapeutic interventions in the future. teng a , ohnmar , kalpana p , chai yh , umapathi t . yong loo lin school of medicine, national university of singapore, singapore; department of neurology, national neuroscience institute, singapore. we present an intriguing diagnostic puzzle, that was eventually cracked serendipitously. a -year-old man was seen for bilateral ptosis. evaluation for myasthenia gravis was negative. nevertheless, a diagnosis of ocular myasthenia gravis was made and he was put on pyridostigmine. he did not respond. the ptosis progressively worsened. he sought a second opinion. at this evaluation, he was noted to have complex ophthalmoplegia without diplopia, bilateral facial weakness and mild bulbar weakness. he had no sensory complaints. the limb examination was remarkable for slightly reduced reflexes, normal strength, and other than increased vibration threshold at the toes, intact sensory examination. repeat serological and electrodiagnostic work-up for myasthenia gravis was negative. a myopathic disorder such as chronic progressive external ophthalmoplegia was considered. serum creatine kinase and lactate were normal. he underwent biceps muscle biopsy which showed increased cox-negative and sdh positive fibers, supporting the then clinical impression of a mitochondrial cytopathy. at this point, he underwent blepharoplasty to improve his vision. routine histological examination of the levator palpebrae muscle showed amyloid deposits. this prompted a review of the earlier biceps biopsy, which revealed amyloid deposits that were not appreciated before. at this point, the significance of the patient and his son's history of lattice corneal dystrophy became apparent. he also reported that his late mother had similar facial appearance as his. the patient's nerve conduction study showed length-dependent sensory axonal polyneuropathy, right carpal tunnel syndrome and bilateral facial neuropathy. he had no definite symptoms of autonomic neuropathy. cardiac evaluation was unremarkable. the final diagnosis of familial gelsolin amyloid polyneuropathy was made. genetic confirmation for the patient and his family is being planned. we highlight the key clinical features of gelsolin neuropathy. the symmetric cranial neuropathy can resemble a muscle or neuromuscular junction disorder and the relative sparing of the cardiac muscle, somatic and autonomic nerves contrasts with transthyretin-related amyloid polyneuropathy. neuropathy is one of the most common long-term complications of diabetes. furthermore, % to % of diabetic neuropathy patients will develop neuropathic pain. the pathophysiology of neuropathic pain in diabetic peripheral neuropathy is complex and not fully understood. a potential mechanism is a change in voltage gated sodium channels, such as nav . . loss of function mutations in this channel cause insensitivity to pain, whereas gain of function mutations have been linked with different pain syndromes including small fiber neuropathy. in a cohort of patients with diabetic peripheral neuropathy we investigated whether mutations in nav . were associated with diabetic neuropathic pain. twelve nav . variants were identified in nine participants all within a cohort of participants with painful diabetic peripheral neuropathy. five of these variants were previously associated with pain disorders: v l, m l; w r, r h, l v. among the other variants two of them met the criteria of potential pathogenicity based on predictive algorithms and were further studied. functional analysis by whole cell patch clamp showed that one of these variants (m t) drastically impairs the inactivation of the channel by shifting the steady-state fast-inactivation towards more depolarizing potentials. there were no phenotypic difference between those participants with pathogenic variants and those participants without pathogenic variants. no rare nav . variants were found in participants with painless diabetic peripheral neuropathy. these observations suggest that mutations in nav . may contribute to painful diabetic peripheral neuropathy. tholance y , rosier c , f bouhour , psimaras d , kuntzer t , taieb g , créange a , delmont e , camdessanché jp , antoine jc . university hospital, saint-etienne, france; university hospital, lyon, france; university hospital, paris, france; university hospital, lausanne, switzerland; university hospital, montpellier, france; university hospital, creteil, france; university hospital, marseille, france. dysimmune sensory neuronopathies (snn) encompass paraneoplastic snn and snn associated with systemic autoimmune diseases such as sjögren syndrome, lupus or inflammatory bowel or rheumatic diseases but also a number of apparently idiopathic cases. biomarker antibodies are well-known in paraneoplastic snn but are lacking in non paraneoplastic cases. from a mono-center retrospective study we identified in anti-fgfr antibody as a potential biomarker of dysimmunity in patients with idiopathic or systemic autoimmune disease associated sensory neuropathy. the identified patients were more frequently women and had a non lenthg dependent neuropathy suggestive of snn. anti-fgfr antibody was the only immunological marker in / of cases at initial work-up although / of patients eventually developed with time systemic autoimmune disease. to confirm the incidence and the clinical pattern of patients with anti-fgfr antibodies we launched a prospective multicenter french study including patients with a sensory neuropathy suspected to be a snn of no paraneoplastic, genetic or metabolic origin. we present here the results on the first included patients compared to healthy blood donors. anti-fgcr antibodies were searched by elisa using the trk intracellular domain of the protein (invitrogen©). we found patients positive for anti-fgfr antibody ( . %). these patients were women and men aged . years as a mean ( - ). the neuropathy was acute and subacute in one patient respectively and progressive in the others. six patients fulfilled the diagnosis criteria of snn and the last one had a sensory neuropathy in the lower limb with abnormal sensory action potentials in the four limbs suggesting snn without reaching the requested criteria. one patient developed uveitis which is a new symptom with anti-fgfr ab. an unclassified dysimmune context was present at the initial work up in patients and one patient developed sjögren syndrome with follow-up. as a whole the clinical pattern of these patients is consistent with that of the initially published series. the lower prevalence of positive sera may be due to more stringent criteria used for elisa but needs to be confirmed on the complete prospective series. peripheral neuropathy research registry (pnrr) neurological assessment was scored using the total neuropathy score clinical version (tnsc), comprising pinprick and vibration sensibility, deep tendon reflexes, strength and patient symptom report. compound sensory action potential (csap) amplitudes were recorded antidromically at the lateral malleolus, stimulating the sural nerve at the mid-calf. of the total sample, % reported lower limb neuropathy, with % of patients reporting 'quite a bit' or 'very much' severity of tingling and numbness in their feet. the average sural csap amplitude was . ± . v and % of patients had sural amplitudes below the lower limit of normal for age. the total tnsc score correlated with the pro fact-gog ntx score (r = −. , p<. ) and sural amplitude (r = −. , p <. ). vibration sensibility correlated with the overall fact-gog ntx score (r =−. , p <. ), and sural amplitude (r = −. , p <. ). sural amplitude correlated with patient reported severity of numbness and tingling in the lower limbs (r =−. , p <. ) but not with the overall fact-gog ntx score. patient reports of neuropathic symptoms in the lower limb correlate with both objective neurophysiological and clinical measures of neuropathy severity. identifying links between objective neurophysiological markers and patient reported outcomes are critical to assess the impact of clinical interventions. tomaselli pj , gouvea sp , nyshyama kfs , nicolau n jr , lourenço cm , marques w jr , . division of neuromuscular diseases, department of neurosciences and behaviour sciences, clinical hospital of ribeirão preto, university of são paulo, ribeirão preto, brazil; neurogenetics, department of neurosciences and behaviour sciences, university of são paulo, ribeirão preto, brazil. mutations in the gab junction beta -protein gene (gjb ) are the second most frequent cause of charcot-marie-tooth disease (cmt), accounting for approximately % of cmt cases worldwide. the gjb codes for connexin protein (cx ). in the peripheral nervous system, the cx is expressed in the schwann cells and allows intercellular traffic of ions and small molecules between opposed cells. we analysed retrospectively detailed clinical and neurophysiological data of five families carrying novel gjb mutation submitted for testing at our neurogenetics laboratory. mutations were identified by bidirectional sanger sequence analysis of gjb coding region. we identified a total of subjects from five different kindreds with novel mutations (p.a v, p.l w, p.l q, p.f s, p.r l). these five novel mutations segregate with phenotype, are located in highly conserved amino acids among gjb and other gab junction protein sequences and among different species, are not present in any public database (exac, dbsnp and genome database), and were not found in normal brazilian controls. in silico analysis, predict these variants to be pathogenic, there was no male-to-male transmission; males were more severely affected than females. four out seven female have subclinical neuropathy and were only identified after clinical and electrophysiological evaluation. the conduction velocities were in the intermediated range in the males patients and higher in the females included in this study. we describe five new pathogenic mutations causing cmtx in a brazilian population and expand the number of causative mutations in the gjb gene. funded pure neural leprosy (pnl) is a slowly progressive, predominantly sensory patchy neuropathy presenting with positive and/or negative sensory symptoms, which are usually followed over time by distal asymmetrical weakness. despite rare, monomelic involvement in leprosy has already been reported. we sought to describe the clinical and electrophysiological patterns of an unusual leprosy neuropathy presentation. clinical data were retrospectively collected from nine patients who had monomelic involvement and were referred for further investigation to the emg lab. seven out nine patients were male. four patients had a brachial plexus like presentation and five have a lumbosacral plexus like presentation. the initial complaint was hypoesthesia in four patients, tingling in two patients and hypoesthesia with tingling in two patients. severe pain was observed in just one patient. all individuals from the group of patients with lumbosacral plexus-like presentation and three with brachial plexus-like presentation had no sensory nerve action potentials (snaps) for all nerves tested in the affected limb with or without motor involvement. one patient with brachial plexus-like presentation had focal slowing of conduction velocity with temporal dispersion of both median and ulnar nerves in the affected limb. one patient with plexus-like presentation had snaps with low amplitude of all nerves in the affect limb. the diagnosis of leprosy was confirmed by nerve biopsy findings, anti-pgl antibody, and positive response to specific treatment. nerve biopsy was performed in four patients, and the bacillus was found in two. the anti-pgl antibody was positive in four patients. plexus mri was performed in two patients and was normal. we found the distribution of motor and sensory symptoms were restricted to on limb in this group of patients. as a typically patchy disorder pnl may affect any nerve, although the reason why damage are restrict to only one limb has to be elucidated. the description of these cases increases the clinical spectrum of leprosy neuropathy. this possibility should be considered in the differential diagnosis of patients with plexopathy from endemic areas after excluding other causes. funded ataxia with oculomotor apraxia type (aoa ) is a very complex disorder characterized by an early-onset progressive cerebellar ataxia with cerebellar atrophy and peripheral neuropathy and it is caused by recessive mutations in the aprataxin gene (aptx ). when the neuropathy is present, it has been described in % of cases as primarily axonal. we describe a case of aoa due compound heterozygous mutations in aptx associated with demyelinating neuropathy. the patient was born from healthy and non-consanguineous parents and presented in the first decade with progressive cerebellar ataxia, multidirectional ophtalmoparesia, oculomotor apraxia, choreiform movements of limbs and peripheral neuropathy. he had normal cognition and stopped walking at age of . blood tests were unremarkable with normal levels of leucocytes, serum proteins, immunoglobulin, cholesterol, vitamin e, and alfa-feto protein. brain mri showed severe cerebellar atrophy. the motor conduction velocity in the upper limbs was slow with preserved amplitudes. the distal latencies and the minimal f wave latencies were prolonged. there was no evidence for superimposed acquired demyelinating neuropathy. direct sequencing of the aptx gene revealed two variants, c. - a>g, p? and c. g>a; p.w x. the first variant is novel and affects a highly conserved acceptor splice site of exon . the other variant is the most common portuguese variant, the nonsense mutation w x is located in exon . parental dna was tested and confirmed the variants were in different alleles. the presence of a demyelinating neuropathy in aoa suggests that phenotypic variability in this condition may be larger than previously considered. at the same time, it increases the differential diagnosis of inherited conditions with cerebellar ataxia and demyelinating neuropathy. finally, this finding opens the functional effects of the aptx gene. funded by: cnpq, fapesp, faepa, pronas (ministry of health). topa a , spina e , iodice r , tozza s , ruggiero l , dubbioso r , esposito m , santoro l , manganelli f . university of naples "federico ii", naples, italy. we report our -year experience of subcutaneous immunoglobulin (scig) in a cohort of patients with chronic inflammatory demyelinating polyneuropathy (cidp) from a tertiary care neuromuscular center. we analyzed data from cidp patients ( males and females, mean age: ± . years; mean age at onset: . ± . years; disease duration: . ± . years) treated with scig and with a follow-up period of months. all patients were previously responders to intravenous immunoglobulin (ivig). eight patients had a typical cidp and five patients had an atypical variant of cidp. five patients switched to weekly maintenance scig therapy (continuous regimen) because of short-lasting response to ivig therapy. eight patients with a longer lasting response to ivig received scig with a pulsed regimen similar to that used for ivig (from to cycles per year); seven of them because of difficulty in hospitalization and one for allergic reaction to ivig. changes in clinical status were assessed over the period of follow-up by using clinical evaluation of muscle strength, modified rankin scale, overall neuropathy scale and inflammatory neuropathy cause and treatmentsum score. in patients we evaluated also six minute walking test, hole-peg-test and meter walking test. all the five patients treated with a continuous regimen of scig remained clinically stable throughout the follow-up period. among the patients receiving pulsed scig treatment, out of ( %) responded to scig similarly to ivig, while three patients ( . %) worsened and needed to be treated again with ivig and the other one ( . %) stopped any therapy. subcutaneously administered immunoglobulin were well tolerated and no patients complained of adverse events. in conclusion, our findings confirm that continuous scig therapy is efficacious in maintaining clinical stability in patients with short-lasting response to ivig. moreover, our data suggest that pulsed therapy with scig may represent an alternative therapeutic option for the treatment of a subset of cidp patients. touvier t , ferri c , mastrangelo r , glimcher l , , wrabetz l , , , d'antonio m . myelin biology unit, division of genetics and cell biology, san raffaele scientific institute, dibit, milan, italy; department of cancer immunology and virology, dana-farber cancer institute, boston, usa; department of medicine, harvard medical school, boston, usa; hunter james kelly research institute, university at buffalo, buffalo, usa; department of biochemistry, university at buffalo, buffalo, usa; neurology, jacobs school of medicine and biomedical sciences, university at buffalo, buffalo, usa. mpz glycoprotein is an abundant product of terminal differentiation in myelinating schwann cells. the mutant mpzs del causes charcot-marie-tooth (cmt) b disease in humans and a similar demyelinating neuropathy in transgenic mice. mpzs del protein is retained in the endoplasmic reticulum (er) of schwann cells and induces an unfolded protein response (upr) characterized by activation of perk, atf and ire /xbp pathways. we have previously reported that activation of chop and gadd , two downstream mediators of perk, is pathogenetic in mpzs del mice (pennuto, ; d' antonio, ) but the role of the other upr branches remains to be investigated. in this study, we investigated the role of the er stress sensor enzyme ire and of xbp -a transcription factor specifically activated by ire -in mpzs del pathogenesis. we generated a new mouse model with schwann cells-specific ablation of xbp and in parallel we exploited mpzs del dorsal root ganglia (drg) explant cultures in which xbp signaling is modulated by gain/loss of function approaches. we observed that absence of xbp dramatically worsens hypomyelination and electrophysiological/locomotor parameters in young and adult mpzs del neuropathic animals. interestingly we observed that perk, atf and ire -mediated ridd signalings are upregulated in neuropathic animals lacking xbp . this suggests that activation of xbp targets have an essential role in limiting mpzs del toxicity, which cannot be compensated by other stress responses. moreover, we demonstrated in mpzs del drg cultures that inhibition of xbp splicing by u c (cross, ) decreases myelination whereas activation of xbp splicing by quercetin (wiseman, ) slightly ameliorates myelination. altogether, these data demonstrate that xbp pathway has a critical adaptive role in mpzs del neuropathy and suggest that activation of this pathway may be beneficial for cmt b and perhaps for a broad range of neuropathies characterized by upr activation. tozza s , bruzzese d , iodice r , esposito m , dubbioso r , ruggiero l , topa a , spina e , santoro l , manganelli f . department of neuroscience, reproductive sciences and odontostomatology, university of naples "federico ii", naples, italy; department of public health, university federico ii of naples, naples, italy. in cmt a patients, the clinical impairment progressively increases over time and correlates with the axonal loss. evidence has suggested that the decline of physical performance in cmt a patients may reflect a process of normal ageing. the aim of our study was to describe, by a case-control cross-sectional design, the progression of physical impairment with ageing in cmt a patients. we enrolled cmt a patients ( m; range - years) and sex-and age-matched healthy controls. to assess physical performance, all patients and controls underwent -meter walk test ( mwt), -minute walk test ( mwt) and -hole peg test ( hpt) of their dominant (d) and non-dominant (nd) sides. moreover, to assess clinical disability, impairment and quality of life in the cmt a group we used the charcot-marie-tooth neuropathy score (cmtns), the mrc sum score and the short form- (sf- ) questionnaire. the linear regression model was used to evaluate the changes over time of clinical measures in patients and controls. the chow test was used to determine whether the ageing had a different impact on clinical measures for the two groups. physical performance worsened with ageing in both patients and controls, but with a greater slope for cmt a patients [difference in slopes: mwt, . (c.i. . to . ), p< . ; mwt, − . (c.i. - . to − . ), p< . ; hpt-d, . (c.i. . to . ), p< . ; hpt-nd, . (c.i. . to . ), p< . ]. the rate of deterioration of physical performance was not different between patients and controls until the th year of age. after the th year of age the rate of deterioration became greater in cmt a group [difference in slopes: mwt, . (c.i. . to . ), p< . ; mwt, − . (c.i. - . to − . ), p< . ; hpt-d, . (c.i. . to . ), p< . ; hpt-nd, . (c.i. . to . ), p< . ]. moreover, in cmt a patients also cmtns, mrc sum score and sf- worsened with ageing and with a greater rate of deterioration after the th year of age. our study demonstrates that clinical decline in cmt a patients goes parallel to the normal ageing process until the th year of age, whereupon the clinical deterioration accelerates. tsouni p , devic p , moura b , planque e , bédat-millet al , devaux j , steck aj , delmont e , hottinger af , kuntzer t . dcn, chuv, lausanne, switzerland; centre de référence maladies neuromusculaires, hospices civils de lyon, lyon, france; cabinet médical, epinal, france; département de neurologie, chu de rouen, rouen, france; cnrs, crn m-umr , université aix-marseille, marseille, france; centre de référence maladies neuromusculaires et sla, hôpital la timone, marseille, france. new therapeutic options in immuno-oncology have allowed significant progress in the management of melanomas. treatment usually consists of a combination of two monoclonal antibodies targeting cytotoxic lymphocyte-associated protein and programmed cell death- . as a result, the immunologic barrier protecting tumor cells is overcome allowing an antitumor response. we report cancer patients with immune checkpoint inhibitor-induced neuropathies as a complication of this immunomodulating oncologic treatment. case reports: our index patient developed severe myalgia days after introduction of ipilimumab-nivolumab followed by painful paresthesias of the face and extremities day after the nd cycle of treatment. generalized areflexic quadriparesis (mrc ) with gowers'sign and distal loss of vibratory sensation were found. a -day course of ivig plus corticosteroids (cs) had no effect. three monthly ivig courses were necessary to improve the deficits at months. a survey of sfnp members revealed other patients with similar acute courses but with phenotypes varying from sensorimotor deficits with areflexia and myalgia to purely sensory ataxic forms following immunomodulating treatment. work-up including anti-nodal antibodies were negative in patients. from the other , had abnormal csf and had necrotizing myopathy. detailed repeat ncs demonstrated signs of nerve hyperexcitability and of demyelination or conduction blocks. evolution was slowly favorable following ivig and cs. discussion: our report underscores that atypical acute generalized demyelinating neuropathies are induced by these novel treatments. they may be associated with severe myalgia or other systemic toxic effects. discontinuation of the oncologic treatment depends on severity of symptoms. outcome was slowly favorable following ivig or cs, albeit slower than in the case of primary inflammatory neuropathies, probably given the long half-life of the monoclonal antibodies. gap junctions (gjs) are membrane channels found in most tissues connecting adjacent cells or different cell compartments as in schwann cells. they are involved in electrical connectivity and metabolic homeostasis allowing the passage of small molecules such as ions, second messengers, nucleotides and peptides. an important functional role of peripheral nerve connexins is suggested by their involvement in x-linked inherited neuropathy as well as in acquired neuropathy caused by oxaliplatin. although gjs play a role in electrical connectivity their specific role in the formation of the sciatic nerve compound action potential (cap) remains unclear. the aim of this study was to investigate the role of peripheral nerve connexins in the electrical responses of the mouse sciatic nerve under normal and stress conditions. for this purpose we used sciatic nerves of three different mouse models, the cx knockout (ko), cx ko and the cx /cx double knockout (dko) mice. using our ex vivo model for extracellular recordings we exposed sciatic nerves from different genotypes to three different gj blockers: octanol, -beta-glycyrrhetinic acid (gra) and octanoic acid (oa) and recorded the cap. amplitude and duration of the cap were used as an indication for the effects of the different blockers on the cap formation. all gj blockers caused a gradual decrease of the cap without any changes in the duration of the cap in all genotypes, suggesting progressive disturbance of axonal membrane excitability in the absence of one or two gj proteins. comparison of the three genotypes showed that cx may play a dominant role in the maintenance of the cap formation since nerves from cx ko mice proved to be more sensitive to the gj blockers compared to the cx ko nerves showing a faster decline of the cap amplitude. moreover the effect of gj blockers was similar in cx ko and dko nerves. finally, the effect of gj blockers on the dko nerves implies the presence of another gj protein. in conclusion, our results confirm the direct functional involvement of cx gj channels and cx hemichannels in the cap formation and indicate the existence of at least one more connexin in peripheral nerve. ca( +)-dependent anti-ganglioside antibody in seronegative guillain-barrÉ syndrome uchibori a , gyohda a , chiba a . kyorin university, tokyo, japan. we have reported ca + -dependent igg anti-ganglioside gq b antibodies in gq b-seronegative patients with fisher syndrome and its related disorders (fs-rd). in patients with fs-rd who were gq b-seronegative in conventional assays using phosphate-buffered saline without ca + , % turned seropositive for gq b-related antigens in assays using ca + -added tris-buffered saline. objective: we investigated whether ca + -dependent anti-ganglioside antibodies was present also in ganglioside-seronegative patients with other clinical disease types of guillain-barré syndrome (gbs) other than fs-rd. methods: the subjects were the following: patients with final clinical diagnosis as gbs (acute motor axonal neuropathy [aman], n = , and acute inflammatory demyelinating polyradiculoneuropathy [aidp], n = ), and patients with final clinical diagnosis as sensory ataxic neuropathy (san), n = . all subjects were ganglioside-seronegative in the conventional assays. we assayed serum igg antibodies against various gangliosides (including asialo-gm ) in elisa using tris-buffered saline as a basal buffer in both ca + -added and -non-added conditions. increase of the optical density (od) more than . in ca + -added condition compared with ca + -non-added one was taken significant, i.e. positive for ca + -dependent antibody. results: ca + -dependent antibody was negative in all aman and san patients. in aidp, the antibody titers (ods) against gainac-gd a were significantly increased in patients, and those against asialo-gm were increased in other patients in ca + -added condition. however, the titiers of those ca + -dependent antibodies were all at low level. conclusions: in clinical disease types of gbs other than fs-rd, ca + -dependent igg antibodies against ganglioside were detected in a few patients with aidp, but the positive rate and the antibody titers were low compared with case of gq b-seronegative fs-rd. ca + -dependent antibodies against ganglioside are considered to be more specific for gq b. the aim of the study was to investigate the relationship between median sensory conduction of median nerve and ulnar nerve in patients diagnosed with carpal tunnel syndrome. two hundred and eighty-six hands with carpal tunnel syndrome and hands in control group were investigated. patients were staged clinically and electrophysiologically. diagnosis of carpal tunnel syndrome was made according to the presence of paresthesia, pain in the innervation area of the median nerve, weakness and atrophy in the median nerve innervated muscles, positive phalen and tinel tests. median motor and sensorial nerve conduction study, including first, second, third finger and palm, and ulnar motor and sensorial nerve conduction of fifth finger studies were performed to all patients and control group. the ratio of distal latency and velocity of nerve conduction of first, second, third and palmar branches to fifth finger was calculated. distal latency of first, second, third finger and palm of patients with cts are longer and velocity is more slowly than controls. in addition to these findings, the velocity of fifth finger is also slower and distal latency of this one is longer than healthy subjects. the most sensitive method of classifying the carpal tunnel syndrome as normal, mild and moderate is the ratio of distal latency and velocity of second finger (p< . ). carpal tunnel syndrome is the most common encountered neuropathy. in nerve conduction studies can be used the ratio of distal latency and velocity of second finger as determine the degree of carpal tunnel syndrome. the most surprising finding of this report is the nerve conduction studies of fifth finger. the subclinical susceptibility of fifth finger can be explained by overusing of wrist. ursino g , gemelli c , grandis m , reni l , bellone e , geroldi a , gotta f , mandich p , ferrara m , schenone a dinogmi university of genoa, genoa, italy; irccs-aou san martino hospital genoa, genoa, italy. charcot-marie-tooth (cmt) neuropathy represents a clinically and genetically heterogeneous group of hereditary peripheral neuropathies characterized by chronic motor and sensory impairment. to date mutations in up to genes may cause cmt. the aim of this study is to describe our large population of cmt patients, and, within this, highlight specific phenotypes. the cmt clinic in genova, started in . during the years, patients underwent complete neurological, rehabilitative, neurophysiological examinations and genetic testing. the patients were routinely tested for common genes (as pmp , gjb , mpz, mfn ), while in specific cases we followed the candidate gene approach testing single genes based on the genotype-phenotype correlation. however, the ngs techniques were used when routine genetic testing was negative and a clear genotype-phenotype correlation could not be identified. cases are present to date in our database of cmt patients. in ( . %) patients, in spite of a clinical diagnosis of cmt, a genetic diagnosis is still lacking; ( . %) patients had alternative diagnosis (i.e hereditary spastic paraparesis etc.). instead, in patients ( . %) a defined genetic diagnosis was reached, of them being females ( . %) and males ( . %). among these, except for the more common cmt a, hnpp and cmt x phenotypes, we frequently observed patients affected by cmt b and cmt f. according to most literature data, we observed ( . %) patients with cmt b and patients ( . %) affected by cmt f. at the first visit, the cmt b phenotype was clearly length-dependent: . % patients showed impairment of the lower limbs and saving of the upper limbs; in terms of severity of the neuropathy, the mean cmtns was . and the mean age was . years. similarly, % of patients affected by cmt f, present with the same phenotype; the mean cmtns at the first visit was . and the mean age was . years. in conclusion, based on the experience of the genova cmt clinic, we describe a large population of cmt patients and a specific phenotype in cmt b and f patients, characterized by involvement of the lower limbs and selective sparing of the upper ones, which may help in addressing the diagnostic algorithm. non-freezing cold injury (nfci) develops following sustained exposure to cold temperatures, resulting in tissue cooling but not freezing. this can result in persistent sensory disturbance of the hands and feet including numbness, paraesthesia and chronic pain. both vascular and neurological aetiologies of this pain have been suggested but remain unproven. we prospectively approached patients referred for clinical assessment of chronic pain following non-freezing cold injury between february and november . of patients approached consented to undergo detailed neurological evaluations including: questionnaires to detail pain location and characteristics, structured neurological examination, quantitative sensory testing, nerve conduction studies and skin biopsy for intra-epidermal fibre assessment. of the study participants all had experienced nfci whilst serving in the united kingdom armed services and the majority were of african descent ( . %) and male ( . %). many patients reported multiple exposures to cold. the median time between initial injury and referral was . years. pain was principally localised to the hands and the feet, neuropathic in nature and in all study participants associated with cold hypersensitivity. clinical examination and quantitative sensory testing were consistent with a sensory neuropathy. in all cases large fibre nerve conduction studies were normal. the intra-epidermal nerve fibre density, however, was markedly reduced; ⋅ % of subjects having a count at or below the ⋅ centile of published normative controls. using the neuropathic pain special interest group (neupsig) of the international association for the study of pain (iasp) grading for neuropathic pain % had probable and ⋅ % definite neuropathic pain. chronic non-freezing cold injury is a disabling neuropathic pain disorder due to a sensory neuropathy. why some individuals develop an acute painful sensory neuropathy on sustained cold exposure is not yet known but individuals of african descent appear vulnerable. screening tools, such as the dn questionnaire, and treatment algorithms for neuropathic pain should now be used in the management of these patients. funded by the wellcome trust and the uk ministry of defence guillain-barré syndrome (gbs) is highly heterogeneous regarding clinical presentation, course, electrophysiological subtype and outcome. in part this variety is associated with differences between geographical regions, although this had not been investigated in a single comparative study. one aim of igos is to define the influence of the geographical origin on the heterogeneity of gbs. in igos all gbs patients within weeks of onset can participate, independent of age, variant, severity and treatment. in february , patients were included from countries. the first inclusions in igos were used in this analysis. seventy-five patients ( %) were excluded because of alternative diagnoses (n= , including a-cidp), protocol violations (n= ) or missing data (n= ). of the remaining patients, % were males and % female with a median age of years (iqr - ). at entry % presented with tetraparesis and % with paraparesis. during follow-up % needed mechanical ventilation and % died. of all gbs patients % received treatment ( % ivig, % pe). the remaining % received supportive care only (mild gbs or low social-economic status). antecedent events were reported in % of patients, including upper respiratory tract infection ( %) and gastro-enteritis ( %) as the most frequent events. the pure motor form was the predominant subtype in patients from bangladesh ( %). in europe/americas and asia (without bangladesh) the predominant subtype was the sensorimotor form ( % in europe/americas and % in asia). in asia (without bangladesh) there was a relatively larger proportion of patients with mfs/mfs-overlap syndrome ( %) than in other regions ( - %, p< . ). the proportion of patients able to walk unaided at months after follow-up was % in europe/americas, % in bangladesh and % in asia (without bangladesh). kaplan-meier analysis comparing electrophysiological subtypes of gbs (as reported by neurologist) showed that patients with inexcitable nerves or axonal neuropathy needed more time to regain the ability to walk unaided than the patients with demyelinating or equivocal result (log-rank test, p < . ). these findings demonstrate the extensive geographical differences in gbs. future igos studies will investigate the role of genetic and environmental factors that additionally could explain these differences. van den bergh pyk , attarian s , grapperon am , nicolas g , cassereau j , rajabally ya , delmont e , woodard jl , piéret f and the university of louvain gbs electrodiagnosis study group * . corticosteroids are considered as one of the first line treatments for chronic inflammatory demyelinating polyneuropathy (cidp). different types of corticosteroids are used and there are no comparative studies assessing the improvement rates, remission rates, tolerability and the side effect profiles of these treatment regimens. in addition, there are currently no reliable predictors of favorable treatment response to steroids, which would greatly ease the choice of first line treatment. in this retrospective study we will compare efficacy, tolerability and safety of three different corticosteroid regimens used as first line treatment in three large cidp centers in netherlands, serbia and italy. treatment naïve cidp patients who received either pulsed dexamethasone, pulsed methylprednisolone or daily prednisone will be included in the study. data will be extracted from patient charts. the primary outcome is the percentage of treatment responders at months after start of first treatment, in which treatment response is defined as subjects who improved after treatment and are either without treatment after six months or are still being treated with the first chosen therapy. secondary endpoints include the remission rates and in case of a relapse, the mean duration of remission to relapse; the discontinuation rate within months of treatment due to inefficacy, adverse events or intolerance; and the frequency of adverse events and serious adverse events (sae) during treatment or within month after stopping treatment. furthermore, we will explore the value of previously reported potential predictors of treatment response. results will be presented at the peripheral nerve society meeting . van lieverloo g , , musters a , adrichem m , esveldt r , doorenspleet m , klarenbeek p , van schaik i , de vries n , eftimov f . academic medical center, department of neurology, amsterdam, the netherlands; academic medical center/university of amsterdam, department of clinical rheumatology and immunology, amsterdam, the netherlands. following reports that pathogenic antibodies are present in a minority of patients with chronic demyelinating inflammatory neuropathy (cidp), we studied whether oligoclonal expansions of b-cell clones are present in patients with cidp. recently, we developed a new method for b-cell receptor (bcr) repertoire landscaping based on high throughput sequencing (hts) of rna extracted from blood. bcr repertoire was analyzed in patients with cidp: patients with active disease and starting treatment (group ), patients with stable disease using intravenous immunoglobulin (ivig) treatment in which treatment withdrawal was attempted (group ), and patients in remission (i.e. no treatment in the last months, group ). clinical parameters and sampling was performed at baseline (group , and ), at months after start of treatment (group ), at months or earlier in case of relapse in group and at baseline only in group . most cidp patients had highly expanded bcr clones, regardless of disease activity and response to treatment. however, in group , the most expanded b-cell clones at baseline showed no overlap with the expanded bcr clones after improvement. based on these preliminary data expanded bcr clones are observed in the peripheral blood of most cidp patients, regardless of disease activity (active, stable disease or remission/cure). functional characterization of these expanded clones remains to be performed. van rijs w , fokkink wjr , tio-gillen ap , brem md , jacobs bc , huizinga r . erasmus mc, university medical centre, rotterdam, the netherlands. myeloid cells, including monocytes, macrophages and dendritic cells, are critically involved in the induction of adaptive immune responses, clearance of pathogens and in the initiation of tissue repair. in the guillain-barré syndrome (gbs), macrophages are present in the peripheral nerve, where they phagocytose (damaged) myelin and axons. dendritic cells (dc) are increased in the cerebrospinal fluid of patients with gbs. however, the composition and phenotype of monocytes and dc subsets in the peripheral blood is unclear and it is unknown if these cells can be used as biomarker to monitor disease activity or response to treatment. here we investigated the frequency and phenotype of six myeloid subsets in the peripheral blood mononuclear cells (pbmc) using advanced -color flow cytometry. pbmc were isolated from patients with gbs, before and after immunomodulatory treatment, and age and gender-matched, healthy controls. the frequency of total monocytes, determined as percentage of cd + cells, was increased in gbs patients compared to controls (p< . ). the monocyte population was skewed towards more intermediate (cd +cd +; p< . ) and less non-classical (cd -cd +; p< . ) monocytes. classical (cd +cd -) and intermediate monocytes as well as cd c+ dc expressed significantly higher levels of cd compared to healthy controls. in contrast, the expression of cd and siglec- was significantly higher in the non-classical monocytes of gbs patients compared to controls. no differences were observed in the expression of cd , cd , cd and siglec- . immunomodulatory treatment strongly reduced the frequency of non-classical monocytes and all dc populations in cd + pbmc. the expression of cd , cd c and hla-dr was reduced in classical monocytes after treatment. in addition, siglec- expression was reduced in several monocyte and dc populations after treatment. in summary, our data identify significant changes in the monocyte compartment in gbs. the decrease in non-classical monocytes may suggest that these cells have migrated to peripheral tissues, promoting the differentiation of classical monocytes into intermediate monocytes. further analysis should reveal whether these changes are related to preceding infections, disease severity and response to treatment. in mme. the two mutations were absent from control databases (e.g. exac), affected highly conserved aminoacids and were predicted to have deleterious effects by in silico analysis. unfortunately, both parents were deceased and we were therefore unable to prove that the two mutations reside on separate alleles. mme encodes the metalloprotease neprilysin whose role in peripheral nervous system is still unclear. higuchi and colleagues have described japanese cmt families with late-onset sensory-motor axonal neuropathy and recessive loss-of-function mutations in mme. we report two novel missense mutations in mme in a case of late-onset cmt . we hypothesize an autosomal-recessive mode of inheritance as most likely given the clinical phenotype and the absence of a family history. anti-contactin- (cntn ) antibodies were recently identified in a subgroup of patients with chronic inflammatory demyelinating polyneuropathy (cidp) showing acute/subacute onset of severe sensory-motor neuropathy and poor response to intravenous immunoglobulin (ivig) and corticosteroids. these antibodies belong to the igg isotype and interact with cntn -neurofascin (nf ) complex at paranodes leading to loss of nodal integrity. a -year-old man presented with acute onset of distal weakness in the lower limbs, four limbs paraesthesias and sensory ataxia. at clinical examination ankle swelling was also observed. nerve conduction study showed a demyelinating polyneuropathy and cerebrospinal-fluid examination revealed cyto-albuminologic dissociation. sural nerve biopsy disclosed diffuse loss of myelinated fibres. at routine blood test serum albumin was reduced and proteinuria was gr/ hours, thus leading to the diagnosis of nephrotic syndrome. kidney biopsy showed changes consistent with membranous glomerulonephritis, together with sub-epithelial deposits of immune complexes and complement deposition. treatment with ivig and corticosteroids did not improve neurological status, while membranous glomerulonephritis showed moderate response to ivig. a six-month course of cyclophosphamide was started leading to normalization of renal function and muscle strength and partial improvement of sensory ataxia. the patient did not require any further treatment and after years his condition remains stable. cntn antibodies were tested on a recently collected patient's serum and resulted positive on both elisa and cell-based assay. the patient here reported showed the typical clinical features of anti cntn -associated cidp including older age, acute onset, severe motor impairment and sensory ataxia. the contemporary occurrence of membranous glomerulonephritis was reported in only one other case. contactin- is expressed at low levels in the kidney and a direct damage of anti-cntn antibodies could be hypothesized. alternatively, renal damage might have been secondary to unspecific immune complexes deposition. a good response to anti-cd rituximab was recently reported in patients with cidp associated with anti-cntn and anti-nf antibodies. notwithstanding this single-case observation, our report suggests that also cyclophosphamide may be considered an effective therapy in anti-cntn antibodies-associated cidp and membranous glomerulonephritis, leading to persistent clinical remission. velasco r , , besora s , santos c , sala r , izquierdo c , simó m , gil-gil m , , jiménez l , pardo b , calvo m , palmero r , clapés v , bruna j duloxetine is the only agent demonstrated effective in treating pain related with chemotherapy-induced peripheral neuropathy (cipn). patients with symptomatic cipn treated with duloxetine were retrospectively collected in a single-institution. aim of the study was to evaluate the drug's efficacy and rate of compliance. only patients with cipn with distressing positive symptoms (pain, numbness and/or paresthesia), and non-progressive disease were included. cipn was graded employing the total neuropathy score (tns © ) and national cancer institute-common toxicity criteria. response to duloxetine was assessed with patient global impression of change (pigc) scale ( : no benefit; : excellent response). consecutive first one-hundred cipn patients treated with duloxetine were analyzed. median age was ( - ). , , and received platinum, taxane, bortezomib and vincristine-based regimen, respectively. median tnsc © was ( - ). severity of neuropathy was grade ( %), grade ( %), and grade ( %). sixteen patients were on treatment with other analgesic agents. median time from finishing chemotherapy to duloxetine initiation was months [ - ]. median pigc score was [ - ]. among responders, . % and . % scored low ( - ) and high ( - ) benefit, respectively. fifty-seven ( %) patients discontinued early duloxetine due to intolerable side effects (n= ) the goal of this study was to determine whether predicted fork stalling and template switching (fostes) during mitosis deletes exon in peripheral myelin protein kd (pmp ) and causes a gain-of-function mutation associated with peripheral neuropathy in a family with charcot marie tooth disease type e. two siblings previously reported to have genomic re-arrangements predicted to involve exon of pmp were evaluated clinically and by electrophysiology. skin biopsies from the proband were studied by rt-pcr to determine the effects of the exon re-arrangements on exon mrna expression in myelinating schwann cells. transient transfection studies with wild type and mutant pmp were performed in cos and rt cells to determine the fate of the resultant mutant protein. both affected siblings had a length-dependent demyelinating neuropathy with severely slow nerve conduction velocities (< m/sec). rt-pcr studies of schwann cell rna from one of the siblings demonstrated a complete in frame deletion of pmp exon (pmp delta ). transfection studies demonstrated that pmp delta protein is retained within the endoplasmic reticulum and not transported to the plasma membrane. our results confirm that that fostes mediated genomic rearrangement produced a deletion of exon of pmp , resulting in expression of both pmp mrna and protein lacking this sequence. in addition, we provide direct experimental evidence for endoplasmic reticulum retention of the mutant protein suggesting a gain-of-function mutational mechanism consistent with the observed cmt e in this family. pmp delta is another example of a mutated myelin protein that is misfolded and thus likely to contribute to the pathogenesis of the neuropathy. in poems and cidp, distal limb nerve conduction studies are limited in identifying demyelination and detecting treatment effects in severely affected patients. blink reflex r latency may help to not only identify demyelination but also provide a meaningful treatment outcome measure especially in severely affected patients. poems and cidp patients having undergone routine nerve conductions and blink reflex testing were identified. correlation between r latency, limb nerve conduction studies and neuropathy impairment scores (nis) was calculated with treatment. blink reflexes were performed in patients ( poems, cidp; nis range: - points). overall, r latency prolongation occurred in . % of patients ( . % poems, . % cidp). patients with r prolongation (> ms) had more severely affected nerve conductions in both poems (ulnar cmap . mv vs . mv, p= . ) and cidp (ulnar cmap . mv vs . mv, p< . ). r latency correlated with nis severity in poems better than cidp (r = . vs . , p=< . vs. . ). follow-up nis and r latency evaluations after treatment were available in patients ( poems, cidp). the r latency changes were concordant with the nis changes in % of poems and % of cidp patients. in severely affected patients [ulnar cmap amplitude ≤ . mv ( . %: / )] except for one, all had prolonged r (> ms), allowing for treatment follow up and initial diagnosis. blink reflex r latencies are valuable in defining demyelination in severely affected poems and cidp patients, but also provide a sensitive, early treatment outcome measure among those same severely affected patients. watson dj , martinez c , wallenhorst c , hubsch a , shebl a , simon tl . csl behring llc, king of prussia, usa; institute for epidemiology, statistics and informatics gmbh, frankfurt, germany; csl behring ag, bern, switzerland; csl behring gmbh, marburg, germany. chronic inflammatory demyelinating polyneuropathy (cidp) rarely occurs in children. clinical trials in pediatric patients have not been performed and there are little data on therapy with intravenous immunoglobulin (ivig). we performed an observational trial to investigate the risk of adverse events of special interest (aesi) with ivig, i.e. hemolytic anemia, aseptic meningitis, acute renal failure, thromboembolic events and anaphylactic reactions. the study cohort was derived from the us premier perspective database and consisted of patients < years with a diagnosis of cidp (icd- cm diagnosis code . ) treated with the ivig privigen ® (csl behring, bern switzerland) between jan and dec . we identified pediatric cidp patients: preschool children (age - years at first treatment for cidp), children ( - years) and adolescents ( - years); females and males; white, one black and two allocated to "other race". six patients had a history of other ivig use for guillain-barré syndrome, one patient for myasthenia gravis and one for immunodeficiency before the diagnosis of cidp. the mean privigen ® dose calculated from the cumulative quantity of privigen ® per treatment episode and the corresponding age-and gender specific median of the us population body weight estimate was . g/kg body weight. the number of recorded treatment episodes per patient ranged from to . using an at-risk period of days for hemolytic anemia, and days for other aesi after each privigen ® administration, no aesi were observed in the patients with cidp with a total of person-days at-risk for ha and person-days at-risk for other aesi. this observational study shows that ivig (privigen ® ) is used for treatment of cidp in pediatric patients with or without concomitant conditions and revealed no particular safety issues in this patient group. hsn- is a peripheral neuropathy most frequently caused by missense mutations in the sptlc or sptlc genes, which code for two subunits of the enzyme serine palmitoyltransferase (spt). spt catalyzes the first and rate limiting step of de novo sphingolipid synthesis. it has been shown that mutations in spt cause a change in enzyme substrate specificity which results in the production of two atypical sphinganines, deoxysphinganine (dsp) and deoxymethylsphinganine (dmsp), rather than the normal enzyme product, sphinganine (sp). levels of deoxysphingolipids are elevated in the blood of hsn- patients and this has been shown to cause the peripheral nerve damage characteristic of the disease, which affects both sensory as well as motor axons. however, the underlying pathomechanism of how deoxysphingolipids damage neurons remains elusive. here, dsp and dmsp-mediated neurotoxicity was examined in primary mouse motor and sensory neurons, by assessing cell survival and neurite outgrowth following exposure to different concentrations of sp, dsp or dmsp. the abnormal enzyme products were found to have a rapid and dose-dependent neurotoxic effect in primary neurons. we also explored the potential mechanisms that underlie deoxysphingolipid neurotoxicity, by characterizing mitochondrial function and changes in calcium handling. we found that mitochondrial dysfunction and calcium handling deficits may be key mediators of abnormal sphingolipid neurotoxicity, in both motor and sensory cell models. specifically, we revealed mitochondrial abnormalities, signs of endoplasmic reticulum stress and dysfunction of store-operated calcium channels. we propose that early deficits in mitochondria and calcium handling may underlie deoxysphingolipid neurotoxicity and thus present potential therapeutic targets for hsn- . months. one patient was excluded because the decrease in hba c was not contemporaneous with weight loss. the mean and median decrease in bmi per month was . and . respectively. the mean and median interval between surgery and decrease in hba c was . and . days respectively. records of these patients were scrutinized and classified as: 'probable tind': acute painful neuropathy and acute dysautonomia with temporal relationship to the decrease in hba c; 'possible tind': acute painful neuropathy or acute dysautonomia or temporal relationship to decrease in hba c is uncertain; 'unlikely tind': when an alternative explanation exists for symptoms. only one patient was classified as 'possible tind': a -year-old woman who developed neuropathic pain in both lower limbs month after a rapid hba c decline of . % and about months after bariatric surgery. she had no documented autonomic symptoms. our study is limited by small cohort size, retrospective design and reliance on hospital records. nonetheless, it demonstrates that besides nutritional neuropathies, tind should also be considered in dm patients who develop peripheral neuropathy after bariatric surgery. in another study, we found tind is uncommon in a general cohort of diabetic patients. the occurrence of 'possible tind' in only dm patients corroborates earlier data that weight loss may act in tandem to increase the risk of tind. woolums bm , tabuchi m , sung h , sullivan jm , mamah c , yang m , blum id , wu mn , sumner cj , lloyd te . johns hopkins university, baltimore, usa. dominant missense mutations in the gene encoding the cation channel transient receptor vanilloid, family member (trpv ) cause inherited neuropathies including charcot-marie-tooth disease c (cmt c). in vitro, mutations in trpv that cause cmt c cause a gain of trpv channel function and increased intracellular calcium which subsequently leads to cellular toxicity. however, the mechanisms by which cmt c mutations in trpv lead to neuronal dysfunction in vivo remain poorly understood. we generated transgenic drosophila that express either wild-type or a cmt c causing trpv mutant (trpv r c ) to assess the effect of trpv r c on neuron function in vivo. selective expression of trpv r c in drosophila ccap neurons (n ccap ) results in a failure of drosophila wing expansion that is blocked by genetically inactivating the channel pore, demonstrating the requirement of channel function in mediating this phenotype. perforated patch clamp analysis of n ccap reveals that trpv r c causes a calcium-dependent increase in n ccap neuronal excitability. this hyperexcitability is restored to control levels by application of a trpv selective antagonist. high level expression of trpv r c causes synaptic and dendritic degeneration, both of which are rescued genetically by inactivating the channel pore or pharmacologically by feeding larvae a trpv selective antagonist. we conducted a genetic screen in n ccap and found that camkii knockdown potently suppresses the trpv r c mediated wing expansion phenotype and selectively rescues degeneration of synapses but not dendrites. we also find that trpv r c , but not controls, disrupts mitochondrial transport in axons by increasing the number of stationary mitochondria. our data demonstrate that trpv r c elevates neuronal intracellular calcium which disrupts mitochondrial transport and mediates neurodegeneration through compartment-specific calcium-mediated signaling pathways, and supports the further investigation of trpv antagonists as potential therapeutics for the treatment of cmt c. mood disorders, including anxiety and depression, are commonly observed among chronic pain patients with prevalence estimates ranging from to %. comorbidity between mood and chronic pain disorders has been linked to altered limbic regulation of the hypothalamic-pituitary-adrenal (hpa) axis. stress activates the hpa axis and can initiate and/or exacerbate symptoms related to both chronic pain and mood disorders. previous studies from our laboratory have investigated the influence of early life stress on mechanical pain hypersensitivity, visceral hypersensitivity and behavioral evidence of mood disorder later in life. here, we are testing the hypothesis that chronic stress exposure in adulthood can increase somatic and visceral sensitivity and anhedonic behaviors in a mouse strain with a genetic predisposition to anxiety. adult, female a/j mice were exposed to repeated foot shock stress for continuous days and tested for alterations in mechanical sensitivity, sucrose preference, visceromotor response (vmr) during urinary bladder distension, and serum corticosterone levels. mice that underwent shock stress had a significantly decreased mechanical withdrawal threshold in the hind paw compared to their baseline and sham group measurements. sucrose preference was measured prior to shock exposure and throughout the shock paradigm as an indicator of anhedonic behavior. mice that underwent shock stress displayed a trend toward decreased sucrose preference, indicating anhedonia, in comparison to mice in the sham group that did not display anhedonia. mice that underwent shock stress displayed significant increases in vmr during bladder distension compared to sham mice. finally, serum corticosterone levels were significantly higher in the mice that underwent shock stress compared to sham mice, indicating a stress-induced increase in hpa axis output. together these data suggest that chronic stress exposure can induce mechanical allodynia, visceral hypersensitivity, and depression-like behaviors in an anxiety-prone mouse strain. future studies will incorporate gene expression in the hypothalamus, amygdala, and hippocampus, as well as investigation of possible downstream peripheral neuroimmune modulation and neuronal morphology changes. guillain-barré syndrome (gbs) is an acute monophasic immune-mediated neuropathy. as prognostic markers of gbs, modified erasmus gbs outcome score (megos), erasmus gbs respiratory insufficiency score (egris), and Δigg have been reported. however, the proportions of subtypes of gbs are known to be different between the western countries and japan, it remains to be elucidated whether those markers can also be applied to gbs in japan or not. we here investigated retrospectively gbs patients and determined the megos and the egris of those cases. among them, Δigg could be obtained in cases. we evaluated the prognosis using gbs outcome score (functional grade: fg) at months; we called good prognosis when fg at months was less than and poor prognosis when that was or more. as a result, in cases with higher score than at megos on admission cases ( %) had poor prognosis and in cases with higher score than at megos at day of admission cases had poor prognosis. in cases with higher score than on egris cases ( %) needed the mechanical ventilator. patients with good prognosis had higher Δigg(average: mg/dl) than patients with poor prognosis(average: mg/dl). we calculated the cut-off value of Δigg in patients, which was mg/dl. patients with higher Δigg than mg/dl could significantly walk independently at six months (p< . ). patients ( %) had poor prognosis in patients with lower Δigg than mg/dl. ( %) of patients were treated with the single cycle of intravenous immunoglobulin (ivig). other patients ( %) were treated with the combined therapies, such as intra venous methylprednisolone and/or plasmapheresis or the second cycle of ivig in addition to ivig. although there was no difference in prognosis between patients with the single cycle of intravenous immunoglobulin (ivig) and patients with the combined therapies, in the patients who had fg> and megos > on admission, the combined therapies made better prognosis than the single course of ivig (p< . ). we found that megos, egris and Δigg were also available in japan. the efficacy of the combined therapies in severe gbs patients should be investigated in the future large scale prospective studies. yiu em , , , burns j , , , menezes mp , , ryan mm , , and for the paediatric cmt best practice guidelines consortium. royal children's hospital melbourne, melbourne, victoria, australia; murdoch childrens research institute, melbourne, victoria, australia; university of melbourne, melbourne, victoria, australia; university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia. charcot-marie-tooth disease (cmt) often presents during childhood. common symptoms include weakness, limb pain and cramps, foot deformity, and balance impairment. guidelines for the optimal management of common problems experienced by children with cmt do not currently exist. development of these guidelines will provide an evidence base for the management and monitoring of children with cmt. a series of systematic literature reviews utilising the grade (grades of recommendation, assessment, development, and evaluation) approach were conducted to answer pre-specified key clinical questions related to the management of paediatric cmt. these included treatment recommendations for symptoms such as weakness, pain, balance impairment, joint deformity, and impaired upper limb function, and anticipatory monitoring for associated complications such as hip dysplasia. this yielded minimal to no evidence for the pre-specified clinical questions, and evidence-based management recommendations could not be made. consensus-based statements will therefore be formulated via a three-round delphi process, to be conducted in . the delphi panel will consist of local and international medical and allied health professionals who have experience in the management of children with cmt. efficacy of pxt in the treatment of adult patients cmt a (n= ) was shown in a multicenter, randomized, double-blind, placebo-controlled phase ii study (attarian et al. ) . pxt taken x/day, orally, for consecutive months was well tolerated and safe. significant improvement of disability was observed for the highest tested dose, thought indicative for an early, meaningful change in disease course (meta-analysis by mandel et al., ) . this formed the rationale to initiate a multicenter, randomized, double-blind, placebo-controlled pivotal phase iii study (clin-icaltrials.gov: nct ) of pxt in mildly to moderately affected cmt a patients in december . the primary objective is to assess the efficacy of doses of pxt compared to placebo on disability as measured by the mean change from baseline overall neurology limitations scale (onls) score at month and . furthermore, efficacy on the proportion of responders (i.e. improvement of onls), impairment (cmtns-v ), functional tests ( -mwt, qmt, -hpt), electrophysiological parameters (cmap, snap and ncv) and quality of life (eq- d, vas) are secondary endpoints. pursuant this study, patients will be eligible for a -month extension study, in which pxt assigned patients will continue with the previously assigned dose, whereas placebo patients will be randomized to one of the two pxt doses. the study is conducted in investigational sites in countries (eu, canada and us). in december patient randomization was completed (n= ). the screen failure rate was %, as expected ( patients were screened). the independent dsmb recommended to continue the study as planned following a safety analysis on the first patients treated for > months. preliminary baseline characteristics are based on patients (data not cleaned). the study population had a mean age of . ± . years (range - ; male . %) of which . % had a confirmed genetic diagnosis of cmt a. the mean cmtns-v was . ± . and the mean motor nerve conduction of the ulnar nerve was . ± . m/s. ten patients withdrew from the study, due to adverse events unrelated to study treatment. the last patient completing the study is expected in march . zhang g , ghosh p , lin j , ghauri a , sheikh ka . department of neurology, university of texas health science center at houston, houston, tx, usa. assessment of epidermal nerve fiber density and its structure integrity is critical for the diagnosis and evaluating the effectiveness of potential therapies in small fiber neuropathies. currently, skin biopsies, at multiple sites, are most commonly used to assess these diseases. these studies are expensive and time consuming due to cumbersome processing and quantification techniques and serial biopsies over time are often not feasible due to costs and patient acceptance. moreover, vast majority of normative data for skin biopsies in humans are available only for few distal sites and a significant proportion of patients with small fiber neuropathies have focal or regional symptoms not involving the commonly biopsied sites in the leg. live imaging could overcome these limitations and provide a noninvasive real time assessment of epidermal nerve fibers all over the body. we previously found that anti-ganglioside antibody (aga) is an effective neuronal delivery vector for transport of various cargos, such as fluorescent dyes, to peripheral nerves. in the current proof of concept study, we examined whether non-invasive multiphoton microscopy can be used to probe/image the epidermal nerve fibers in living animals after systemic and/or local delivery of fluorescently conjugated aga. we found that the individual nerve endings in skin and cornea are distinctly labeled, and visualized under two-photon microscope. the epidermal nerve fiber labelling by fluorescent-tagged aga was further validated using transgenic mice selectively expressing yellow fluorescent protein in their nervous system. in-vivo multiphoton imaging provide a tool with potential for dynamic longitudinal evaluation of small fiber neuropathies, including nerve degeneration and regeneration, without tissue removal. thus, the use of multiphoton microscopy in conjunction with fluorescently labeled aga as neuronal vector can have many research and clinical applications, such as labeling and live visualization of epidermal nerve fibers to assess small sensory nerve fibers in health and disease. zhou y , tavori h , lee s , al salihi m , fazio s , notterpek l . mcknight brain institute, university of florida, gainesville, florida, usa; knight cardiovascular institute, oregon health and science university, portland, oregon, usa. the majority of hereditary neuropathies are due to abnormalities in peripheral myelin protein (pmp ), whose genetic variants include increased expression (gene duplication), haploinsufficiency (gene deletion), or point mutations. phenotypic heterogeneity in clinical presentation is common among hereditary neuropathy patients even within the same family, the cause of which has not been determined. to investigate the role of pmp in the pathogenesis of the neuropathies we have generated and characterized pmp null (pmp −/− ) mice (amici et al., ) whose peripheral nerves show alterations in lipid metabolism (lee et al., ) . to examine the molecular changes underlying these abnormalities we determined the expression of cholesterol synthesis (srebp pathway), and cholesterol uptake, transport and efflux genes (lxr pathway). in affected nerves, we found the cholesterol synthesis pathway inhibited, while the lxr pathway, and particularly apoe and abca , upregulated at the mrna and protein levels. since pmp is expressed at low levels in the liver, the central organ for the regulation of cholesterol in the body, we studied liver tissue form pmp −/− mice. liver from pmp −/− mice showed significant hepatomegaly, clear features of microvesicular steatosis, as well as marked increase in lxr pathway genes and proteins (abca and apoe), as compared to wt. ultrastructural studies identified lipid droplets and significantly enlarged mitochondria in the liver of male pmp −/− mice, which is not due to mitochondria fusion, as the levels of mfn and remained similar to wt. as disturbed hepatic cholesterol homeostasis induces the activation of kupffer and stellate cells, we determined the extent of inflammation in nerve and liver tissues from pmp −/− mice with leukocyte (cd b) and macrophage markers (cd ). in nerve sections, we detected an increase in the number of cd b+ cells, which was confirmed by western blots. in the liver of pmp −/− mice we found a significant increase in cd -reactive kupffer cells and elevated levels of tnf-alpha. the severe dysregulation of cholesterol metabolism in nerve and liver, including neuroand hepatic inflammation in the absence of pmp − suggest that dysregulated cholesterol metabolism and inflammation may act as a disease modifier in pmp -dependent neuropathies. our aim was to ascertain frequency of and risk factors for tee, arterial (ate) and venous thromboembolic events (vte) in neuromuscular patients receiving regular ivig. we performed a retrospective case-note review of inflammatory neuropathy patients receiving regular ivig treatment. we collected the following data over a month study period this analysis suggests tee incidence is higher in ivig treated patients than comparable population-based rates. examination of tee occurrence in age and vascular risk factor matched ivig-treated and ivig-naïve individuals is required to appreciate the excess risk associated with ivig treatment. référence des maladies neuromusculaires et la sla, hôpital de la timone ha) and whether temporal dispersion (td) parameters are helpful. fifty-eight patients diagnosed according to asbury and cornblath ( ) were prospectively included between january and september . edx and were performed - (mean= ) and - days (mean= ) after disease onset, respectively. there were no differences in classification consistency between ho and ha at edx (p= . ) and edx (p= . ), but more patients were classified as aman when comparing ra with ho and ha at edx (p< . ). at edx , ra classified more patients as equivocal with ho (p< . ) and as aman with ha (p< . ) ) or edx (ho, fe p= ; ra, fe p= . ; ra with td, fe p= . ). gm , gd a, gd b and gq b igg antibodies were tested (willison at edx , only ho showed maybe more antibodies with aman compared to aidp (fe p= . , phi=− . ) and with aman compared to equivocal cases since correlation with factors associated with axonal gbs, in casu rcf and antibodies, is far from exclusive, the usefulness of edx subtype classification using specific criteria sets, remains doubtful. the frequency of rcf indicates that nodal/paranodal alterations may represent the main pathophysiology in more gbs patients than currently thought ruiz m , lessi f , cacciavillani m , riva m , salvalaggio a , campagnolo m , briani c . neurology, department of neuroscience, university of padua, padua, italy; hematology and clinical immunology unit, department of medicine, university of padua, padua, italy; cemes, data medica group, padua, italy.arsenic trioxide (ato) is highly effective in treatment of acute promyelocytic leukemia (apl). it is licensed in italy for treatment of relapsed-refractory apl and for first line chemotherapy in low risk patients. ato most frequent side effects are well described, but less is known on ato induced. we describe apl patients who were treated with all-trans retinoic acid (atra)/ato as first line therapy. the characteristics of ato induced neuropathy was prospectically analyzed by neurological evaluation using both the total neuropathy score, clinical version (tnsc) (a validated scale for chemotherapy induced peripheral neuropathy) and neurophysiological assessment. patients have been evaluated at baseline, at the end of the induction phase, at the end of ato/atra treatment and year after discontinuation of treatment. baseline neurophysiology was performed at the end of induction phase. both patients were men, respectively and -yr-old. none of the patients had previous history of neuropathy. baseline tnsc was (no clinical signs of neuropathy) in both patients. neurophysiological evaluation performed after the end of induction cycle did not reveal signs of peripheral neuropathy in both patients. patient received mg of ato during induction, total , mg. patient received mg of ato during induction, total , . both patients developed leg numbness during consolidation cycles and patient also hand numbness. tnsc at the end of therapy was in patient and in patient . neurophysiology at the end of therapy detected signs of sensitive axonal neuropathy in both patients. they received full doses of ato consolidation ( . mg/kg/day for days/week, on alternate months for total months and tretinoin weeks on weeks off). during the first year of follow-up both tnsc and neurophysiology year after the end of consolidation cycle were consistent with full recovery. our patients developed sensory axonal neuropathy during ato therapy, that clinically manifested during consolidation cycles and improved up to complete recovery during follow-up. published case reports show that outcomes may not be as good as in our patients. a multicenter prospective study evaluating the characteristics of ato-induced neuropathy in apl is ongoing. ruiz m , campagnolo m , salvalaggio a , cacciavillani m , taioli f , fabrizi gm , briani c . department of neuroscience, neurology unit, university of padua, padua, italy; data medica group, emg unit, cemes, padua, italy;mutations in the mitochondrial copper-binding protein sco , cytochrome c oxidase assembly protein, have been reported in several cases of fatal infantile cardioencephalomyopathy with cox deficiency. we identified compound heterozygous variants in sco in two unrelated patients with isolated length dependent axonal sensorimotor polyneuropathy of variable clinical severity (axonal autosomal recessive charcot-marie-tooth disease type , cmt ) by whole exome sequencing. although peripheral neuropathy has been described as a secondary feature in a few cases of fatal infantile cardioencephalomyopathy, the disease onset, clinical phenotype and survival in our patients differ significantly from the previously described cases. our patients developed predominantly motor neuropathy; moreover, they are still alive and they have not developed cardiomyopathy, which is the main phenotype and cause of death at early infancy in reported patients. both of our patients harbor mutations adjacent or near the conserved copper-binding motif (cxxxc), including the common reported pathogenic variant e k and the novel change d g. in addition, each patient carries a second mutation located in the same loop region of the protein, p t and r q. western blots from fibroblasts from these cmt patients showed reduced levels of cox , a subunit of complex iv, indicating cox deficiency. our findings demonstrate that cmt can be the predominant phenotype associated with sco mutations, pointing to a broader phenotypic heterogeneity. the mechanism linking mitochondrial respiratory chain dysfunction to isolated axonal loss of variable severity remains to be elucidated. the muscarinic acetylcholine (ach) type receptor (m r) is a metabotropic g protein-coupled receptor expressed by adult sensory neurons. cholinergic signaling through muscarinic receptors can modulate axonal plasticity in invertebrates and lower vertebrates. we have recently shown that selective (pirenzepine) and specific (muscarinic toxin : mt ) m r antagonists elevate neurite outgrowth and protect from small and large fiber neuropathy in adult sensory neurons in various animal models (calcutt, et al., ) . furthermore, we demonstrated that excessive cholinergic signaling due to m r overexpression caused a significant reduction in neurite outgrowth (calcutt, et al., ) . the mechanism of m r-antagonist driven neurite outgrowth remains poorly understood, however, we have proposed that ach constrains axonal outgrowth via m r activation. cholinergic signaling is mediated via recruitment of trimeric g proteins, of which g alpha- and g alpha- regulate cytoskeleton dynamics by control of tubulin polymerization. activated gtp-bound g-alpha proteins destabilize microtubules by increasing the intrinsic gtpase activity of tubulin. we have therefore tested the hypothesis that cholinergic signaling regulates neurite outgrowth via modulation of g protein mobilization and the dynamics of the tubulin cytoskeleton. we found that over-expression of m r in adult sensory neurons induced dissolution of the tubulin cytoskeleton in distal neurites. g alpha- expression in adult sensory neurons was significantly higher (p< . , -fold) than g alpha- . subsequent knockdown the peripheral neuropathy research registry (pnrr) is a multicenter collaborative research project sponsored by the foundation for peripheral neuropathy to advance the science in distal symmetrical polyneuropathies (dsp). the registry was designed to prospectively characterize clinical phenotype and natural history of patients with dsp and obtain biofluids to identify new causes and genetic modifiers of dsp with careful genotype/phenotype correlations, and to develop biomarkers. the enrollment in the registry is still ongoing but an interim analysis was carried at the end of december . eligible study participants were years or older with a diagnosis of idiopathic, diabetic, chemotherapy-or hiv-induced peripheral neuropathy. they were examined by a physician at one of the six consortium members (johns hopkins university school of medicine, icahn school of medicine at mount sinai medical center, beth israel deaconess medical center, northwestern university medical center, university of utah medical center and kansas university medical center). the collected data set included ( ) a detailed questionnaire, discussing their symptoms, medical history and family history, ( ) a standardized neurological examination form, ( ) electrodiagnostic evaluation and ( ) diagnostic laboratory testing. blood samples (whole blood, plasma and serum) were collected for future biomarker and genotyping evaluations. at the end of , complete data sets and blood samples were collected from patients. % had diabetic pn, % had hiv-associated pn, % had chemotherapy induced pn and % were diagnosed with idiopathic pn. detailed analysis of clinical presentation, examination findings and diagnostic investigations will be discussed at the presentation. standardized phenotyping with linked bio-specimen banking will help establish the minimum data set required for neuropathy diagnosis and support genotype-phenotype correlations with next generation sequencing technologies and development of novel biomarkers. pnrr data will improve our understanding of disease mechanisms paving the way for new therapeutic discoveries in painful and non-painful neuropathies. chemotherapy-induced peripheral neuropathy (cipn) is a major side effect of treatment, typically presenting as a sensory neuropathy. symptoms include pain, paraesthesia, and numbness in the extremities, resulting in functional impairment. increasingly, patient reported outcomes (pros) are utilized to accurately examine the impact of cipn symptoms on patient function. however, the links between objective neurological assessment and pro measures remain ill defined. this study aimed to identify links between neurophysiological measures and pros in patients treated with neurotoxic chemotherapy. assessments were conducted in patients (f= , mean age . ± . years) who had completed neurotoxic chemotherapy on average . ± months previously (platinum-based n= , taxane-based n= or taxane/platinum combination therapy n= ). patients reported the presence and severity of neuropathic symptoms via the fact-gog ntx , a validated patient questionnaire. clinical the most important neurological side effect of a large number of anti-cancer drugs is a painful peripheral neuropathy. mainly chemotherapeutics that interfere with microtubules, including the plant derived vinca-alkaloids such as vincristine, are well known to cause chemotherapy-induced peripheral neuropathies (cipn). to date, few treatments are available and focus on symptom alleviation and pain reduction rather than on preventing the neuropathy all together. for the first time, we highlight the potential of specific histone deacetylase (hdac ) inhibitors as a preventive therapy for cipn, using novel rodent models for vincristine-induced peripheral neuropathies (vipn), characterized by a sensory axonopathy. one reason so few therapies are available, is because the exact pathophysiological mechanisms are poorly understood. mounting evidence proposes axonal transport, a pathway frequently disturbed in neurological disorders, as a major player in the pathophysiology of vipn. proper axonal transport requires dynamic microtubules which are highly modulated by post-translational modifications. since vincristine interferes with the polymerization of microtubules, we reason disturbances in microtubule dynamics, and by extension axonal transport, could contribute to vipn. we illustrate that increasing acetylation of -tubulin after hdac -inhibition, can restore vincristine-induced defects in axonal transport in cultured dorsal root ganglion neurons. also in vivo, -tubulin acetylation was restored in the saphenous nerve and dorsal root ganglia, two sensory tissues that are affected by vincristine. ultimately, this correlates to a reduced severity of the neurological symptoms, both on the electrophysiological and on the behavioral level. moreover, we discovered that hdac -inhibition was not only protective against neurotoxicity, but also reduced tumor progression in a mouse model for acute lymphoblastic leukemia. taken together, our results show that hdac -inhibition is an ideal strategy to prevent vipn with beneficial effects both on the neurotoxicity as well as on tumor growth.approximately two-thirds of cidp subjects need long-term corticosteroids or intravenous immunoglobulins (ivig), with ivig being slightly preferred based on safety profiles. subcutaneous ig (scig) is an alternative option for ig delivery but has not previously been investigated in a large-scale clinical trial in cidp.we performed a randomized, double-blind trial in cidp investigating . and . g/kg weekly doses of scig igpro (hizentra ® , csl behring) versus placebo in subjects for maintenance treatment. ivig-dependent adults with definite or probable cidp according to efns/pns criteria were eligible. the primary outcome was the percentage of subjects with a cidp relapse ( -point deterioration on adjusted incat disability score) or who were withdrawn for any other reason during the -week scig-treatment period. multiple secondary endpoints were assessed. superiority of at least one igpro dose over placebo was tested one-sided using the cochran-armitage trend test for the primary outcome and the jonckheere-terpstra tests for secondary outcomes.the primary outcome occurred in % of high-dose scig, % of low-dose scig, and % of placebo subjects (p < . ); cidp relapse occurred in % of high-dose scig, % of low-dose scig and % of placebo subjects (p < . ), respectively. both scig doses were superior to placebo (low-dose vs placebo p = . ; high dose vs placebo p < . ). median incat score, mrc sum score, and grip strength remained stable in scig subjects. high-dose scig prevented the r-ods decline seen with low-dose scig and placebo (p < . ). all placebo subjects deteriorated on measures of strength and disability.causally related adverse events occurred in ( %) subjects ( % placebo, % low dose, and % high dose).scig igpro was efficacious and safe as maintenance treatment. mutations in metalloendopeptidase (mme) gene have been associated with autosomal-recessive late-onset charcot-marie-tooth type- (cmt ). to date, all patients have had at least one truncating mutation, either in homozygosity or in trans with a missense mutation. more recently, loss-of-function and missense heterozygous mutations were also identified in autosomal-dominant cmt. we report the case of a previously healthy caucasian woman, born to healthy unrelated parents, who presented at the age of thirty-nine with numbness and cold sensation in the lower limbs. subsequently she developed progressive gait disturbance and impaired hand dexterity. her homozygous twin presented at the same age with similar symptoms. the family history was otherwise uneventful, in particular neither neuropathy nor dementia were described. neurological examination at the age of fifty-three revealed a steppage gait, distal upper and lower limb atrophy and weakness, distal sensory loss and bilateral pes cavus. deep tendon reflexes were normal in the upper limbs and absent in the lower limbs. nerve conduction studies revealed an axonal sensory and motor neuropathy. a sural nerve biopsy revealed a reduction in myelinated nerve fibers and active axonal degeneration. targeted sanger sequencing of mpz , gjb , gdap , nefl, fkrp, bscl , hspb and mfn were negative. sureselect focused exome sequencing was therefore performed and identified two missense heterozygous mutations [c. g>a,p.c y;c. t>c,p.y h] or lack of efficacy (n= ). most frequently reported adverse events were cognitive ( %), gastrointestinal ( %) and genitourinary ( %). discontinuation due to perception of lack of efficacy was more frequently reported by men ( % vs % p= . ). women presented higher punctuations on pigc scale compared with men ( . ± . vs . ± . , p= . ). pigc scores were significantly higher in patients receiving taxane ( . ± . ) than platinum ( . ± . ) agents (p= . ). no significant differences according severity of neuropathy neither type of chemotherapy were observed in drop-out and retention rates. patients with long-lasting cipn (> months) reported lower pigc scores ( . ± . vs . ± . , p= . ) and higher frequency of suspension due to adverse events ( % vs %, p= . ) and less rate of continuation of duloxetine ( % vs %, p= . ). more than one-third of patients with disturbing cipn discontinued duloxetine prematurely due to intolerable side-effects. low tolerability, male gender and long-lasting cipn may limit duloxetine usefulness in the treatment of symptomatic cipn. verboon c , jacobs bc , and the igos consortium. department of neurology, erasmus medical centre, rotterdam, the netherlands; department of immunology, erasmus medical centre, rotterdam, the netherlands.the efficacy of intravenous immunoglobulin (ivig) in guillain-barré syndrome (gbs) has only been demonstrated in severely affected patients who are unable to walk independently. although there is no proof that ivig is effective in milder forms of gbs, some neurologists are treating these patients with ivig considering that even milder forms of gbs may result in poor recovery, residual deficits, fatigue or pain.we determined the effectiveness of a single course of ivig ( g/kg in - days) in relatively mild forms of gbs in the ongoing observational international gbs outcome study (igos). the gbs disability score, mrc sum score and patient reported outcome measures (prom) were compared at and weeks. ordinal logistic regression analysis was used to determine the effect of ivig on the gbs disability score, adjusted for previously identified prognostic factors.data were analyzed from the first patients enrolled in igos by december , including patients with mild gbs at entry, of which patients ( %) were treated with supportive care, while patients ( %) received ivig (start ivig after onset of symptoms in days, median , iqr - ). at baseline, patients in the ivig treated group compared to the untreated group less frequently had pure motor gbs ( % versus %, p< . ) and axonal damage or unresponsive nerves ( % versus %, p= . ), but a worse gbs disability scores at nadir (p= . ). the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ). at weeks, the median mrc sum scores and prom were not significantly different between treated and untreated patients. however, more patients in the ivig group showed complete recovery of muscle strength at weeks than patients in the control group ( % versus %) (p= . ) and more frequently showed full neurological recovery on the gbs disability scale ( % versus %, p= . ). additional results will be presented at the conference.based on the results of this interim analysis in observational data, we conclude that patients with a relatively mild form of gbs may benefit from a single course of ivig. despite treatment with intravenous immunoglobulin (ivig), many patients with guillain-barré syndrome (gbs) recover insufficiently. we primarily aimed to determine whether a second ivig course ( g/kg in - days) in patients with a poor prognosis improves outcome on the gbs disability scale after weeks. we included patients from the prospective, observational international gbs outcome study (igos) treated with ivig and who had a poor prognosis on the modified erasmus gbs outcome score (megos). of patients enrolled in igos, patients were eligible; patients ( %) were treated with one ivig course (control group); patients ( %) received an 'early' second ivig course ( - weeks after start first course) and patients ( %) a 'late' second ivig course (within - weeks). one week after study entry, patients receiving an 'early' or 'late' second ivig course had significantly worse gbs disability scores and mrc sum scores than controls, implying the need for adjustment of baseline characteristics. the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ) for the 'early' group, and . ( % ci . - . ) for the 'late' group, suggesting worse outcomes with a second course of ivig compared to controls. at months, patients ( %) in the 'early' second ivig group, patients ( %) in the control group and only ( %) in the 'late' second ivig group were able to walk unaided (p= . ). the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ) for the 'early' second ivig group and only . ( % ci . - . ) for the 'late' second ivig group. in gbs patients with a poor prognosis, we did not find a beneficial effect of a second course of ivig after weeks follow-up. our results suggest that an 'early' administered second ivig course might improve outcome at weeks. given the limitations of this observational study, a randomized controlled trial with a larger number of gbs patients with a poor prognosis being treated early in the disease course is needed to confirm or refute these results. vidal c , bhatt n , agudelo c , mahapatra a , saporta ma . department of neurology, university of miami miller school of medicine, miami, usa.multifocal motor neuropathy (mmn) is an inflammatory demyelinating chronic neuropathy characterized by progressive asymmetric weakness in the distribution of two or more peripheral nerves, without objective sensory loss or upper motor neuron signs. the cardinal neurophysiological finding is conduction block outside the usual sites of nerve compression. supportive clinical criteria include high titers of anti-gm antibodies, good response to ivig, increased cerebrospinal fluid (csf) protein (< g/dl) and magnetic resonance imaging (mri) with diffuse swelling of the brachial plexus. we report a case of a year old woman with a two month history of progressive muscular weakness which began in the right upper extremity, described as difficulty in gripping objects, writing and typing on the computer, followed by progressive lower extremity weakness with difficulty rising from a chair and left foot drop. she also complained of pain in her right trapezius and scapular area. she denied any tingling or numbness. on neurological examination there was right scapular winging and asymmetric weakness predominantly involving the right upper and left lower extremities. reflexes were absent throughout. lumbar puncture revealed albuminocytological dissociation (wbc and protein . g/dl). ncs revealed slow conduction velocities for both ulnar and median nerves and a median nerve conduction block at the forearm segment. sensory nerve action potentials were normal for all tested nerves. needle emg revealed acute denervation of right periscapular muscles. brachial plexus mri showed symmetric bilateral thickening from trunks to peripheral nerves. anti-gm igg/igm were negative. two days after lumbar puncture and the beginning of ivig, patient presented binocular diplopia on extreme left lateral gaze. brain mri with and without contrast showed no intracranial pathologies. after five days of ivig, patient was discharged with significant clinical improvement and recovery of the right scapular winging. diplopia improved a week after discharge. we report an atypical multifocal motor neuropathy case. although this patient fulfills all clinical criteria for mmn, we report some features usually not found in mmn such as scapular winging and a mild and transient left vi nerve palsy. mmn should be included in the differential diagnosis of scapular winging. vlckova e , , raputova j , , srotova i , , sommer c , Üçeyler n , birklein f , rebhorn c , rittner hl , kovalova e , , nekvapilova e , , belobradkova j , olsovsky j , weber p , dusek l , jarkovsky j , bednarik j , . despite many studies addressing biomarkers for pain in diabetic polyneuropathy (dpn), little is known about why it affects only a certain proportion of dpn patients. the somatosensory system plays a key role in the pathophysiology of neuropathic pain (neup) and subgroups with different sensory profiles might respond differently to pain treatment. we aimed to characterize sensory phenotypes of patients with painful and painless diabetic neuropathy and to assess demographic, clinical, metabolic, electrophysiological and psychological parameters related to the presence of neup in a large cohort of well-defined dpn subjects.this observational cross-sectional multi-centre cohort study (performed as part of the ncrnapain eu consortium) of subjects with dpn (non-painful, ndpn, n= ; painful, pdpn n= ) associated with diabetes mellitus of type and (median age years, range - years; women) comprised detailed history taking, neurological examination, laboratory tests, quantitative sensory testing, nerve conduction studies, neuropathy severity scores, and neuropsychological questionnaires. all parameters were analysed with regard to the presence and severity of neup. the presence and severity of neup were positively correlated with severity of neuropathy and thermal hyposensitivity (p< . ). a minority of pdpn patients ( . %) had a sensory profile indicating thermal hypersensitivity; this was associated with less severe neuropathy and better response to pain therapy. the presence of neup was also associated with female gender (p< . ) and with higher cognitive appraisal of pain as assessed by the pain catastrophizing scale (p< . ), while parameters related to diabetes (duration, hba c, microangiopathy) showed no influence on neup presence and severity. this study confirms the necessity of comprehensive dpn phenotyping and underlines the importance of the severity of neuropathy that should be taken into account in the stratification of patients with pdpn for analgesic treatment and drug trials.people with charcot marie tooth disease (cmt) experience slowly progressive muscle weakness, sensory loss and musculoskeletal changes over time. this leads to disability and risk of comorbidities due to inactivity. exercise is important to maintain general health but may also help to improve symptoms of cmt. we conducted a randomised controlled crossover trial of aerobic exercise to ascertain the effect of training on fitness levels, muscle strength, function and general well-being. in addition, we monitored the safety of training and feasibility of participation in this type of exercise in local community gyms. motivation, confidence and barriers to exercise were explored using qualitative interviews. the recruitment target was people. in total people with cmt a were approached to participate and were unable to commit to the trial or did not meet the study criteria on initial screening. thirty-one people underwent more detailed screening but three failed to meet the study criteria, five people withdrew before starting and three withdrew part way through the study. the data for the people who started the study were analysed using a random effects model. there was a % participation level in the training and it was well tolerated with no increases in pain or blood serum creatine kinase. an increase in vo peak (ml/min/kg) was observed in the cmt group with (pre training: n= , . ± . , % ci . to . ; post training: n= , . ± . , % ci . to . ; pre control: n= , . ± . % ci . to . ; post control: n= , . ± . , % ci . to . ) . there was wide between subject variation leading to a small overall effect size with cohen d of . ( % ci:- . to . ). a tentative regression model showed no effect of group or time point. there were no major changes in other measures of impairment, function or patient reported outcome measures. this pilot study showed that a community based model of training had a small effect on cardiopulmonary fitness, and was well tolerated with good participation. ankle-foot orthoses (afo) are commonly prescribed for children with charcot-marie-tooth disease (cmt) to manage foot drop, however the type and severity of functional impairment results in gait deviations that might require alternate orthotic designs. the aim of this study was to identify d gait patterns of children with cmt based on severity of functional weakness (based on heel walk, toe walk and foot drop items during gait using the cmt pediatric scale) to inform a design pipeline for d printed orthoses. d gait data were captured with an -camera vicon nexus motion capture system using the lower body plug-in-gait model in children with cmt ( male; ± . yrs, ± . cm, ± . kg), of various cmt types: cmt a, cmt e, cmt f, cmt a, cmt c, cmtx , cmtx and compared to typically developing children ( male; . ± . yrs, ± . cm, ± . kg). data were subdivided into three groups denoting increasing severity of dorsiflexion and plantarflexion weakness: no difficulty heel or toe walking (cmt nd ), difficulty heel walking (cmt dh ), difficulty toe and heel walking (cmt dth ). the cmt nd group showed a near-normal gait pattern. the only significant differences noted at the ankle were reduced peak dorsiflexion in stance (p< . ), indicating that an orthotic intervention may not be required. in addition to reduced peak dorsiflexion, the cmt dh group demonstrated significantly reduced dorsiflexion in swing (foot-drop) and a reduced dorsiflexor moment in loading response (p< . ). this suggest, the cmt dh group would require a flexible afo to allow activation of the plantarflexors during push-off, prevent foot-drop and restore a heel rocker in loading response. in contrast, the cmt dth group presented with significantly delayed and increased peak dorsiflexion in stance and reduced plantarflexion and power at push-off (p< . ). they also had significantly increased mean knee extensor moment (p< . ) revealing early signs of 'crouch gait'. therefore, the cmt dth group would require a rigid afo to limit the amount of dorsiflexion and assist movement of the ground reaction force anterior to the knee during stance. three distinct gait patterns at the ankle were identified in children with cmt, indicating patient-specific orthotic design pathways to target specific functional impairment. wojciechowski e , , chang a , cheng t , , little d , , menezes mp , , hogan s , burns j , . university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia.children with cmt are often prescribed ankle-foot orthoses (afo) to manage lower limb impairment such as foot drop and foot deformities. they are handmade by plaster cast followed by thermoplastic moulding. this traditional approach provides limited design options, can be costly, with long outpatient wait times. d printing, also known as additive manufacturing, has the potential to transform the way orthoses are prescribed, designed and manufactured. the aim of this review was to evaluate the evidence of d printing afos compared to traditional manufacturing methods, for children with cmt. there are currently no studies evaluating the application of d printing afos for children with cmt. however, a small, but emerging evidence base exists for d printed afo's in adults from studies including healthy participants and populations with rheumatoid arthritis, post-polio syndrome, foot drop and ankle weakness secondary to injury. samples sizes ranged from - participants for studies related to in-shoe orthoses and from - for studies related to afos. the methods of d printing included sterolithography, selective laser sintering and fused deposition modelling using materials such as nylon , nylon , polylactide, polycarbonate and abs. d printed afos were comparable to traditional manufactured orthoses in terms of patient-perceived comfort, temporal-spatial parameters, plantar pressure measurements and d gait analysis. however, the effects of long-term usage and durability of d printed afos has not been investigated. d printing orthoses have potential advantages including increased design possibilities, improved productivity, higher compliance, and reduced labour needs. disadvantages include redesigning clinical pathways, limited evidence base for clinical effectiveness, limited biocompatible materials, occupational safety considerations and a high level of expertise required for software operation and fabrication of devices. further research is required to determine the feasibility of d printed afos for children with cmt, and the most appropriate and effective printing pathway, materials to improve health outcomes of affected patients. wong shj , koh sj , lee bjh , chng ysk , pawa c , subramaniam t , cheng ksa , umapathi t . lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore; yong loo lin school of medicine, national university singapore, singapore; khoo teck puat hospital, singapore.treatment-induced neuropathy of diabetes mellitus (dm) (tind) is a complication of rapid glycaemic control. individuals present with neuropathic pain and autonomic dysfunction within weeks of improvement in glucose control. the use of both insulin and oral hypoglycaemic agents has been associated with tind. its severity is determined by the rate and quantum of hba c decline. other predisposing factors include anorexia and weight loss. we studied the incidence of tind in dm patients who have undergone massive weight loss and hba c decline after bariatric surgery. we screened electronic records of patients ( dm, non-dm) who underwent bariatric surgery between and . dm patients fulfilled the tind hba c criteria of a decrease of ≥ % over months or ≥ % over key: cord- -bie veti authors: nan title: ecc- abstracts date: - - journal: int j antimicrob agents doi: . /s - ( ) -x sha: doc_id: cord_uid: bie veti nan f spain introduction: the prevalence of erythromycin resistance (er-r) in group a streptococci (gas) has increased in spain since early s with current rates exceeding % in some regions. this study determined the emm -types associated to erythromycin resistance in spain. material and methods: isolates belonged to the sauce* surveillance collection. rapid sequence analysis of specific pcr products was used to deduce emm -types corresponding to the majority of the known gas m serotypes. pcr primers used: gasm ( ?-tattgcgct-tagaaaattaa- ?) and gasm ( ?-gcaagttctt-cagcttgttt- ?). sequencing was done with the big dye terminator mix and autosequenator (applied biosystems). dna sequences were subjected to homology searches against the bacterial dna database. results: overall, gas isolates ( er-r) were analysed. three m-types (m , st and m ) accounted for . % of the er-r isolates, whereas they just represented a . % of the ery-s. for er-r isolates the strongest association was seen with m (or / ; % ci . Á/ . ), and m was second after m only in the last temporal period of the study ( Á/ ) . no homogeneous distribution of er-r m-types by centres was seen. conclusions: few m-types (leading by m ) are responsible for the er-r in spain. but for m , the remaining er-r m types (st , m and m ) did not show a temporally nor geographically homogeneous distribution. *sauce is an acronym standing for 'sensibilidad a los antimicrobianos utilizados en la comunidad en espana' (susceptibility to the antimicrobials commonly used in the community in spain ) and is the spanish word for the willow tree. significant increase in the prevalence of erythromycin-resistant, clindamycin and miocamycin-susceptible (m-phenotype) streptococcus pyogenes in spain ( ( Á/ purpose: a variety of methods is used for a molecular typing of enterococcus spp. and related gram-positive bacteria. these include dna-based methods such as macrorestriction analysis using pulsedfield gel electrophoresis (pfge), ribotyping, and amplification-based methods such as rapid amplification of polymorphic dna (rapd) and amplified fragment length polymorphism (aflp). we used a homogeneous strain collection of transconjugants resulting from filter-matings with different antibiotic-resistant e. faecium and a recipient isolate from our lab. the influence of transferred antibiotic-resistance determinants on the outcome of different typing methods was investigated. results: fragment patterns resulting from pfge indicated minor differences between the transconjugants and the recipient. in respect to different primers used for rapd, none or only a single fragment shift was detected in the resulting fragment patterns. aflp clusters all transconjugants into a group of major relatedness, but the result was strongly dependent on the mathematical method used for cluster analysis. fragment patterns of digested plasmids showed the possession of different or only widely related plasmids in the transconjugants. conclusions: the results of this study clearly show that under certain situations typing methods commonly used for enterococci and related gram-positive bacteria come to their limits. the sasss network aims to set up a national surveillance study to obtain standardized information on antimicrobial susceptibility to various bacterial pathogens. currently, hospitals are participating in the project from different geographical regions in saudi arabia. during the st year ( ), the sasss focused on setting up this network. overall, high frequencies of resistance to antibiotics to different bacterial pathogens in saudi arabia were seen. geographical variations of resistance were noticed, which could be related to different prescribing practices. approximately, and % of escherichia coli and k. pneumoniae , respectively, were extended spectrum â-lactamases (ebls) producers. resistance of enterobacteriacae group to carbapenem and pipracillin/tazobactam is low. resistance of pseudomonas aeurginosa to various anti-pseudomonal antibiotics including carbapenem is high and alarming. methicillin resistant staphylococcus aureus (mrsa) comprised % of s. aureus isolates. no vancomycin intermediate s. aureus (visa) was detected. high level resistance to gentamicin in enterococcus were seen in % of the isolates and only % of enterococcus facieum were glycopeptide resistant. resistance of streptococcus pneumoniae to penicillin ranged between % to almost %. surveillance of antibiotic resistance on a national level is necessarily to give guidance to practicing physicians on the best agents to use. the world-wide problem of betalactam resistance (r) in streptococcus pneumoniae (sp) has been complicated by increasing r to macrolides and some older fluoroquinolones (fq) (ciprofloxacin cip). aim of our study was to evaluate rate of acquisition of resistance to different fq: cip, sparfloxacin (spx) and levofloxacin (lev) of sp strains with different levels of susceptibility to penicillin (p). fifteen strains were serially and daily passaged in subinhibitory concentrations of these four antibiotics by a gradient plate method until acquisition of resistance. clinical strains isolated from children in day-care centers were used. five strains were susceptible (s) to penicillin (p) (one reference strain, four clinical isolates: p micsb/ . mg/l): five were intermediate (i) to p (one reference strain: p mic . mg/l, four clinical strains p mics: . Á/ mg/l), five were resistant (r) to p (p mics . mg/l). mean of number of passages (n ) necessary to reach i or r level with each fq as selecting agent are in the following table: spx and lev induced resistance but more slowly than cip. our results show that rate of acquisition of resistance to fq is strongly related to alteration of susceptibility to p, probably by modification of cell wall. these results are concordant with clinical results. clinical relevance of phase variation in pneumococcal opacity: nasopharyngeal (np) colonization in children from day care centers (dcc) soa . carsenti h, mancini g, bensoussan m, dunais b, pradier ch, dellamonica p. archet hospital, infectious disease, nice, france streptococcus pneumoniae (sp) adherence to nasopharyngeal (np) epithelium is a prerequisite for induction of otitis transparent sp (t) have been shown to colonize the np of infant rats better than opaque (o) sp. opaque sp has proven more virulent than the t form during systemic infection in a mouse model. aim of this study was to evaluate phase variation in the nasopharynx of children. sp strains were isolated during a winter epidemiology study of np samples in children from family dcc. mics determinations were performed by e -test for penicillin (p), amoxicilline (amx) and ceftriaxone (cro). serotypes were performed using the quellung reaction. upon oil immersion microscopic examination short chains of six to eight cocci were noted as , '/, '/'/, '/'/'/ for absence, , , !/ chains by field, respectively. phase variation was detected on catalase trypticase soja plates, amx and cro mics and bactericidal activity was determined for pairs of o and t variants with different serotypes and susceptibility to penicillin. seventy strains of sp were screened for phase variation. nine out of with chain length , '/ had o variants while out of strains with chain length '/'/ or '/'/'/ showed o variants. proportion of o variants was predominant when chain length increased. serotype f was prevalent. bactericidal activity of o variants showed a four-to eightfold increase of mbc. o variants may be present in np of children while t are predominant form for colonization. these virulent variants with lower level of autolysis showed less susceptibility to killing by antibiotics. they may persist in np and explain the absence of eradication by active molecules. antimicrobial resistance among clinical strains of s. pneumoniae isolated from patients with community-acquired respiratory tract infections (carti) in russia soa . kozlov rs a , bogdanovitch tm a , sivaya ov a , agapova ed b , ahmetova li b , furletova b , gudkova lv b , gugutsidge b , ilyina vn b , marusina b , multich ig b , ortenberg ea b , schetinin ev b , shturmina purpose: to determine the antimicrobial resistance of pneumococci causing carti in different russian cities. methods: a total of non-duplicate strains isolated in russian cities in were studied. antimicrobials tested included penicillin (pen), amoxicillin (amo), erythromycin (ery), azithromycin (azi), clarithromycin (cla), midecamycin (mid), spiramycin (spi), clindamycin (cli), levofloxacin (lev), vancomycin (van), rifampicin (rif), tetracycline (tet) and co-trimoxazole (sxt). susceptibility testing was performed by broth microdilution with interpretation of the results according to nccls guidelines ( ) a map of bacterial resistance in a hungarian region soa . farkas a a , juhasz a b , orosi p a , miszti c c , balogh m d . a kenezy teaching hospital, hygienie, debrecen, hungary , b kenezy teaching hospital, laboratory, debrecen, hungary , c university of debrecen, microbiology lab, debrecen, hungary , d regional hospital berettyóújfalu, laboratory, debrecen, hungary background: regional trends of microbiological resistance pattern constitute basic data and qualifying criteria for effective infection control. purpose: the aim of our study was to establish an internationally compatible regional database in a hungarian county hajdú -bihar. methods: our model is the national nosocomial infections society publications' format from the u.s. published in . it contains data regarding various icu types, ambulatory patients and hospitalised patients. the same format is used for antibiotic utilisation data and device related infections' rates as well. we collected cleaned data of years Á/ from all the microbiological laboratories of our county. results: ciprofloxacin p e. coli . . . Á/above susceptibilities not significantly different from u.s. data were as follows: mr cns, streptococcus pneumoniae /penicillin and rd generation cephalosporin, pseudomonas aeruginosa /piperacillin and enterobacter spp. and escherichia coli /ceftriaxon. conclusions: this database proved to be a very useful tool for choosing primary wards of active surveillance including places for infectious disease physician's visit (icu, rehabilitation unit). additional analysis is needed at an individual institution's level for other heavily used (or useable) antibiotics and bacteria as well (aminoglycosides, beta-lactam Á/beta lactamase inhibitor combinations, nd generation cephalosporins, corynebacteria . to compare epidemiological, clinical, and immunological features of add before and after haart introduction, between the patients (p) diagnosed in Á/ , and the p detected since , in a case-control study. though the mean number of newly diagnosed aids p had a sharp drop in the haart era, from p/year in Á/ to p/year since (p b/ . ), the distribution of add and underlying immunodeficiency showed limited changes. when excluding a greater frequency of tuberculosis (tb) (p b/ . ) and wasting syndrome (p b/ . ), all other add did not show a different frequency before and after . a tendency towards a higher mean cd count at aids disease was noticed: vs cells/ml (p b/ . ), with a significant difference for candida esophagitis , toxoplasmosis, kaposi sarcoma and tb (p b/ . Á/b/ . ). the limited variation of clinical and immunological presentation are attributable to the poor impact of haart before aids recognition: . % of p detected since did not receive haart or had insufficient compliance to antiretrovirals, so that . % of p were aids presenters. during the haart era, an increase of mean age and sexual transmission was found (p b/ . ). notwithstanding the effects of haart on the natural history of hiv disease, the consequences on add distribution and related immunodeficiency were negligible, since most p could not benefit from haart before aids onset. a high clinical suspicion for add should be maintained when facing p with missed or undertreated hiv disease. radata */communication internet platform management of resistance analysis guided haart switch for implementation in clinical practice of hiv-infected individuals soa . paech v, lorenzen t, stoehr a, plettenberg a. ifi, interdisciplinary infectiology and immunology, hamburg, germany purpose: hiv-resistance analyses are indicated to prepare switch of haart in hiv-infected individuals with failure to ongoing haart regimen. specialists at several responsible sites often feel lack of complementary informations if interpretation of resistance analyses is done independent from each other. clinical benefits from resistance analysis assays are sigfnificantly higher for those physicians, who can access external advice from hiv-experts for possible treatment options. the database concept 'radata' (www.radata.de) was developed in germany to generate expert advice for implementation in haart switch. results: fifteen hiv-treatment centres, seven laboratories and high ranked authorities in hiv-medicine contribute to radata database since it is started in january in germany. hiv-infected subjects are eligible to participate at the project after presentation of failure to haart (viral load !/ c/ml). expert advice is generated after all data are evaluated and based on recommendations of Á/ external hiv-experts. observation after therapy switch is scheduled for a period of months. conclusions: radata is a novel database concept with features for evaluation of data and availability of complementary information to participating sites. the project is designed to provide its proficiency to patients and centres from germany and foreign countries. further information will be provided after the number enclosed subjects have enlarged. in vitro effects of hiv infection on abc transporter expression and antiretroviral drug efficacy soa . therefore, we evaluate in primary cultures of human monocytederived macrophages (mdm) and lymphocytes, effects: ( ) of retroviral infection and haart on the expression and activity of p-gp and mrp; and ( ) of specific inhibitors of these host proteins on antiretroviral activities of nrti, non-nrti and ip. results: on the one hand, we evidenced a transitory increase of p-gp mrna expression in lymphocytes and mdm in response to in vitro hiv infection. this was correlated to an increased p-gp cell surface expression and activity, and an increased tnf-alpha production and mrna. in contrast, no significant modulation of mrp was observed. on the other hand, psc and probenecid potentiated in vitro the anti-hiv activity of azt and indinavir. these effects were accentuated when psc and probenecid were combined. conclusion: these results showed that: ( ) hiv infection by increasing abc transporter expression could favorise the efflux of antiretroviral drugs and decrease their pharmacological effects; and ( ) specific inhibitors of these transporters could reverse these deleterious effects. effects of interferon alpha plus ribavirine therapy on frequencies of hcv, hiv and cmv specific cd -t-cell responses in peripheral blood of hiv/hcv coinfected patients after months of treatment soa . methods: two groups of patients with chronic hcv infection were studied: hiv coinfected progressors with antiretroviral therapy and hiv-negative controls. twelve hcv/hiv and hcv patients have already reached months of ifn-alpha'/ribavirine therapy. virusspecific cd -t-cells in peripheral blood were analyzed by ifngamma-elispot-assays using hiv-p , one cmv and three hcv (core, ns , ns ) antigens. results: ( ) at baseline, hcv-specific cd -th -cells frequencies were significantly lower than hiv-and cmv-specific ones; ( ) frequencies of cd -th -cells against hcv as well as against cmv were similar in the two groups; ( ) in hcv'//hiv'/, hcv specific cd -t-cell frequencies did not change between baseline and th month of anti-hcv treatment, decreased in three and increased in only one case. hiv-and cmv-specific frequencies were decreased in seven patients. similar results were observed in hiv-negative group. conclusion: ( ) hcv-specific immune responses might be more prone to tissue compartmentalization than hiv-specific ones; ( ) immune defects induced by hiv infection might not be responsible for the low level of hcv-specific responses observed in hiv-progressors; ( ) ifn-alpha'/ribavirine therapy influence on hcv-and hivspecific cd -t-cell frequencies after months of treatment will be discussed. frequencies of hiv- -p specific th cells (elispot) are correlated with plasma hiv- viral load in a cohort of lt-np and slow progressors soa . martinez v a , alatrakchi n a , costagliola d b , bonduelle o a , agut h c , autran b a , alt study group a . a hopital pitié-salpêtrière, laboratoire d'immunologie cellulaire, paris, france , b faculté de medecine saint-antoine, inserm sc , paris, france , c hopital pitié-salpêtrière, laboratoire de virologie, paris, france background: hiv- -specific t helper- cell responses have been associated with long-term-non-progression (lt-np) in hiv infection but the correlation between frequencies of hiv- -p -specific th cells and viral load has not yet been studied. we prospectively quantified these frequencies by using an ifn-gamma elispot assay in a cohort of lt-np. methods: a cohort of lt-np and slow progressors (infection !/ years and cd counts !/ /mm ) was analysable. hiv- -p specific t cells were analyzed using: proliferation, ifn-gamma eli-spot assays and ifn-gamma production in cell supernatants. results: wide ranges were observed in the frequencies of hiv- -p -specific cd th cells as assessed by elispot ( - sfc/ pbmc) with a median of sfc/ pbmc. these frequencies were negatively correlated with viral load (r /(/ . , p / . ) but not with cd counts and associated with a low level of t cell activation assessed by cd on cd cells (r /(/ . , p / . ). similar results were obtained with t cell proliferation and ifn-gamma production. conclusion: interestingly, the numbers of hiv- -p -specific th cells correlate with plasma viral load, independently of cd counts indicating that: ( ) the defect in hiv- -specific cd th cells does not reflect the global cd depletion; and ( ) these responses are strongly correlated to the control of virus replication. impact of drug Á/drug interactions on therapeutical management of active tuberculosis in hiv infected patients soa . martinez v a , truffot c b , caumes e c , katlama c c , jarlier v b , bricaire f c , jouan m d . a department of infectious and tropical diseases, pitié salpêtrière hospital, institut pasteur, unité de génétique mycobactérienne, paris, france , b department of bacteriology, pitié salpêtrière hospital, paris, france , c department of infectious and tropical diseases, pitié salpêtrière hospital, paris, france , d institut pasteur, unité de génétique mycobactérienne, paris, france since and the use of haart, management of hiv patients with active tuberculosis raised the question of drug Á/drug interactions and therapeutical management of both infections. retrospective cohort study: follow-up of hiv patients with active tuberculosis diagnosed between and . studied data included evolution of tuberculosis and hiv, cd cell counts, plasma hiv viral loads, antituberculosis and antiretroviral regimens. ninety-four percent of patients were treated by quadruple combination antituberculosis drug with rifabutin for five patients. fourteen patients were treated by double antiretroviral therapy of nucleoside reverse transcriptase inhibitors (nrtis) and by triple or more drugs (nrtis and/or nonnucleoside reverse transcriptase inhibitors nnrtis and/or protease inhibitors pis). the median follow-up was months. there was no difference on cd cell counts and viral loads in the two groups and between the patients treated by nnrtis and pis at the diagnosis of tuberculosis, at the time of antituberculosis drug discontinuation and concerning cure rates of tuberculosis. on the other hand, the plasma hiv viral load was significantly better controlled in patients with nnrtis than with pis (p b/ . ). in hiv patients with active tuberculosis receiving haart, antiretroviral combination including nnrtis allows a better control of viral replication than regimen including pis without impact of the use of rifampin or rifabutin. adherence is essential to the effectiveness of antiretroviral therapy. a pharmacy visit would improve the patient advisement. a survey was carried out over a period of months in the u.m.i.t. a self-report was distributed to patients. ninety were evaluated. for %, information's given by the clinician were sufficient and for % the treatment advice cards were useful. however, % of them would like to attend a pharmacy visit. the topics they would prefer to be tackled were drug interactions ( %), side effects ( %) and effect of forgetting ( %). the treatment was well accepted and tolerated for, respectively and % of the patients. the viral load and the cd count were well known by, respectively and %. however, inaccurate pattern of treatment was frequent ( !/ %) and bad adherence was observed: treatment forgotten occasionally ( %), regularly ( %) or inadequate attitude when the treatment was forgotten ( %). number of pills, dose frequency, length of the treatment would be risk factors of nonadherence. for the majority of patients, a pharmacy visit is necessary and beneficial. the first result shows a better understanding of the treatment, an improvement of the adherence and an enhancement of plasma concentration of antiretroviral drugs. in vivo activity of glycopeptides against s. aureus infection in a rabbit endocarditis model: is mic predictive for in vivo efficacy? soa . asseray n, caillon j, lemabecque v, jacqueline c, batard e, potel g, bugnon d. laboratoire antibiologie, faculte de medecine, nantes, france we have studied the in vivo efficacy of vancomycin (v) and teicoplanin (t) against five staphylococcus aureus (sa) strains: two methicillin-susceptible (mssa and ), two methicillin-resistant (mrsa and ) and one glycopeptide-intermediate (gisa ) strain, in a rabbit endocarditis model. mics of v and t (v/t) were . / . , / . , / , . / . , and / , for mssa , mssa , mrsa , mrsa , and gisa , respectively. the animals were randomly infected with one of these strains, then treated for days by v or t. a continuous infusion of v, simulating a mg/kg/ h human dose was used. t was infused as a continuous infusion allowing simulating a mg/kg human dose, following an initial bolus. these regimens achieved clinically relevant serum steady-state concentrations of glycopeptides ( !/ mg/ l). results were as follows: expressed in log cfu/g of vegetations (mean /sd, followed in parenthesis by the number of rabbits used). purpose: to determine the prevalence of the decreased glycopeptides susceptibility among clinical isolates of staphylococcus aureus collected from patients hospitalized in strasbourg university hospital between / / and / / . the susceptibility to glycopeptides of s. aureus isolates collected from hospital environment during approximately the same period was also investigated. methods: the susceptibility to glycopeptides was studied among the mrsa isolates, using: Á/ detection of the decreased susceptibility to glycopeptides using agar plates containing mg/l teicoplanin, Á/ detection of hetero-visa strains using agar plates containing mg/ l vancomycin, Á/ determination of the mics of vancomycin and teicoplanin using the agar dilution and the e -test strips methods. results: thirty-nine percent of s. aureus clinical isolates ( out of strains) are mrsa. no visa or hetero-visa strain was detected. six percent mrsa isolates are teicoplanin intermediate s. aureus strains. in the environment, % s. aureus isolates are methicillinresistant (five out of ). the five strains are all susceptible to glycopeptides. conclusion: the results regarding vancomycin are reassuring. however, the high rate of mrsa and the presence of teicoplanin intermediate s. aureus isolates prove that prevention and control measures need to be improved. comparative investigation of polymerase chain reaction and a conventional methods for detection of methicilin resistant staphylococcus amont clinical isolates soa . kantardjiev tv a , vacheva-dobrevski rs b , panajotov sv a , bachvarova am a , velinov ti a , levterova vs a . a national center of infectious and parasitic diseases, microbiology, sofia, bulgaria , b military medical academy, clinical microbiology, sofia, bulgaria purpose: identification on methicillin resistant staphylococci has a great clinical implication and significant impact of antibiotic therapy. the aim of this study is to compare the disc-diffusion test (ddt), oxacillin agar screen test (oast) and pcr for detection of mec a gene. fifty selective clinical isolates ( staphylococcus aureus and nine s. epidermidis ) determined as methicillin resistant by ddt were enrolled in the study. ddt was performed with oxacillin disk ( mkg/ml) on mueller-hinton agar (mha) without nacl (nccls, ) . oast was performed on mha with % nacl, oxacillin mkg/ ml, t c. these strains was genotypically characterized for the mec a gene presence by pcr method using the mec a - ?-aaa atc cat ggt aaa ggt tgg c- ? and the mec a Á/ ?-agt tct gca gta ccg gat ttg c- ? primers (gibco, brl) . results: in the group of mrs isolates, detected by pcr, positive results were as follow: s. aureus and six s. epidermidis . for six s. aureus isolates ddt and oast were positive; pcr-negative. for two s. aureus and two s. epidermidis isolates pcr was positive; phenotypic methods-negative. conclusions: accurate and rapid detection of mrs is a constant challenge for laboratories. the pcr assay (first time in our country) appears to be more reliable than routine susceptibility testing for the rapid diagnosis of mrsa infections at hospitals, particularly due to the heterogeneous resistance of many strains. . / . ( ) . / . ( ) . / . ( ) . / . ( ) . / . ( ) v . / . * ( ) . / . ( ) . / . * ( ) . / . ( ) . / . ( ) t . / . * ( ) . / . ( ) . / . * ( ) . / . ( ) . / . ( ) distribution and antibiotic susceptibilities of bacteria isolated from suspected urinary tract infections of inpatients in hungary soa . rozgonyi f, csukás z, kamotsay k, szabó d, ostorházi e, berek z, maródi c. institute of medical microbiology, semmelweis university, budapest, hungary between l january and december , a total of , urine samples were cultured % as native urine (nu) and % as uricult-plus (up) (orion diagnostica, finland) . cultivations were negative in % of nu and % of up specimens, while contamination was revealed in % of nu and % of up. in the clinical bacteriologically evaluable positive nu and up cultures, the distribution of the gram-negatives was very similar with the predominance of escherichia coli ( and %) followed by enterobacter spp. the distribution of gram-positives differed significantly according to the types of specimens. nu resulted in % group-d and % group-b streptococcus while up did and . %, respectively. third generation cephalosporins and fluoroquinolons were equally very effective against e. coli strains, while ampicillin inhibited growth of % only. carbapenems, cefepime and the fluoroquinolons were the most active against enterobacter strains. interestingly, trimethoprim'/ sulfarmetoxazole combination could inhibit more than % of enterobacteriaceae strains. piperacillin'/tazobactam ( %), imipenem ( %), and ciprofloxacin ( %) could be the drog of choice against pseudomonas aeruginosa . enterococcus strains were highly sensitive to glycopeptides ( %), nitrofurantoin ( %), imipenem ( %) and amoxicillin'/clavulanic acid ( %). antimicrobial susceptibility levels of escherichia coli isolates cultured from urine at a tertiary care teaching hospital. temporal trend and comparison between community-acquired and nosocomial urinary tract infection soa . nanetti a, manfredi r, valentini r, calza l, chiodo f. uni versity of bologna, infectious diseases, bologna, italy in order to assess the local temporal trend of antibiotic sensitivity of the most common urinary tract bacterial pathogen, all urine-cultured escherichia coli isolates were reviewed as to susceptibility profile, and specimen source (community-versus hospital-acquired infection). when evaluating sensitivity levels of community-acquired pathogens ( Á/ ), a significant resistance rise was limited to cotrimoxazole (p b/ . ) and nalidixic acid (p b/ . ), while a tendency towards increased resistance regarded norfloxacin (p / . ) (fig. ) . when community-acquired e. coli isolates were compared with nosocomial strains (tested in the years Á/ ), a greater susceptibility of community-acquired e. coli isolates was limited to cotrimoxazole versus all other compounds in the year (p b/ . ), while it was extended to amoxicillin, cephalotin, nitrofurantoin and piperacillin in the year (p b/ . ) (fig. ) . on the whole, e. coli showed an elevated sensitivity rate ( !/ % of tested strains) to nitrofurantoin, gentamicin, amikacin, and nd-and rd-generation cephalosporins, while only amoxicillin and piperacillin had a mean resistance rate !/ %, regardless of the community or nosocomial origin. a permanent surveillance of sensitivity levels of the most common pathogens responsible for infectious diseases enables to identify local antimicrobial activity and its temporal variations, and plays a key role in starting empiric therapy, pending bacterial identification and in vitro assays. conclusion: in uti, the antimicrobial agents such as st cg combined with aminoglycosides are recommended as initial treatment as well as the rd cephalosporin generation at monotherapy. in addition, the fluoroquinolones and aminoglycosides are effective in uti. prevalence of resistance mutations to antirretrovirals and relation to virological failure s . garcia f a , suarez s a , alvarez m a , martinez nm a , valera b b , pasquau j b , hernandez quero j a , maroto mc a . a hospital san cecilio, microbiology, granada, spain , b hospital virgen nieves, microbiology, granada, spain purpose: to investigate the prevalence of resistance mutations in the reverse transcriptase (rt) and protease (p) genes of hiv and to relate it with the type of virological failure (vf), we have studied patients ( % naïve or pregnant women, % were first vf, % were second vf, % more than two vf. resistance mutations were investigated using trugene hiv- genotyping kit (visible genetics). results: global prevalence of resistance mutations for rt inhibitors (rti) has been !/ % for m l, d n, k n, m v, l w, t yf, and for l i, m i, l p, a vt, l m for p inhibitors (pi). the prevalence of resistance mutations for the naïve patients studied was very low (a g, v i for rti and l i, m i, m i */all n / */and l p n / for pi). for patients on first vf only k n, m v, t yf (rti) were !/ %, as well as l i, d n, l p (pi); when patients on second vf were studied, then m l, e d, k n, m v, g a, l w, t yf, k qe (rti) and m i, l p, a t (pi) were !/ % prevalence; finally, when patients with more than two vf were studied, the following resistance mutations were !/ %: m l, d n, k r, k n, v i, y c, m v, g a, l w, t yf (rti), and l i, m i, m il, l p, a t, l m (pi). conclusions: the prevalence of primary resistance in the population studied is very low; the prevalence of mutations in the reverse transcriptase and protease genes increase in parallel to the type of virological failure. genotypic resistance in hiv- rna from patient plasma compared with rapid virus isolation and phenotypic resistance in patient pbmcs s . stuermer m, groeschel b, cinatl j, doerr hw. institute for medical virology, university clinic frankfurt, frankfurt, germany objective: to compare hiv- virus isolation in the presence of antiretroviral drugs with plasma hiv- genotyping. materials and methods: hiv- genotyping was performed using the viroseqtm vers. from applied biosystems. interpretation of genotype was done according to international standards. cd -cells were purified from patient plasma and cultivated in microtiter plates coated with anti-cd and anti-cd antibodies in the presence of different concentrations of antiretroviral drugs. virus production was measured using a p antigen assay. phenotypic activity was expressed as % reduction of p concentrations. results: seventeen samples were analyzed. for samples results were obtained from both methods, two samples could not be analysed by phenotyping and four samples not by genotyping. only / samples showed total and / samples partial concordance, / samples showed discordance between the two assays. in discordant samples the genotype gave a definite interpretation. conclusion: hiv- virus isolation and phenotyping from pbmcs may overcome the problem of currently used resistance assays, which analyse only the reverse transcriptase and the protease gene of hiv- . possible mutations in other regions may influence viral fitness and therefore contribute to the growth of the virus population present. the lack of concordance between the two assays is related to the different blood compartments used. the clinical value of resistance tests using pbmcs is under investigation. interleukin- co-operates with a new type i ifn, ifn-tau to inhibit early steps of hiv-i biological cycle s . rogez c a , clayette p a , martin m a , dereuddre-bosquet n a , martal j b , dormont d c . a cea, drm, fontenay-aux-roses, france , b inra, physiologie animale, jouy-en-josas, france , c cea, crssa, ephe, drm, fontenay-aux-roses, france background: type i interferons (ifn) exhibit efficient antiviral activities notably against hiv, but severe side effects restrict their clinical uses. ifn-tau is an ovine or bovine non-cytotoxic type i ifn which displays higher inhibitory effects towards hiv replication than ifn-alpha, particularly in human monocyte-derived macrophages (mdm). the antiretroviral activity of ifn-tau seems to involve antiviral and immunomodulatory mechanisms: il- synthesis is increased in dose-dependent manner in mdm treated with ifn-tau and a specific inhibition of il- biological activity decreases its antiretroviral efficiency. results: after a -h infection, a significant decrease of intracellular hiv rna amount was found in mdm treated with ifn-tau. in parallel, no additive inhibition was observed with ifn-tau during the elongation of proviral dna. these results suggest either an inhibition of hiv nucleocapsid uptake or an immediate hiv rna degradation, and the expression of ?, ?-oas, mxa protein and pkr was then measured. ifn-tau induced the expression of these three host cell factors. the role of il- on these different steps was evaluated and we showed that il- co-operates with ifn-tau during the very early step of hiv biological cycle. conclusion: altogether, these results evidence that ifn-tau uses the same antiretroviral pathway as others type i ifn in mdm, and that il- takes part to its inhibition of early steps of hiv biological cycle. actinomycin-d as a modulator of resistances due to cell-wall active agents like bacitracin (bc) and lysozyme (lz) s . chakrabarty an a , dastidar sg b . a calcutta university, medical microbiology, calcutta, india , b jadavpur university, pharmaceutical technology, calcutta, india it was observed that development of lzr in the lzr mutants took placed at three different levels and was accompanied with unselected, distinctive and elevated levels of bcr. similarly, bcr in bcr-mutants were also detected at three different levels. although the levels of bcr ( / mg/ml) in the bcr mutants could be raised only by persistent efforts, an increase in the levels of lzr (as cross-resistance) in the same mutants could be easily achieved. a correlation of actinomycin-d resistance with lzr and bcr of the mutant bacteria and the effects of lipase treatment on the same showed a Á/ -fold rise in actinomycin-d resistance of the lzr and bcr mutants of gram-positive bacteria compared with their correspondence wild-types. these findings suggest that lzr and bcr are controlled by several genes accounting for reduced cell-wall and cell-membrane permeability and indirectly, by phenotypic alteration of the lipid content of the cell-wall. thus, the alteration of cell-walls and membranes and a phenotypic extra lipid layer can work in conjunction with the efflux pump mechanisms finally determining the levels of drug-resistance. experimental development of drug resistance to non-antibiotics: a role of alteration of membrane fluidity and efflux systems s . dastidar sg a , mazumdar k a , asok kumar k a , chakrabarty an b . a jadavpur university, pharmaceutical technology, calcutta, india , b department of medical microbiology, calcutta university, calcutta, india drug resistance among clinical strains was studied by selecting mutants resistant to promazine (pr) and methdilazine (md). the results showed that successive step-up mutants of pr and md developed cross-resistance to several unrelated drugs, which in subsequent steps had broader resistance spectra with higher levels of resistance. experiments on the membrane fluidity or permeability of bacterial cells using diphenyl hexatrine (dph), a fluorescent probe on md-mutants showed that three was marked reduction in the membrane fluidity and permeability. when several analogues of the basic phenothiazine structure, e.g. -chlor-methyl-n -methyl-pyrrolidine (cmp), methyl- -methyl- -pyrrolidone- -carboxylate (mmpc), hydroxymethyl-n -methyl-pyrrolidine (hmp) and md with final substitution were tested for antibacterial function on different strains, highest activity was observed with respect to md. with anaerobic bacteria the resistance(s) dependent on efflux pumps showed higher levels of resistance even to md. we have found the non-antibiotic agents triflupromazine, trimeprazine and diclofenac sodium have high degree of activity against vibrios, staphylococci and pseudomonads. the explanation of such a phenomenon in terms of possible efflux pumps will be discussed. csf, plasma and urine pcr in lyme neuroborreliosis s . pícha d a , moravcová l a , lásiková Š a , marešová v a , Ž ïárský e b . a charles university, nd medical school, st clinic for infectious diseases, prague, czech republic , b department of cellular and molecular biology, charles university, rd medical school, st clinic for infectious diseases, prague, czech republic the main reason for high diagnostic value of pcr in neuroborreliosis (nb) is the direct way of spirochete detection. two sets of primers in nested pcr were used: one for plasmide gene encoding ospc protein and second for chromosomal gene s rdna. so far patients with clinically manifested involvement in nb were enrolled into the prospective designed study (being continued). the main including criterion was positive prove of intrathecal specific antibody secretion (in patients) and pcr positivity in csf (in ). all patients were repeatedly examined by neurologist and samples of csf, plasma and urine were taken: ( ) before treatment; ( ) after treatment; ( ) after months. before treatment were patients pcr positive in csf, six in plasma, and in urine. five were parallel positive in csf and plasma and four in all three body fluids. urine after treatment was positive in seven ( %) cases and completely negative after months. the pcr has had relative high sensitivity ( %), but does not rich the sensitivity of antibody index ( %). supported by grant mzcr ; . consumption of imipenem correlates with b-lactam resistance in pseudomonas aeruginosa s . lepper pm a , hö gel j b , trautmann m a , grusa e c . a department of medical microbiology and hygiene, university of ulm, ulm, germany , b department of biostatistics, university of ulm, ulm, germany , c hospital memmingen, central pharmacy, memmingen, germany purpose: in the present study we investigated the monthly consume of three anti-pseudomonas-active antibiotics, namely imipenem, piperacillin/tazobactam (pt) and ceftazidime during a period of years ( Á/ ) . the use of these antibiotics was correlated to the rate of resistance in pseudomonas aeruginosa . results: inspection of the time series for use of imipenem, ceftazidime, and pt, and the corresponding time series for resistance (each available from july to july ) indicates a remarkable coincidence between use of imipenem and resistance against the three antibiotics mentioned. pearsons's coefficient of correlation for the use of imipenem and the resistance against imipenem was . (p b/ . ), between imipenem use and pt resistance was . (p b/ . ), and between imipenem use and ceftazidim resistance . (p b/ . ). we found positive regression coefficients quantifying an association with imipenem use in the same month (p b/ . ) and with the use during the preceding month (p b/ . ). the same was true when checking dependence of ceftadizime resistance (p b/ . ) and pt resistance (p b/ . ) on imipenem use observed during the same month. neither the use of ceftadizime nor of pt could be identified as factors creating resistance to one of the three antibiotics under consideration within a reasonable period of time. conclusion: there might be a strong pressure towards resistance created by carbapenems. this could limit the use of carbapenems for initial empiric therapy. treat hard and fast: short course antibiotic treatment and its relation with patient compliance and effectiveness s . perez-gorricho bpg a , ripoll m b , pechere jc c . a niño jesus hospital, infectious diseases, madrid, spain , b insalud, outpatient consult, madrid, spain , c university of geneve, microbiology, geneve, switzerland 'treat hard and fast ': short course antibiotic treatment and its relation with patient compliance and effectiveness. finding the important implications for the way in which physicians manage patients with mild Á/moderate respiratory tract infections, and the relation of this management with the perception of antibiotic effectiveness, and the compliance with the antibiotic regimen has been the main purpose of the research. in a pan-european market research study of more than patients, designed to determine behaviour to the antibiotic management of mild-moderate respiratory tract infections, patient expectations of antibiotic therapy were identified, particularly those aspects that relate to efficacy and compliance. the study identifies three key drivers of patients perceived antibiotic efficacy: length of antibiotic course, time to onset of symptom relief and time to complete resolution of symptoms. the results demonstrate that once daily treatment for short periods is perceived by patients to be significantly more effective than longer antibiotic courses and thus better meets patient expectations of therapy. in this study, a macrolide, azithromycin, was selected as the drug therapy of shortest course, being the antibiotic with the shortest dosage schedule for common outpatient infections. the perception of efficacy with short course therapy also correlates with overall satisfaction with management by the physician and with patient compliance with antibiotic therapy. purpose of the study: group b streptococci (gbs) remain a major cause of neonatal infections. consensus guidelines have recommended an intrapartum antibioprophylaxis by amoxicillin, which has reduced the incidence of early-onset neonatal gbs infections. however, an increased incidence of beta-lactam-resistant gram-negative neonatal sepsis has been reported. the aim of our study was to analyse the consequences of this antibioprophylaxis on the intestinal microbial colonization of newborns. a study of the fecal flora was carried out on stools samples from days-old newborns divided into groups: group a intrapartum treated mothers (n / ); and group b untreated mothers (n / ). both groups were matched with regards to known factors affecting intestinal microbial colonization: gestational age, type of delivery and feeding. results: colonization by enterobacteria and enterococci was not significantly different and occurrence of amoxicillin-resistant enterobacteria was similar ( / and / in groups a and b, respectively). however, the colonization by clostridia was modified: the number of newborns colonized was significantly less important in group a than in group b (group a: / and group b: / p b/ . ). conclusion: in our study, intrapartum antibioprophylaxis did not affect intestinal colonization by aerobes but reduced significantly colonization by clostridia, potentially anaerobic pathogens. impact of an antibiotic policy restricting the use of b-lactams and macrolides on the incidence of clostridium difficile associated diarrhoea in general medical, renal and elderly patients s . boswell tc a , pacey s b , broomfield s c , westmoreland d c , yates c c . a nottingham city hospital, microbiology, nottingham, uk , b nottingham city hospital, pharmacy, nottingham, uk , c nottingham city hospital, infection control, nottingham, uk the purpose of the study: to investigate the short-term impact of a new antibiotic policy for the treatment of urinary and respiratory infections on the incidence of clostridium difficile associated diarrhoea (cdad) in hospitalised medical, elderly care and renal patients. the results obtained: a policy restricting the use of b-lactams (except parenteral penicillin), and promoting alternative antibiotics including levofloxacin for pneumonia, and doxycycline for non-pneumonic respiratory infections, was launched in july . as a result there was a significant and sustained reduction in use of aminopenicillins, cefuroxime and macrolides, with a corresponding increase in doxycycline and levofloxacin. the incidence of cdad was determined during the st months of the new policy and compared to the last months of the old policy. the incidence of cdad fell from . to . per patients, and from . to . per in-patient days (p b/ . ). in contrast, there was no change in the incidence of cdad in other specialties (surgery, oncology etc.) that had not introduced the new policy. there was no change in the incidence of nosocomial bacteraemia with quinolone-resistant coliforms or mrsa, despite the increased use of levofloxacin. conclusions: hospital-wide reduction of b-lactam and macrolide use in medical patients can result in a significant and immediate reduction in cdad. longer follow-up will determine if this effect is sustained. use of imipenem/cilastatin i.v. (tienam i.v.) for the treatment of low respiratory tract infections in intensive care units s . izzo l a , orsetti r b , boschetto a a , binda b a , della casa u a , caramanico l a , la mazza a a . a department of surgery, universitá degli studi di roma 'la sapienza','p. valdoni', rome, italy , b s. camillo-forlanini, intensive care unit, rome, italy ventilator associated pneumonia (vap) is considered the most frequent infection in the intensive care unit (icu), occurring in Á/ % of patients intubated for longer than h besides nosocomial pneumonia is a common complication in the critically ill surgical or trauma patient. inadequate treatment can lead to the complications of acute respiratory distress syndrome (ards), empyema, and lung abscess. the most important aetiological agents both in vap and in pneumonia which arise as complication in surgical or trauma patients are bacteria, whit a marked predominance of staphylococcus aureus and pseudomonas aeruginosa . the authors present their experience ( cases) on the employment of imipenem/cilastatin i.v. (tienam i. v.) as initial empirical monotherapy at the dose of mg)/ /day or g)/ / day for the treatment of the serious lower respiratory tract infection in an icu. tienam is a well tolerated broad spectrum antibacterial agent that is effective against the majority of gram-positive and gram negative aerobic and anaerobic bacteria including most pseudomonas species. except one patient deceased for causes related to his very poor general conditions and three cases in which has been necessary the addition of an aminoglycoside, in all the other patients the imipenem/ cilastatin (tienam) monotherapy has shown satisfactory clinical and bacteriological responses. clinical auditing of the impact of recommendations on antibiotic treatment s . kinoo j a , david-ouaknine f a , hacquard b a , echard y a , decazes jm b . a centre hospitalier lagny marne la vallée, lagny sur marne, france , b hospital saint louis, paris, france the aim of this study was to assess the impact of curative antibiotic recommendations on suitable prescriptions at lagny-marne la vallée hospital (general hospital, beds). two prospective exhaustive audits were made (all complete hospitalizations, excluding psychiatry, february Á/may and ) of the detailed curative antibiotic prescriptions, before and after distribution of internal recommendations. the same methodology, designed by a multidisciplinary team, was used for both periods. the same antibiotics were available at the pharmacy. the prescriptions were assessed by an infectious diseases specialist and a pharmacist using pre-established criteria: literature recommendations ( audit), internal recommendations ( audit). six hundred and fifty-six prescriptions for patients were collected and analysed in , for patients in . exhaustivity of the recovered prescriptions was over %. patient characteristics, infection sites and microbiological findings were similar for both groups. suitable prescriptions were significantly increased ( Á/ %, p b/ . ). unsuitable prescriptions (economic reasons, too short or too long course, incorrect administration, or underdosage) were significantly reduced. prescriptions for incorrect indications were unchanged and necessary combined treatment not being prescribed, increased. local recommendations improved prescriptions, but efforts have to be done in order to go on the improvement of the practice behaviour. cost-effectiveness analysis of antibiotic therapy in hospitalized patients with copd exacerbations (ae-copd) s . beghi g, aiolfi s, maghini l, patruno v, aiolfi e. s marta hospital, pulmonary rehabilitation unit, a.o., rivolta d'adda, italy antibiotic costs represent a high burden of total drug costs for hospital administrations. a scientific approach considering also the economic aspects of each therapeutic decision may gain optimal treatment objectives at pondered costs. in our study we retrospectively evaluated the clinical effectiveness and costs of antibiotic therapy in patients with ae-copd. from to , our retrospective study results support previous pharmaco-economic considerations according which in choosing an antibiotic regimen for ae-copd we must take into consideration the expected clinical and microbiological results without forgetting to consider the economic burden of our decisions. significant increase in fungaemia due to non-albicans candida species s . shah pm. klinikum der j.w. goethe universitaet, schwerpunkt infektiologie, frankfurt am main, germany until , predominant candida species in blood cultures was candida albicans . it accounted for . % of all candida species cultured from blood. since then we have observed a gradual increase in number of non-albicans candida species. from , onwards nonalbicans candida species out-number c. albicans . this observation is especially important as non-albicans candida species are generally non-susceptible to azole derivatives and empirical use of azoles in suspected candidaemia should not be recommended. amphotericin b is uniformally active against almost all candida species. echinocandin may be an alternative. see figure below. a search for newer antifungal chemotherapeutics s . chakrabarty an a , dastidar sg b , saha b b , basu l b . a calcutta university, medical microbiology, calcutta, india , b jadavpur university, pharmaceutical technology, calcutta, india fungal infections due to the mucor-rhizopus (m-z) group present formidable problems due to lack of appropriate and effective drugs against them, as seen in increasing number of clinical situations; death due to mycoromycosis is nearly inevitable. we analysed the biological 'weak-spots' of the mucor-rhizopus group and attempted to devise suitable drugs using their weak-spots. we have noted that like many free-living fungi, the m-z fungi are facultatively chemoautotrophic (can grow on simple sources of carbon and nitrogen and a solution of mineral salts), like the human pathogenic chemoautotrophic nocardioform bacteria. we devised a minimal medium based on that of davis and mingioli, supplemented with simple chemical compounds as sole sources of carbon and nitrogen. the key chemical here was diphenylamine with trypan blue (dpa Á/tb) and other similar sources of c and n. we found that while media free of these chemicals (controls) allowed good growth of different strains of m-z fungi, a mixture of dpa Á/tb completely prevented their growth over a wide concentration range. experiments with immunocompromised mice showed that these drugs at the concentrations used are well tolerated; mice experimentally infected with several clinical isolates of m-z fungi and receiving these chemicals showed that these fungi could not grow in vivo. in vitro activity of newer fluoroquinolones against multi-drug resistant salmonella typhimurium s . nolones resistance is being also reported. we have studied the in vitro activity of b-lactams and fluoroquinolones against multi-drug resistant s. typhimurium from human sources. material and methods: fifty multi-drug resistant s. typhimurium were tested against cefazolin, cefuroxime, cefotaxime, cefepime, ofloxacin, levofloxacin, and moxifloxacin, by the agar dilution method according nccls guidelines. results and conclusions: all the strains were resistant to four or more of the following antibiotics: ampicillin, tetracyclines, chloramphenicol, streptomycin, sulphonamides and nalidixic acid. a high proportion of strains were intermediate or resistant to amoxicillin/clavulanate. we found no resistance to cephalosporins. nevertheless, % were intermediate to first and/or second gen. cephalosporins. cefotaxime and cefepime were the most active cephalosporins (mic : . mg/l). though increasing fluoroquinolones resistance has been described among this kind of strains, no resistance to fluoroquinolones was found here. levofloxacin was the most active fluoroquinolone (mic : . mg/l), followed by ofloxacin (mic : . mg/l) and moxifloxacin (mic : . mg/l). high rates of resistance to antibiotics by salmonellae from diarrhoeic children in zliten-libya s . ghenghesh ks a , ben ali m b , abuhelfaia a b , dufani ma a . a faculty of medicine, al-fateh university, medical microbiology, tripoli, libyan arab jamahiriya , b faculty of arts and sciences, el-ghomes university, biology, el-ghomes, libyan arab jamahiriya salmonellae are major bacterial cause of diarrhoea in libya particularly in children. included in the present study salmonella species isolated from children with diarrhoea in zliten city-libya. the children aged between a few days to years. the organisms were tested for their susceptibility to antibacterial agents using the disc diffusion method. of the isolates examined, ( %) were resistant to ampicillin, ( . %) to amoxicillin Á/clavulanic acid combination, ( %) to cefoxitin, ( . %) to chloramphenicol, ( . %) to doxycycline, ( . %) to gentamicin, ( . %) to nalidixic acid, ( . %) to trimethoprim Á/sulphamethoxazole and none ( . %) were resistant to norfloxacin. a strong relationship was observed between the availability of antibiotics in the pharmacies of the city and resistance of the isolated salmonellae to these drugs. the misuse of the antibiotics by the community may be an important factor (among others) in the emergence of these high rates of resistance by the salmonellae examined. effect of ceftriaxone along with probiotics administration on intestinal ecosystem and betalactamase activity s . bertazzoni minelli e a , benini a a , zoppi g b . a department of medicine and public health-pharmacology section, university of verona, verona, italy , b department of paediatrics, university of verona, verona, italy oral bacteriotherapy during antibiotic treatment is a much debated topic. aim: to study whether different probiotics can prevent imbalance of the intestinal ecosystem (dysbiosis) in children during therapy with ceftriaxone (cx). methods: fifty-one children (mean age . years) with febrile respiratory tract infections were treated with cx mg/kg/day iv, alone (therapy ) and along with the following preparations: saccharomyces boulardii ( ); enterococcus spp. ( ); lactulose ( ); l. casei gg ( ); l. rhamnosus , l. bifidus and l. acidophilus ( ); b. bifidum and l. acidophilus ( ); and a mixture of various lactobacilli and bifidobacteria at high concentrations ( ). faecal samples, collected before and after treatment, were analysed for microflora composition, cx concentration, and beta-lactamase (bl) activity. results: cx causes intestinal dysbiosis. no c. difficile was found. faecal bl increased after therapy in all treated groups. cx alone increased bl activity in % ( / ) of children (no activity before treatment); a higher incidence ( Á/ %) was found in groups and . after therapies , , , , and , bl activity was found in or more children. cx was detected in % of faecal samples. conclusions: the probiotics administration seems to protect against dysbiosis caused by cx and to contain the increase in faecal bl activity. the effects differ according to the probiotic administered and are peculiar to certain bacterial species. these preliminary data need further studies. comparative study of initial and acquired drug resistance in pulmonary tuberculosis in iran s . mansoori d, arami s, mirabolhasani z. nritld, infectious disease, tehran, islamic republic of iran purpose: resistant to anti-tuberculosis agents particularly multiple drug resistant (mdr) is a major obstacle in treatment tuberculosis in the world. between september and march for smear and culture positive pulmonary tuberculosis patients (old / , new / ) pretreatment susceptibility tests of isolated bacilli to inh, rif, emb and stm were performed by standard proportional method and the results were attributed to three groups: (i) newly diagnosed without any history of treatment; (ii) patients with history of treatment for one course; (iii) patients with history of treatment for two or more courses supposed to be mdr cases. the results were collected for each drug individually and different combinations of two, three and four medications. results: resistance to one, two, three and four drugs was significantly increased in group iii comparing to groups ii and i, also in group ii compared to group i. we observed a high rate of primary resistance to inh and stm in groups i and ii and a high rate of mdr (inh and rif resistance) in groups ii and iii. conclusion: the duration of bacilli exposure to antituberculosis agents in the past is a major factor in developing resistance. in contrast to who's guideline, due to high rate of primary resistance especially to stm in our area, we do not recommend addition of stm for treatment of patients whose initial four-drug regimens have been failed (group ii). donors, to understand host interactions with this bacteria, to develop new methods of diagnosis and define new vaccine candidates. nineteen tb patients and seven healthy donors were enrolled in french hospitals. cellular immune responses were evaluated by lymphoproliferation and ex-vivo quantification of specific th cells by elispot-ifn-gamma assays. four recombinant proteins of m. tuberculosis were tested: esat- , b, erp and tb b . and compared with tuberculin. we confirmed that b (but not esat- in our study) gives higher responses in tb patients compared to donors according to the results in proliferation assay (p / . ). in addition, frequencies of th cd specific cells for esat- and tuberculin were statistical different between the two groups (p / . and . , respectively). . % for b and . % for esat- of the patients tested were responders in elispot-assay versus . % for both in proliferation assay. for the new antigens erp and tb b . , no difference was observed between the two groups. in conclusion, b and esat- are recognised by a large number of our patients. they seem to be promising antigens for the development of new methods of diagnosis or for the development of new vaccines. erp and tb b . are not preferentially recognised by tb patients. other exported antigens will be tested. the incidence of tuberculosis (tb) is increasing worldwide. in recent some years, geographical differences in the incidence of tb in former yugoslavia have been observed. an important rise in tb cases was registered in the bordering region of bosnia. it is likely that poorer living conditions, influenced by war and emotional stress, may promote such rising incidence of tb. renal tuberculosis was diagnosed in patients (female , male ) from district brcko in bosnia, during the period of years, Á/ . at the same time none patient had active pulmonary tb lesions, fibrous lesions were noticed in patients, but we did not diagnose any signs of previous pulmonary tb in seven patients. seven patients developed relapse of renal tb after Á/ years of previous treatment. guided by clinical parameters, precisely done renal echosonography enabled early suspicion and searching for renal tb, by radiological and other methods. bacteriological diagnosis was performed by detection mycobacterium tuberculosis on loewenstein Á/jensen medium in patients. pcr as simple, fast and highly sensitive method enabled diagnosis in incipient stadium of disease, so antituberculous therapy could be instituted some months earlier. prompt diagnosis of renal tuberculosis (using pcr, besides standard methods) is necessary, otherwise delayed diagnosis may be dangerous. a study on resistance to first generation anti-tuberculosis drugs in mycobacterium kansasii s . mirsaeidi sm, farnia p, mohammadi f, mansoori sd, jabbari r, taghizadeh r, masjedi mr, velayati aa. national research inistitute of tuberculosis and lung diseases, infectious diseases and immunology (nritld ), tehran, islamic republic of iran purpose: this research has been performed to determine antibiotic resistance of atypical mycobacteria especially mycobacterium kansasi . results: twenty-three pigmented colonies which indicated atypical agents from nritld's mycobacterium culturebank were selected, they then underwent type identification and antibiogram for inh, rif, etb, stm. nine samples were m. kansasi and were other non-mtb, was m. gordonei , m. xenopi , mac, m. bovis , m. tererra , m. asiatioum , m. marinum and . mean age in m. kansasi cases was '/ . year and in non-kansasi cases '/ . year and in whole society ntm was . '/ . . frequency of resistance in kansasi group were to inh ( %), to rif ( %) to etb ( %), to stm ( %) and prevalence of mdr was ( %) and in non-kansasi group frequency of resistance were ( %) to inh, to rif ( %), to etm ( %) and to stm was ( %), to mdr was ( %). conclusion: a significant difference was seen between the age groups of patients who are affected with m. kansasi and non-kansasi (p b/ . ), also in frequency of resistance to first generation anti-tb drugs. m. kansasi is detected as the most common atypical mycobacterium agent in pulmonary infections and attention to antibiogram is recommended before treatment. buruli ulcer caused by mycobacterium ulcerans , is the third most common mycobacterial after tuberculosis and leprosy in west africa. nowadays, the only effective treatment is surgery. it consists in a large excision of the lesions, often followed by a skin transplant. in this study, the effectiveness of rifampin, amikacin and their combination were estimated in the treatment of mice, which were infected experimentally by m. ulcerans . after weeks of treatment with rifampin, amikacin or their combination, no more viable bacilli were found in infected tissues. the animals were kept for other months. among the mice treated with rifampin alone, two mice out of relapsed. the minimal inhibitory concentration of these isolated strains went from . to mg/ml. the dna sequence, obtained from a -pb of the rpob gene from these strains, showed a missense mutations, which affect a ser- replaced by a phenylalanine. this modification on the gene leads to an important inefficacy of treatment when rifampin was used alone. this study showed that rifampin and amikacin have a bactericidal action on m. ulcerans and that a combination of these antibiotics is necessary to avoid the selection of resistant mutants. histopathologic and electron microscopy studies of a severe isolated hiv enteropathy detected in an aids presenter. favorable response to haart introduction or . manfredi r, calza l, chiodo f. university of bologna, infectious diseases, bologna, italy advanced hiv infection was detected in a heterosexual female with a -year history of chronic diarrhoea and severe wasting, as expressed by a body weight of kg, a cd '/ count of cells/ml, and a plasma viraemia of . million copies/ml. a malabsorption syndrome was confirmed by d-xylose test, but repeated pathogen search tested negative at stool examination and light microscopy, scanning electron microscopy (em), and transmission em study of enteric mucosa. em assays detected an ultrastructural modification of duodenal mucosa never reported to date: an extensive thinning of enterocytic microvilli, disappearance of glycocalix, and large vacuolization of the enterocyte cytoplasm. two weeks after starting an indinavir-based haart, diarrhoea disappeared and our patient significantly gained body weight: kg after months, kg after , and kg after year, paralleling a cd '/ increase to cells/ml, and undetectable hiv viraemia. the subsequent -year follow-up confirmed absence of gut disturbances, a stable body weight, a cd '/ count of Á/ cells/ml, and hiv viraemia persistently b/ copies/ml. repeated endoscopy and related histopathologic and em assays documented a notable improvement of mucosal damage, with complete cure reached after years of haart. a direct intestinal localization of hiv may be responsible for severe diarrhoea, malabsorption, and wasting, though the morphological features of hiv enteropathy are still unclear. haart acts favourably also against isolated hiv-related enteropathy. kaposi's sarcoma in a non-hiv immunocompetent adult: relapsing due to the development of a squamus cell carcinoma or . sioula e a , magira ee a , georgopoulou c a , rontogiani d b , gounaris t a . a evagelismos, internal medicine and infectious diseases, athens, greece , b evagelismos, pathology, athens, greece a -year-old heterosexual hiv negative girl was diagnosed with cutaneous kaposi's sarcoma. the disease was started years earlier with the appearance of lesions on the left feet and on the right knee. absolute number of cd and cd were and cells/dl, respectively with a decreased lymphocyte proliferation. human herpesvirus type had been detected in biopsy specimens and she placed on recombinant interferon alpha- b. follow up few months later the lesions decreased in size. two years after the onset of the disease the patient readmitted because of a mass on the left paratracheal region along with mediastinitis. her body temperature was increased. the patient underwent thoracic ct scan, which demonstrated mediastinal well defined soft tissue infiltration associated with mediastinitis and a well-defined mass in the left paratracheal region. the mass biopsy revealed squamous cell carcinoma. several violaceus lesions were observed on the arms, hands and face. severe bilateral lymphedema of the legs with a reddish papules nodules and tumors from . to cm in diameter on the soles, toes and calves were present. this case illustrates the significant relapsing of the cutaneous kaposi's sarcoma soon after the appearance of and the carcinoma and the mediastinitis. a -year-old male patient with insulin-dependent diabetes underwent cardiac surgery for aortocoronary bypass years ago. two weeks after surgery he developed mediastinitis and sternal osteomyelitis caused by methicillin-resistant staphylococcus aureus (mrsa). twice, revisions and plastic surgery for sternal osteomyelitis were performed. the patient received initially treatment with vancomycin. then the patient received intravenous outpatient treatment with teicoplanin for weeks followed by by treatment with fusidic acid and then trimethoprim/sulfametrol. the fistula was closed. four months later he presented again with substernal pain and purulent discharge. the culture revealed the growth of staphylococci which were first mistaken for coagulase-negative staphylococci. after closer investigation these staphylococci were identified as small variant mrsa. computer tomography (ct) revealed multiple mediastinal abscesses. the patient was treated with intravenous linezolid mg bid for days and then switched to oral linezolid mg bid. the oral therapy was pursued for weeks under close surveillance. the patient improved substantially, the purulent discharge disappeared. the mediastinal abscesses were not detected any longer by ct at the end of treatment. the treatment with linezolid was well tolerated. platelets decreased initially but rose to normal values without treatment modification. nosocomial pneumonia due to stenotrophomonas maltophilia in a profound granulocytopenic patient hospitalized for community-acquired staphylococcus aureus severe sepsis or . radulescu a a , sasca n b , lupse m c , tatulescu d c . a university of medicine and pharmac, epidemiology, cluj-napoca, romania , b the teaching hospital of infectious diseases, laboratory, cluj-napoca, romania , c university of medicine and pharmacy, infectious diseases, cluj-napoca, romania objective: to present the diverse opportunistic infections in immunocompromised patients and treatment difficulties. findings: a -year-old women was admitted to the teaching hospital of infectious diseases cluj with a -day history of fever, myalgia, lumbar pain, hemorrhage syndrome. severe sepsis was diagnosed and the conditions that evolved in it were paronychia in a patient with chronic leukemia having prolonged and profound granulocytopenia due to aggressive treatment with ifn. the condition at admission was critical due to trombocytopenia ( b/ platelets/ml) and hemorrhage syndrome. the evolution was favorable under antimicrobial treatment (imipenem), blood and platelet transfusion, intravenous immunoglobulins, granulocyte colony-stimulating factor, antifungal prophylaxis and supportive care. blood and pus cultures revealed mssa. in the th day of hospitalization she developed bronchopneumonia and respiratory failure. the sputum culture was positive for stenotrophomonas maltophilia susceptible to ceftazidime, fluoroquinolones. treatment was unsatisfactory until introducing ticarcilline Á/clavulanate and ciprofloxacine. she had uneventful recovery despite remaining granulocyto-trombocytopenic. conclusions: treatment of infections with emerging agents in immunocompromised patients is difficult, guidance by results of susceptibility testing being misleading with a poor correlation between the tests and treatment outcome. early disseminated listeriosis in a liver transplant recipient (ltr): a rare case due to an in vitro multiresistant strain or . manfredi r, de ruvo n, vivarelli m, bellusci r, montalti r, la barba g, abtueli aden a, cucchetti a, attard l, calza l, cavallari a. university of bologna, infectious diseases, bologna, italy a ltr receiving cyclosporin, azathioprine and steroids, developed an extraordinary episode of sepsis and pleural effusion due to a multiresistant listeria monocytogenes (lm) isolate. a lm strain serov. showing the same, extensive resistance pattern (all penicillins and stand nd-generation cephalosporins), was isolated from multiple blood cultures and pleural fluid weeks after surgery, while stool exam was negative. our p spent her life in countryside and bred some animals, but denied consumption of uncontrolled food. iv cotrimoxazole administration achieved a complete clinical and microbiological cure in days. underlying immunodeficiency may prompt unusual/severe lm infection, but because of its usual community-acquired origin, lm disease remains infrequent in hospitalized p. only seven anecdotal reports of lm infection were described in ltr, Á/ , all but occurring months Á/years after surgery. an early respiratory and systemic infection caused by a community-acquired lm strain which proved resistant to first-choice antibiotics, but had a favorable response to cotrimoxazole (used only once in a ltr with lm sepsis), characterized our episode. an epidemiological survey retrieved the possible source of this usually community-acquired infection. lm should be regarded as an emerging opportunistic pathogen in ltr, and specific risk factors should be seeked. when immunodeficiency is of concern, the unpredictable sensitivity of lm should prompt in vitro assays to adjust antimicrobial therapy problems for discussion hbv Á/hcv and liver carcinogenesis: where does the viral influence end? or . pappas ga. university hospital, internal medicine, ioannina, greece hepatocellular carcinoma (hcc) is a major clinical problem worldwide, usually evolving over a long-standing liver pathology, in the latter stages in the form of cirrhosis. hbv and hcv chronic infection is a common etiology of cirrhosis, and hence, hcc. a number of studies have attempted to clarify the role of these viruses into the progression towards hcc. does their end in the stage of cirrhosis? is progression towards hcc independent of the etiology of cirrhosis (since alcoholic cirrhosis also proceeds to hcc)? do the trials with interferon alfa for patients with hbv or hcv cirrhosis exhibit a favorable result due to the antiviral properties of interferon, or is interferon exhibiting anti-oncogenic potential?, and is hcc cytokine and hormone sensitive (view ongoing trials with somatostatine analogues versus hcc)? (hence, if we treat alcoholic cirrhosis patients with interferon could we have a favorable response?). which patients with hbv or hcv cirrhosis are eligible for interferon treatment?: interferon treatment is a potential hazard for those with thrombocytopenia. how ethical is it to conduct a randomised trial where one leg of cirrhotic patients is left without antiviral therapy? and on the basis of which classification system should the two legs of such a trial be separated? moreover, do viral proteins with oncogenic potential exist (the controversy over the recently discovered hbv protein is still, unresolved)? a major topic awaiting for a major debate. to assess the role of hiv-associated campylobacteriosis (c) according to haart availability, patients with positive culture were identified since . compared with the Â/ hiv-infected p followed in the last decade, no epidemiological differences were shown, save a greater sexual exposure to hiv (p b/ . ). the introduction of haart caused a drop of frequency of c (from . to . episodes per p-year; p b/ . ), and modified clinical features, with disappearance of dissemination and mortality, reported in and patients before (p b/ . ). hiv-related immunodeficiency and disease stage were significantly related to c features before and after haart availability: p b/ . for cd and neutrophil count, p b/ . for aids diagnosis. most cases ( ) were community-acquired, but alimentary or environmental risk factors were never found. ten patients received cotrimoxazole prophylaxis (nine before ; p b/ . ), while no relationship occurred with steroid or antibiotic use, caused cases out of . a % sensitivity was found to quinolones, followed by cephalosporins ( . %), gentamicin ( . %), macrolides ( . %), and cotrimoxazole ( . %). a Á/ -day antimicrobial therapy cured p , but relapses caused by similar strains occurred in patients within Á/ weeks, all in the pre-haart era (p b/ . ). c still occurs in the haart era, probably due to its varied mode of transmission. the frequency of c is greater in hiv-infected patients, but less frequent visceralization, recurrences, and mortality characterized the haart era. objective: to determine the incidence and risk factors for nosocomial viral respiratory infections (nrvi) and involvement of human coronaviruses (hcov) in a neonatal and pediatric intensive care unit. methods: prospective observational study. nasal samples were obtained by cytological brush at admission and weekly thereafter for all hospitalized infants. nasal samples were taken monthly from staff. virological studies were performed, using immunofluorescence for respiratory syncitial virus (rsv), influenza viruses, paramyxoviruses, and adenoviruses; both immunofluorescence and rt-pcr were used for hcov detection. results: during , hcov related nrvi were detected in nn and six in children. three hcov-related outbreaks were observed (february, august and december), associated with a high prevalence of infection in staff. during august outbreak, hcovinfected nrvi were detected over hospitalized infants. seventy-five of hospitalized preterm nn with gestational age under weeks and . % of staff members were infected. risk factors for nrvi in nn were birth weight, gestational age, ventilation, oxygenation and hospitalization length. ninety-two percent of infected preterm nn were symptomatic, mainly with bradycardia and respiratory worsening. conclusions: these data provide additional evidence for a significant role of hcov in nrvi occurring in hospitalized preterm nn. strain typing and screening of dna targets to assess echinococcus sp transmission in new and old geographic endemic foci or . bart jm a , piarroux r a , dia l b , benchikh-elfegoun mc c , vuitton da a , bardonnet k a . a serf, parasitology, besancon, france , b national centre of veterinary study, parasitology, nouakchott, mauritania , c university of mentouri, parasitology, constantine, algeria purpose: cystic echinococcosis is due to echinococcus granulosus. parasite cycles depending on the main intermediate host species involved in different foci have been described promoting mixed infection in the same definitive host. strain typing is a tool to identify the main intermediate host involved via the dogs in the human infection route and to focus the control measures. many dna targets have been used to compare samples and to access the parasite cycle in different countries. but no study has compared the value of each of these targets. eight targets have been tested in mauritania where echinococcosis is an emergent disease, and in algeria where strain typing has never been done. thirty-five cyst samples from human, ovine, camel and bovine have been tested with six nuclear and two mitochondrial targets. results: the two mitochondrial targets and four out the six nuclear targets have allowed to discriminate the different foci. two strains have been found infectious to human : the 'sheep' strain in algeria and the 'camel' strain in mauritania. conclusion: although overlapping geographically sometimes, this raises the question of the respective genetic evolution of the different strains and of their involving in human infection. alveolar echinococcosis in france: an update or . bardonnet k, bresson-hadni s, bartholomot b, gérard a, watelet j, beytout j, saurin jc, piarroux r, vuitton da, who centre collaborating for prevention and treatment of human echinococcosis, university of franche-comté, besançon, france introduction: the highest prevalence rate for alveolar echinococcosis (ae) in europe has been found in france. in , a french observatory of human ae was done in order to get data that could be used to evaluate presentation, evolution and management of ae. material-methods: french cases were collected for the period Á/ . registration of every case was performed with the subject's agreement. a questionnaire was filled in by referring to the patients' medical files or to practitioners or to patients themselves. completeness of the collection of cases was ensured by multiplying the sources of information. results: two hundred and sixty nine french patients were registered. sex ratio averaged . mean age at diagnostic was . years. . % of diagnosis was performed in 'echinococcosis free' french areas. symptoms, but not always specific liver symptoms, were present at diagnosis in . % of cases. the liver was the main location of lesions in . % of cases. a wide spectrum of management of the patients was observed, accounting for regional differences. conclusion: this french observatory of human ea will facilitate a better management of the disease at the national level. it shows new epidemiological trends, and especially an extension of the endemic area. can coins and paper currency transmit bacillus anthracis ? or . ghenghesh ks. faculty of medicine, al-fateh university, medical microbiology, tripoli, libyan arab jamahiriya anthrax is an often fatal bacterial infection caused by bacillus anthracis . recent events that began in september in us has gained the organism worldwide attention and heightened awareness of and concern about anthrax. many cases of anthrax with a number of deaths have been reported as a result of contact with envelopes, sent through postal mail, containing b. anthracis endospores. a number of studies have shown that currency is colonized with bacterial organisms, that include enteropathogens (e.g. shigella sp.), other enteric flora (e.g. escherichia coli ) and potential pathogens (e.g. staphylcoccus sp. , pseudomonas sp. and bacillus sp.). furthermore, methicillin-resistant s. aureus (mrsa) isolates that produced enterotoxin (seb) and toxic shock syndrome toxin- also been reported. all of these studies do agree on that currency may be considered as a method of spreading potentially pathogenic and pathogenic bacteria in the community. therefore, currency could also be a vehicle for spreading other highly pathogenic organisms that include b. anthracis . in addition, the introduction of the 'euro' could also allow such bacteria greater freedom to travel across the euro zone. the threat of using currency, particularly paper notes, in spreading lethal organisms should be investigated and proper measures to prevent the use of such a method by terrorists should be implemented. salvage of temporary femoral catheters for haemodialysis using antibiotics in ambulatory patients or . gerasimovska v, oncevski a, dejanov p. department of nephrology, clinical centre, skopje, the former yugoslav republic, macedonia the stay of femoral catheters (fc) for haemodialysis is typically short-term for several days. we used fc as a temporary vascular access (va) for a longer period of time in outpatients going on regular ambulatory haemodialysis, who had a problem with their permanent access. we analysed patients who were discharged from hosptal with fc. duration time of fc was between and days (average . days) with cummulative total of days. the incidence of bacteriaemia was . episodes/ catheter days. in six patients we had signs of infection, so according to our protocol we took blood cultures from peripheral vein, and from catheter (at same time) and started with antibiotic therapy (ab) systemically and locally (ab was 'locked' in catheter) with different duration of time. dominant microorganism was staphylococcus coagulasa negative, and much less staphylococcus aureus , and enterococcus.ab that were frequently used were: cefotaxim, vancomycin and ciprofloxacin. at one of six patients we removed catheter at once without trying to save the catheter. catheter tip was sent for microbiological analysis too. criteria for catheter-related bacteriemia (crb) was found in only one patient, and for possible crb in five patients after we removed the catheters. in the absence of clinical signs of infection, ab treatment was not provided for positive tip culture alone or for positive blood culture of the catheter with negative blood culture from peripheral vein. advances in meningitis education or . holt de a , tait mi b , cavanna al b , worgan-brown s a , hart b a . a the meningitis trust, stroud, uk , b the computer-aided learning unit, school of health science, university of wales, swansea, uk background: meningitis remains an important cause of death worldwide despite improvements in diagnosis, treatment and prevention. clinical and lay awareness of the disease relies on education, however educational delivery has changed and the introduction of material suitable for computer and internet application is now necessary. we have developed educational material on cdrom and on the internet applicable both at tertiary university and secondary school level. application: a computer-aided learning program on cdrom, covering all aspects of meningitis has been produced. it is suitable for undergraduate teaching of healthcare professionals from student nurse and doctors to pharmacists. in order to reach school children in a form acceptable to both pupils and teachers, we have developed a curriculum-linked website. these applications are simple to use and can be incorporated into existing courses of study, so that issues raised can be discussed with tutors and group peers. comment: the introduction of new methods of teaching and learning mean that compatible educational material must be produced. we believe that these applications, focusing on meningitis, are the first of their kind and that they offer tutors the opportunity to progress their teaching of the disease both in methodology and content. brivudin compared to famciclovir for improved therapy of herpes zoster: effects on acute disease and postherpetic neuralgia or . potentially treatment-related adverse events occurred in . % of the brivudin recipients and in . % of the famciclovir recipients (p / . ). conclusions: in zoster patients ]/ years, brivudin )/ mg and famciclovir )/ mg showed equivalent effects on prevalence and duration of phn. brivudin is as effective as famciclovir in stopping viral replication in acute herpes zoster. brivudin offers the advantage of a once daily dosage regimen while being as well tolerated as famciclovir. activity of complexes of pt(ii) and pd(ii) with pyridine- -carbaldehyde thiosemicarbazone (hfotsc) (acta virol., , , ) with selectivity index (si) . times higher than that of acyclovir (acv). in order to evaluate virus specific response and structure Á/activity relationships we continue our investigations with three pt(ii) and three pd(ii) complexes. the activity was evaluated against sensitive to acv hsv (strain bja) and resistant strains r- (hsv ) and pu (hsv ) and compared to that obtained against strain victoria (hsv ) infection. si was indicative for activity. the virus specific response was demonstrated by the fact that viruses sensitive to acv were also sensitive to pt(hfotsc) ]cl , while acv resistant viruses were sensitive to [ptcl(fotsc) ]. the structure Á/activity relationship was proved by the fact that the less active against hsv infection was [pd(fotsc)]. influenza diagnosis, treatment, and the impact of new antivirals on current treatment behaviours during influenza outbreaks or . schaetz l a , sessa a b , a hoffman-la roche f. basel, switzerland , b italian college of general practitioners, italy introduction: annual influenza epidemics severely affect individuals, families, health care systems and society. the availability of new and specific antivirals provides an opportunity for better management of influenza. methods: during the / and / influenza seasons, physicians ( Â/ /country) and public ( Â/ /country) in the usa and europe were interviewed to determine perceptions of influenza and behaviours for its treatment. results: patients recognise influenza illness as severe and identify it by symptoms of fever, muscle aches/pains and cough. physicians use these symptoms to diagnose influenza clinically ( % fever, % muscle aches/pains, % cough); their main treatment objective being to reduce complications. antibiotics for influenza treatment are broadly recommended/prescribed by about % of european physicians, whereas currently available antivirals are only recommended by %. the recommendation of antivirals by us physicians increased from % (season / ) to % ( / ) and markedly decreased antibiotic use (from to %). experience from the two influenza seasons shows that influenza antivirals are only used while the virus is circulating and that the volume of use is proportional to the size of the outbreaks. conclusions: experiences in the usa show that with prompt outbreak information antivirals can be used appropriately in times of influenza activity. influenza treatment with oseltamivir: costs and benefits for the individual as well as for society or . objective: to evaluate the effects of treatment of influenza with antivirals (oseltamivir) on health outcomes and costs to patients and society. methods: based on clinical trial data and data from the literature a simulation model has been developed. the underlying clinical pathway covers morbidity and mortality due to influenza and its specified complications. health outcome data and costs were attached to events in the model. the model compares various scenarios, which are defined by treatment schemes within defined populations and other parameters. application of the model is shown using uk unit cost data simulating an otherwise healthy adult population comparing oseltamivir with usual care. results: early treatment results in reduced morbidity, which translates into faster recovery and return to normal activities ( . days). lower morbidity and mortality make this a cost-effective intervention from a societal perspective. the analysis covers more than different scenarios and the incremental cost effectiveness ratios will be discussed. conclusion: antiviral treatment appears to be effective in terms of health outcome and cost for otherwise healthy adults from the perspectives of both the individual patient and society. however, this effect is very sensitive to time when treatment is started and the accuracy of the diagnosis of influenza. oseltamivir is well tolerated by all patient groups or . thakar b a , dutkowski r b , froelich e c , gilbride j a , ward p a . a roche global development, welwyn, uk , b f hoffman-la roche, nutley, usa , c f hoffman-la roche, basel, switzerland introduction: oral oseltamivir, the ethyl ester pro-drug of a potent inhibitor of influenza virus neuraminidase, is licensed for the treatment and prophylaxis of influenza in the usa. patients and methods: safety data [adverse events, laboratory safety evaluations] derived from clinical trials involving !/ subjects (including Â/ children and Â/ high-risk adults) and healthy volunteers in a large study investigating ecg parameters. spontaneous event reports from medwatch or yellow-card reports following use by Â/ individuals worldwide. an observational case-control study of !/ subjects with influenza-like illness treated with oseltamivir. results: oseltamivir was well tolerated in clinical trials; drug-related side-effects were limited to transient gi effects occurring in / : exposed individuals. these resolved spontaneously and caused drop out in b/ % of treated subjects. no effects on ecg parameters were noted at doses ]/sixfold above the licensed regimen. oseltamivir had no adverse effects on pulmonary function. no additional effects were identified among high-risk adults or children, or following prolonged dosing for prophylaxis. occasional reports of liver dysfunction have been documented post-marketing but causal association has not been established. conclusions: oral oseltamivir is an effective and safe antiviral suitable for influenza management in all patient groups. the decision to stop the vaccination against smallpox and the loss of specific immunity of a high proportion of the population made apocalyptic the perspective of a natural or provoked re-emergence of smallpox. therefore, it is important to improve the current capacities to prevent or to treat the orthopoxvirus infections. uracil dna glycosylase (udg) is one viral enzyme indispensable to the replication of poxviruses. udg of the copenhagen strain of vaccinia virus (vv) was characterized with the aim of defining specific inhibitors susceptible to be used as a new class of active antiviral substances on the viruses of the orthopoxvirus genus. the activity of this enzyme was analysed in real time, in an original method, on a pcr quantitative instrument by digestion of amplified dna revealed by fluorescent intercaled molecules. this technique was used to screen and select several active antiviral substances on udg. moreover, the antiviral activity was estimated by the cytopathic effect of the vv on infected vero cells. the cytotoxicity was determined by inhibition of trypan blue exclusion. the specificity of action of each tested compound was estimated by the selective index ( % cytotoxic dose/ % effective dose). two antiviral compounds were selected for their inhibitory effect on udg activity and on vv replication in vero cell culture : ('/)- -iodo- ?-deoxyuridine and -chlorouracil. these compounds are candidates for the chemotherapy of poxvirus infections. objective: to study the efficacy and tolerance of russian antiviral drugs produced from dna in a limited resources context. results: the drug derinat was produced from salmons' milt. mm of dna was Á/ kda, hyperchrome effect !/ %, protein content b/ . %. the conjugation of the dna with fe '/ resulted in a new drug named ferrovir which influences dna and rna synthesis during early stages of hiv- replication by blocking the virus's action on cells' metabolism and reduces cytomegalovirus titre in fibroblast cells for . Á/ . ig tcid . a protective effect of ferrovir against fatal herpes encephalitis mice was found. the drugs are not toxic. ic !/ mg/ml. ec of ferrovir against hiv- was mg/ml. in limited clinical trials patients received mg of drugs twice daily ( Á/ days). administration was well tolerated and no side effects were observed. derinat in . % cases of herpesvirus infection ( patients) improved the healing and shortened duration of illness. hiv-infected patients ( ) treated with ferrovir showed sustained, elevated cd '/ counts and a significant reduction in hiv- viral load (median . ig). the apparent remission was found in patients with concomitant hiv and herpes virus infection. conclusions: antivirals show good antiviral potency against rnaand dna-viruses; are well tolerated by patients and are useful in case of mixed infections; low price makes them accessible to populations with low financial resources. ortho total hcv core antigen assay can aid early prediction of response in patients treated with interferon/ribavirin or . lunel f a , veillon p b , payan c b . a ahu angers, laboratoire de bacterio-virologie, angers, france , b chu angers, laboratoire de bacterio-virologie, angers, france aim: evaluate the predictive value of total hcv core antigen assay and viral kinetics in patients with chronic hcv. methods: one hundred and twenty two patients infected by genotype , , or pretreatment viral load (bdna . , chiron) !/ meq/ml, with no previous treatment, received mu interferon (ifn) during months (m). ribavirin was given with ifn after months therapy, for months in patients with detectable rna. viral load was expressed as log (ui/ml) and hcv ag as log (pg/ml)/ ). results: pretreatment ag values were correlated with viral load (r / . ). we observed a rapid decrease of ag ( . log pg/ml) and viral load ( . log ui/ml) after m in sustained responders (sr). in patients who relapsed (rr) after ifn alone, the fall was less important ( . log pg/ml, . log ui/ml) during m . in sr and rr to combination therapy, the decrease of ag and viral load at m was, respectively, (ag: . and . log pg/ml; rna: . and . log ui/ml). we did not observed significant variation of ag and viral load in nonresponders. the negative predictive value of hcv rna and ag after m of treatment were %, and positive predictive values were and %. after month of ifn alone, the hcv ag decrease was highly predictive of sr, correlated with rna negativation and early reduction of hcv rna ( !/ log). conclusion: early measurements of total hcv core antigen are useful to predict long-term response to treatment. lamivudine in the treatment of acute hepatitis b or . vincenti a, meini m, luchi s, de gennaro m, ricciardi l, moneta s, scasso a. infectious diseases department, infectious diseases, lucca, italy acute hepatitis b is a self-limiting infection, but in some cases its course may be particularly severe. we report a case of a -years-old woman affected by acute hepatitis b treated with lamivudine. on admission in the hospital the alanino-aminotransferase was u/l, the aspartate-aminotransferase u/l, bilirubin , mg/dl, hbsag, hbcigm and hbeag were positive, hbv dna was . copies/ml. during the following days, the levels of ast and alt gradually rose; on the th day prothrombine time was %, bilirubin mg/dl and the patient developed signs of encephalopathy. four plasmapheresis were practiced without benefit, so the patient was treated with lamivudine, mg/day. after days of therapy, lamivudine was discontinued because of the appearance of diffuse maculopapular rash. at this time the results of liver function tests were normal; after four months hbsag and hbv dna were no longer detectable. in our patient lamivudine prevented an acute hepatic failure. our experience suggests a promising role of lamivudine in the treatment of acute hepatitis b, but how long such therapy have to be practiced and in which patients? prospective, controlled, clinical studies using lamivudine in patients with acute-hepatitis b are necessary. the cost-effectiveness of amantadine versus symptomatic care in the treatment of influenza or . morris s a , carman wf b , barber j c . a city university, london, uk , b west of scotland specialist virology centre, glasgow, uk , c alliance pharmaceuticals, chippenham, uk aim: to assess the cost-effectiveness of amantadine versus best symptomatic care in the treatment of influenza in the uk. methods: we constructed an economic model populated with parameters from the published literature. the model structure is the same as that used in the economic evaluation of zanamivir published by the national institute for clinical excellence in the uk. we conducted a cost-utility analysis (incremental cost per qaly gained) of amantadine versus best symptomatic care. the analyses are conducted for all adults (average-risk group) and the at-risk population (high-risk group), based on the prevalence of influenza over an average season and when the virus is circulating. the perspective is that of the nhs. results: in the average-risk group the incremental cost per qaly gained of amantadine relative to best symptomatic care is uk£ , during an average influenza season and uk£ , when the virus is circulating. for high-risk individuals the figures are uk£ , and uk£ , , respectively. the results are sensitive to the hospitalisation rate. conclusions : if the threshold for cost-effectiveness is £ , per qaly gained amantadine represents value for money in the treatment of influenza in a variety of scenarios, including the baseline for both average-risk and high-risk groups when the virus is circulating. background: surveillance studies all over the world have revealed an extraordinary increase in the prevalence of penicillin resistant streptococcus pneumoniae . the newer quinolones are believed to have broad activity against s. pneumoniae . methods: a total of penicillin resistant clinical strains isolated from patients at hacettepe children's hospital, ankara, turkey between and were tested for their in vitro susceptibility to various antibiotics that are commonly used in the treatment of respiratory tract infections. the minimum inhibitory concentrations (mics) of the penicillin, amoxicillin/clavulanic acid, doxycycline, azithromycin, clarithromycin, ceftriaxone, ciprofloxacin, levofloxacin, moxifloxacin and gemifloxacin were determined using the nccls recommended procedure for e -test. results: the range of mics, mic and mic values for all agents tested against the strains are shown in the table. gemifloxacin and moxifloxacin had the highest in-vitro activity among the quinolones tested. all strains tested were susceptible to b/ . mg/ml gemifloxacin, b/ mg/ml moxifloxacin and mg/ml levofloxacin. conclusions: there is some degree of resistance to all the drugs except the newer quinolones which were active against all isolates studied. purpose: stenotrophomonas maltophilia prevalence is growing, mainly in some hospital areas. s. maltophilia is frequently multidrug resistant. fluoroquinolone (fq) resistance varies from one to another study, but in whole resistance is moderate to high. gyra and parc qrdr partial codes have been recently described. we have studied correlations between fq-resistance and mutations in these sequences in s. maltophilia clinical strains. material and methods: gyra and parc qrdr regions from six fqresistant and two fq-susceptible s. maltophilia clinical strains were amplified and sequenced. mics of ciprofloxacin (cfx), gatifloxacin (gfx) and clinafloxacin (cnfx) were determined by the agar dilution method, according guidelines defined by nccls for p. aeruginosa . results and conclusions: mics ranges of cfx, gfx and cnfx for resistant strains were Á/ , Á/ and . Á/ mg/l. susceptible strains had mics of cfx, gfx and cnfx of , . and . Á/ . mg/l, respectively. most susceptible and resistant strains had no significant mutations in the fragments sequenced. only one resistant strain (mic of cfx mg/l) and one susceptible strain (mic of cfx mg/l) had a significant gyra mutation, the same in both strains (ile Á/val). thus, fq resistance in s. maltophilia shall derive from changes in other areas in the topoisomerases or probably from other mechanisms of resistance, such as efflux pumps. purpose: corynebacterium urealyticum is the cause of encrusted cystitis and other inespecific utis and systemic infections. it is frequently multi-drug resistant, with a high rate of resistance to fluoroquinolones (fq). the mechanisms of resistance to fqs have not been described in c. urealyticum . we describe the c. urealyticum parc gene qrdr region and its relationship with quinolone resistance. materials and methods: the activity of ciprofloxacin (cfx), levofloxacin (lfx), gatifloxacin (gfx), clinafloxacin (cnfx) and moxifloxacin (mfx) against c. urealyticum clinical strains was determined following nccls guidelines for enterococci. we amplified and sequenced their parc qrdr by standard methods. results and conclusions: five strains ( . %) were cfx-susceptible (mic . Á/ . mg/l), had mics Á/ mg/l and ( . %) were highlevel cfx-resistant (mic Á/ mg/l). cnfx was -fold more active than cfx. mfx and gfx had mics of and mg/l. all the strains, including the type strain, showed a c to t change at the position referred to wild type s. aureus parc gene, leading to a ser- -phe change, described as the main parc change in fq-resistant s. aureus . this finding suggest that this mutant sequence, as compared with parc sequences from other grampositives, might be the wild-type for this species, and might explain in part its high resistance rate, and its apparent lightness to develop high-level resistance. purpose: during routine surveillance, we identified ciprofloxacinresistant (mic!/ mg/l) pneumococcal isolates and compared clinical details and resistance patterns. results: they were isolated from sputa ( ) and blood cultures ( ) from adults, most with heart or lung disease. hospital admissions were common; half had been inpatients in the previous months. nineteen patients received quinolones in the preceding months, in part reflecting the local policy (introduced in ) of penicillin and ofloxacin for first line treatment of pneumonia. thirteen patients had radiological signs of pneumonia and were pyrexial with raised inflammatory markers. agar dilution mics for quinolones, including norfloxacin with and without reserpine, penicillin and erythromycin were performed. an increase in norfloxacin mics was noted over the period ( Á/ mg/l) to ( mg/l). fluoroquinolone efflux was suggested in three isolates. resistance to moxifloxacin (mic Á/ mg/l) was noted from onwards. all isolates were serotype v and resistant to penicillin (mic!/ . mg/l). thirty-one were resistant to erythromycin (mic!/ mg/l). conclusion: the policy of using quinolones may have contributed to the development of quinolone resistance and this cluster of isolates. the increasing levels of quinolone resistance observed raise concerns about the future use of newer quinolones for the treatment of respiratory infections. s. maltophilia has emerged in the last years as an important nosocomial pathogen, inherently resistant to most of the antimicrobial agents. new quinolones has been proposed as a treatment of choice because their enhanced activity, but several parameters (t a , atmosphere, method) can affect the results of mics. methods: we have performed mics using two different methods (agar dilution and microdilution) and different conditions: and c of temperature; atmosphere of o and co , and incubation times of , and h. a total of strains were assayed with nine quinolones following standard nccls. comparisons were made between results with Á/ and Á/ h using the x -test (a / . ). results: no differences were found between Á/ and Á/ h results with agar dilution, except with atbs in the case of mics at c co . on the contrary, almost all the atbs showed significant differences in the results with and h using microdilution method, at any condition of ta or atmosphere. comparison of mics (p values, significance level / %) with incubation times of and h at different procedure conditions. the incubation time is a parameter that seems to affect significantly the results of mics of quinolones when microdilution method is used, whereas only few differences can be encountered with the agar dilution method. results: breakpoints were used as proposed by nccls . during the study period the pneumococci resistance was noted as follows: % to pc, % to em and % to sxt. the rank order of activity of the five fqs against multi-drug resistant pneumococci was: cip (mic : mg/l), ofx (mic : mg/l), lvx (mic : mg/l), grx (mic : . mg/l), tvx (mic : . mg/l). conclusions: in romania, fluoroquinolones represent alternative treatment to beta-lactams and macrolides for first-line empirical treatment for respiratory tract infections caused by pneumococci but, continued vigilance for emerging resistance to fqs is further indicated. introduction and material/methods: susceptibility testing (semiautomated broth microdilution method, sensititre, trek diagnostics, usa, following nccls recommendations) was performed with six different quinolones to streptococcus pneumoniae isolates with a ciprofloxacin-cip */mic ]/ mg/l collected in two consecutive sauce$ surveillances in spain ( Á/ / Á/ ). nccls resistance (r) breakpoints were used ( ]/ for ofloxacin-ofl and levofloxacin-lev; ]/ for sparfloxacin-spa; ]/ for gatifloxacin-gat */and moxifloxacin-mox), but for gemifloxacin-gem */where ]/ was used. results were as follows. conclusions: for cip-r isolates gem and mox were the most active agents. gem was the only agent not influenced by cip mic increase regarding prevalence of r , with % resistance for strains with cip mic ]/ mg/l. $sauce is an acronym standing for 'sensibilidad a los antimicrobianos utilizados en la comunidad en españ a' (susceptibility to the antimicrobials commonly used in the community in spain) and is the spanish word for the willow tree. in vitro activity of gatifloxacin and seven other antibiotics against respiratory and urinary tract pathogens from the community. first results of the basic */study ps grimm h, on behalf of a european multicenter study group, institute for med. microbiology, weingarten, germany a total of centers in austria, belgium, france, germany, italy, portugal, spain and switzerland are involved in the basic study (bacterial annual susceptibility information collection). the mics of gatifloxacin (gati), ciprofloxacin (cipro), clarithromycin (clari), benzylpenicillin g (pen), amoxicillin (amox), amoxicillin/clavulanic acid (augm), cefuroxime (cur) and cefixime (cix) were determined using the microdilution method. each center is requested to investigate strains each of the following species: s. pneumoniae (spn), s. pyogenes (spy), s. aureus (sau), e. faecalis (efa), m. catarrhalis (mca), h. influenzae (hin), e. coli (eco), k. pneumoniae (kpn), p. mirabilis (pmi) and p. aeruginosa (pae). so far approximately strains are enrolled. some important mic %/percentage resistance were as follows: from the oral antibiotics tested gatifloxacin has the highest activity and broadest spectrum against all relevant respiratory and urinary tract pathogens. gatifloxacin is a promising alternative for therapy of respiratory tract bacterial infections. in vitro activity of gatifloxacin against bordetella pertussis in comparison with erythromycin, ciprofloxacin and levofloxacin ps bourgeois n, pangon b, ghnassia jc, doucet-populaire f, de versailles ch. microbiologie, le chesnay, france purpose of the study: bordetella pertussis infections are far more common in adults and adolescents than is generally estimated. however, they are often not recognised. infected or colonised adults can act as a reservoir of infection, passing it to children. fluoroquinolones are currently recommended for the treatment of respiratory tract infection in adult patients, which is usually empirical. gatifloxacin is a novel -methoxyquinolone, with a potent activity against both gram-negative and -positive bacteria. the in vitro activity of gatifloxacin was compared with those of erythromycin, the drug of choice for both treatment and prophylaxis of pertussis, ciprofloxacin and levofloxacin, against clinical isolates strains of b. pertussis including erythromycin resistant strains. results: we used the agar dilution method on mueller Á/hinton medium supplemented with % horse blood to determine the mic of each antibiotic. gatifloxacin (mic , . mg/l) was as active as ciprofloxacin and levofloxacin (mic , . mg/l) against both sensitive erythromycin (mic , . mg/l) and resistant erythromycin (mic , !/ mg/l) strains. conclusion: gatifloxacin may be an effective drug in the treatment or prophylaxis of adults with suspected or confirmed pertussis. ex vivo serum activity (killing rates) after gemifloxacin mg versus trovafloxacin mg single doses against ciprofloxacin-susceptible andresistant streptococcus pneumoniae ps calvo a a , giménez mj b , alou l a , gó mez-lus ml a , aguilar l b , prieto j a . a microbiology department, universidad complutense, madrid, spain , b glaxosmithkline, medical department, tres cantos, madrid, spain serum bactericidal activity was measured ex vivo after single dose administration of gemifloxacin (gem) mg and trovafloxacin (tro) mg to healthy volunteers in a randomized, cross-over phase i trial. blood samples were collected h (cmax) after dosing and serum killing rates were determined against a serotype penicillin (pen) Á/ciprofloxacin (cip) susceptible strain (s ) (mics of . , , . and . mg/l for pen, cip, gem and tro) and a serotype pen Á/cip resistant strain (s ) (mics of , , . and . mg/l for pen, cip, gem and tro). tubes with . ml of serum sample and . ml broth ( % todd Á/hewitt'/ % hbss) were incubated over h at c. final inocula was cfu/ml. mean colony counts for samples and controls (k ) are shown in the figure: gem exhibited higher colony counting decrease of the initial inocula, versus tro, for both strains. after h incubation, the initial inocula decrease obtained with tro and the cip susceptible strain was similar to that obtained with gem and the resistant strain, showing a lower influence of cip mic increase in the ex vivo bactericidal activity of gem versus tro. urine bactericidal activity after administration of gemifloxacin and trovafloxacin single doses in a phase i study ps garcía-calvo g a , parra a a , giménez mj b , ponte c a , aguilar l b , soriano f a . a fundación jiménez díaz, medical microbiology, madrid, spain , b glaxosmithkline, medical, madrid, spain urine bactericidal activity after o.d. administration of gemifloxacin (gem) mg and trovafloxacin (tro) mg, was assessed in six adult males in a cross-over phase i trial. urine killing rates (ukr) against escherichia coli atcc (mic mg/l of . and . for gem and tro) and s. saprophyticus atcc (mic mg/l . and . for gem and tro) were performed with samples collected at , Á/ , Á/ and Á/ h. a . ml of iso-sensitest broth and . ml of bacterial logarithmic growth were added to ml sample, giving a final inoculum of cfu/ml. colony counting was performed after , , and h incubation. percentages of initial inocula reduction (iir) were calculated. mean urine concentrations measured by bioassay were (mg/l): . , . and . for gem, and . , . and . for tro. against e. coli , an iir of . % was obtained after h incubation with all samples except with tro at Á/ h. against s. saprophyticus an iir of % was obtained after h incubation with all samples except with tro at Á/ h, where bacterial regrowth was found. the maintenance over h of gem urine antibacterial activity suggests its efficacy in the treatment of uncomplicated cystitis. influence of the decreased susceptibility to ciprofloxacin on gemifloxacin versus levofloxacin efficacy in experimental pneumococcal pneumonia in guinea pigs ps garcia-olmos m a , parra a a , gimenez mj b , garcia-calvo g a , ponte c a , aguilar l b , soriano f a . a fundacion jimenez diaz, medical microbiology, madrid, spain , b glaxosmithkline, medical, madrid, spain the efficacy of ciprofloxacin (cip), levofloxacin (lev) and gemifloxacin (gem) in the treatment of pneumococcal pneumonia was assessed in a guinea pig model using three strains (s) with mics (mg/l) of , and . (s ), , and . (s ) and , and (s ) for cip, lev and gem, respectively. intraperitoneal treatments started h after s. pneumoniae intratracheal inoculation, and continued t.i.d up to four doses. ten animals were included in each group. doses (mg/kg) used were , and for cip, lev and gem, respectively, in order to mimic auc - h and cmax obtained in humans after standard doses. animals that survived h after inoculation were sacrificed and colony counts were performed in lungs ( the purpose of the study levofloxacin (lfx) is a fluoroquinolone whose activity against both gram-negative bacilli and gram-positive cocci enables its use in monotherapy for the treatment of nosocomial pneumonia. our aim was to study the pharmacokinetic Á/pharmacodynamic appropriateness of lfx mg iv bid in the treatment of six inpatients with ventilator-associated pneumonia (vap) ( / years; m Á/ f; / kg). blood and urine samples were collected in steadystate conditions at appropriate intervals. lfx concentrations were analysed by hplc. the aetiological agent was identified in all the cases and its in vitro sensitivity to lfx was always assessed. the results obtained mean values ( /sd) of the major pharmacokinetic parameters were: cmax, . / . mg/ml; vdss, . / . l/kg; t / b, . / . h; cl, . / . ml/min/kg; auc -t, . / . mg/ml h. cumulative urinary excretion was . / . %, confirming that lfx clearance is mainly renal. clinical cure and microbiological eradication were obtained in all the patients after a Á/ day therapy. a suprainfection due to acinetobacter anitratus insensitive to lfx occured in case. the major pharmacodynamic parameters of fluoroquinolone efficacy were significantly higher than the proposed threshold (cmax/mic !/ ; auc/mic !/ ) in all the cases. the conclusion reached the findings suggest that lfx mg bid iv may be considered effective in the treatment of vap caused by sensitive bacteria. comparative pharmacokinetics of levofloxacin in patients with lower respiratory tract infections (lrti) being treated with sequential therapy ps pea f a , brollo l a , lugatti e b , di qual e a , dolcet f b , talmassons g b , furlanut m a . a institute of clinical pharmacology and toxicology, dpmsc, university of udine, udine, italy , b division of pneumology, sm misericordia hospital, udine, italy the purpose of the study levofloxacin (lfx) is a fluoroquinolone whose activity against both gram-negative bacilli and gram-positive cocci enables its use in monotherapy for the treatment of lrti. our aim was to study the pharmacokinetic Á/pharmacodynamic appropriateness of a standard switch lfx iv/os regimen ( mg iv od for Á/ days followed by mg os od for Á/ days) in the treatment of seven inpatients with lrti ( / years; m Á/ f; / kg). blood samples were collected in steady-state conditions at appropriate intervals. lfx plasma concentrations were analysed by hplc. the aetiological agent was identified in / cases and its in vitro sensitivity to lfx was assessed. the results obtained absolute oral bioavailability was . / . , with a cmax of . / . vs . / . mg/ml after iv and oral administration, respectively. no significant difference in the main pharmacokinetic parameters was observed between the two routes. the major pharmacodynamic parameters of fluoroquinolone efficacy were significantly higher than the proposed threshold (cmax/mic !/ ; auc/mic !/ ) in the two assessable cases. all the patients were clinically cured after a Á/ day therapy. the conclusion reached the ad interim findings show that lfx mg od may guarantee per os an exposure similar to that achievable after iv administration, suggesting that sequential therapy may be considered effective in the treatment of lrti. levofloxacine in the exacerbations of copd due to pseudomonas ae ps micheletto c, tognella s, pomari c, dal negro r, ospedale orlandi, div.pneumologia, bussolengo, italy development of antibiotic resistance in bacteria is a problem of great concern. gram-negative bacteria, including multidrug-resistant (mdr) pseudomonas aeruginosa (ps), are responsible for a significant proportion of episodes of copd exacerbations, particularly in elderly ( ). aim was to check the susceptibility to common antimicrobial treatments against ps strains isolated from bronchial secretions in patients with severe exacerbations of copd. methods: microbial investigations were conducted on specimens: spontaneous purulent sputum ( . %), and tracheobronchial aspirates ( . %, collected with a protected specimen brush). results: fifty-seven ps pathogen strains ( cfu) were identified ( . %) and tested over a -month period: ps. aeruginosa . %; ps. putida . %; ps. fluorescens . %, and burkholderia cepacia . %. the assessed susceptibility to most common antibiotics was: levofloxacine ( %), ciprofloxacine ( %), ipenem cil. ( %), ceftazidime ( %); amikacin ( %), and piperacillin'/tazobactam ( %). a much lower susceptibility was found for ticarcillin Á/clavulanic acid ( %), gentamicin ( %), and netilmicin ( %). ( ) at present, levofloxacine proves the most effective antimicrobic option for treating copd exacerbations due to ps infection; ( ) a much more efficient policy of antibiotic prescribing should be promoted in order to prevent the selection of resistant strains in these cases. these results confirm the excellent 'in vitro' activity of levofloxacin against nosocomial gram-negative pathogens, including the esbl producing strains ( % of escherichia coli , e. cloacae and k. pneumoniae were inhibited at . mg/l). levofloxacin was more rapid than ciprofloxacin to determine a bactericidal effect particularly against s. maltophilia . moreover, considering the favourable pk/pd profile, levofloxacin can represent a valid therapeutic option for the treatment of severe gram-negative nosocomial infections. moretti f, quiros-roldan e, casari s, chiodera a, viale p, carosi g. university of brescia, institute of infectious and tropical diseases, brescia, italy a -year-old man, ivdu, hiv positive was attended in aur hospital for fever and toracic pain. a x-chest radiography revealed a round lesion of cm near the lingula with central hyper-diaphan area. lymphocytes cd '/ count was cells/mm and viral load . cp/ ml. hospital stay rhodococcus equi was found in cultures of peripheral blood, faecal and sputum specimens. antibiotic treatment with oral rifampin ( mg/qd) and with intravenous imipenem ( mg tid) was started. due to the persisting fever, immodificated radiography and negativity for p. carinii , mycobacteria and bacteria in bal coltures, imipenem was substituted by parenteral vancomycin ( mg bid). after days, because of persisting fever and increase of the diameter of the lung lesion ( cm) vancomycin was sustituted by oral levofloxacyn ( mg bid), continuing rifampin. after a days course of levofloxacyn therapy the fever remitted. the patient was discharged with levofloxacyn ( mg bid) and rifampin and, after months of follow-up, a radiological control pointed-out a remarkable resolution in the lung lesion. we may suppose that levofloxacyn can be effective for the treatment of r. equi infection, even if more studies (particularly controlled studies) are necessary. liberti a a , izzo b a , loiacono l b , calabria g a , patarino t a , izzo e a . a ii department, naples, d. cotugno hospital, italy , b iii department, naples, d. cotugno hospital, italy the increasing prevalence of salmonella typhi strains with reduced susceptibility of chloramphenicol had prompted the search for other antibiotics with the same efficacy.quinolones are a class of antibiotic with an activity in vitro and in vivo against enteropathogenes. we inwestigated the use of levoxacin in two regimens of treatment of typhoid and paratyphoid infection. patients and methods: thirty-two adult patients were incluted in this study from september to april ; patients had positive culture for s. typhi and six had positive cultures for s. paratyphi . all isolated were fully susceptible to levoxacin. we compared treatment with levoxacin for days, mgr b.i.d. (group , patients), with treatment for days, mgr b.i.d. (group , patients). clinical cure was defined as defervescenze of fever by day of treatment, with an absence of complications and no clinical relapse during the followup. results and conclusion: the clinical cure rate was % ( patients) for group and % ( patients) for group ; the difference in these rates was not statistically significant. the blood cultures of all patients were sterile by day of treatment and remained so until the th month of follow-up, no subjects had clinical or microbiological relapse and all stool cultures remained negative, also. the two regimens of treatment was good tollerated and no adverse event was registered; it was concluded that levoxacin treatment for days in enteric fever is not necessary the mulidrug-resistence of s. typhi led to the use of quinolones as the first-line drug in the treatment of enteric fever. pefloxacin in the treatment of patient with acute infectious diarrhoea ps troselj-vukiae tvb a , strahinja v b , poljak i a , stojanoviae d c , nikoliae n d . a department of infectious disease, university hospital center, rijeka, croatia , b glaxosmithkline, marketing, rijeka, croatia , c dept. rijeka, institute of public health, rijeka, croatia , d dept. rijeka, maritime academy, rijeka, croatia the purpose of the study was to investigate clinical and bacteriological efficiency in and days pefloxacin treatment and to compare it with symptomatic therapy. the results obtained in patients treated with pefloxacin the therapy was clinically effective already in the third day while in the control group this happend in the th day. bacteriological eradication was noted in patients ( %) of the first and patients ( %) of second group in the th days of the treatment. they all had negative cultures and weeks after pefloxacin protocols were completed. only patients ( %) in control group had negative stool cultures in the th day of the treatment and all of them weeks after it ended. there was no statistically significant difference in clinical (p / . ) and bacteriological (p / . ) efficiency between and days pefloxacin treatment protocols. both protocols significantly differed in clinical (p b/ . ) and bacteriological (p / . ) eradication from the control group. the conclusion reached is that the efficiency of pefloxacin (quinolones) in the treatment of acute infectiuos diarrhoea and justifies their use in the more severe forms of the disease. dalhoff a a , ullmann u b , schubert s b . a bayer ag, wuppertal, germany , b university of kiel, institute of med. microbiology, kiel, germany background: the antibacterial activity of moxifloxacin (mxf) was compared to levofloxacin (lev), amoxicillin (amx), clarithromycin (cla) and erythromycin (ery) in an in vitro model. method: pharmacokinetics in bronchial mucosa (bm) and serum (s) following single oral doses of mg mxf or cla and mg lev, amx or ery were simulated using a one compartment model. bacteria tested staphylococcus aureus (nos. , ), streptococcus pneumoniae (nos. , ). aliquots were taken ( Á/ h) and plated on to brain heart infusion agar for enumeration. results: s. pneumoniae was eliminated by all agents studied. significant differences were apparent with s. pneumoniae and the s. aureus strains: objective: to compare the safety and efficacy of once-daily moxifloxacin with once-daily ceftriaxone in the treatment of cap in hiv-infected patients (pts). methods and results: in a retrospective survey, oral moxifloxacin ( mg daily)/ Á/ days) was compared to standard regimen of i.v. ceftriaxone ( g daily)/ Á/ days) for treatment of cap in hiv'/ pts. adults pts with clinical signs and symptoms of cap and consistent chest x-ray findings were included. pts had a median age of years (range Á/ and % were male). demographic characteristics were similar in both treatment groups; pts received mxifloxacin and pts ceftriaxone. clinical success rates were % for moxifloxacin and % for ceftriaxone. at a post-study evaluation approximately weeks later, moxifloxacin-treated pts and ceftriaxone-treated pts had relapsed. the adverse events reported were comparable for both treatment groups. there were four-related adverse events ( gi, headache) for moxifloxacin-treated and ( gi, skin) for ceftriaxonetreated pts. conclusion: the results of this study show that moxifloxacin as oral therapy is as effective and well tolerated as i.v. ceftriaxone in the treatment of hiv'/ pts with cap. therapy with moxifloxacin was not associated with any significant clinical or laboratory abnormalities. these data suggest that once-daily oral administration of moxifloxacin is potentially convenient and cost-effective alternative therapy for cap in pts with hiv infection. moxifloxacin in the treatment of acute maxillary sinusitis after first-line therapy failure or acute sinusitis with high risk of complications ps gehanno p a , berche p b , perrin a b , arvis p c . a ent department, bichat claude bernard hospital, paris, france , b microbiology department, necker enfants malades hospital, paris, france , c bayer pharma, medical affairs department, paris, france the efficacy and safety of moxifloxacin (mxf) mg once daily for days was evaluated in the treatment of acute maxillary sinusitis after first-line therapy failure, or acute sinusitis with high risk of complications. in this prospective, multicenter study, a total of patients with acute bacterial sinusitis confirmed by sinus x-ray were valid for efficacy analysis: one hundred and seventy five patients ( . %) had an acute maxillary sinusitis which failed to respond to a previous antibiotic therapy given for a mean duration of . days, and ( . %) had an acute sinusitis with high risk of complications (frontal: , pan-sinusitis: and sphenoid: ). ninety two patients ( . %) were microbiologically valid. clinical cure and continued clinical cure rates at Á/ and Á/ days post-therapy were . and . %, respectively. clinical cure rates at Á/ days post-therapy were . and . % in sinusitis after first-line therapy failure and in sinusitis with high risk of complications, respectively. bacteriological eradication rate during therapy (day Á/ ) was . %. at Á/ days post-therapy. bacteriological success rates were . % in patients who failed to respond to a previous antibiotic and . % of patients who had sinusitis with high risk of complications. . % of patients experienced drug-related adverse events, abdominal pain ( . %), nausea ( . %) being the most frequently reported events. mxf was rapidly effective and a well-tolerated treatment for this kind of infection. neisseria gonorrhoeae with decreased susceptibility to penicillin and ciprofloxacin: novel mutation patterns in the gyr a and par c genes of ciprofloxacin resistant isolates and plasmid profile of penicillin resistant isolates of n. gonorrhoeae in india (delhi) ps chaudhry u, saluja d. dr. b. r. ambedkar center for biomedical research, university of delhi, delhi, india commercial sex workers (csws) serve as the most important reservoir of gonorrhoea. periodic monitoring of the antimicrobial susceptibility profile of neisseria gonorrhoeae in a high-risk population provides essential clues regarding the rapidly changing pattern of antimicrobial susceptibilities. in india, such a surveillance of the in vitro antimicrobial susceptibility of n. gonorrhoeae was established in . significant increasing trend of penicillin and ciprofloxacin resistance with high mic of Á/ and Á/ mg/ml, respectively were found over the years ( Á/ ) . the molecular basis of ciprofloxacin resistance, i.e. mutations in the gyr a and the par c genes of isolates, were analyzed. four isolates (with an mic of mg/ml for ciprofloxacin) harbored triple mutations (ser- to phe, asp- to asn and val- to leu) in the gyr a gene. the third mutation of val- to leu, lies downstream of the quinolone resistance determining region of the gyr a and has not been described before in gonococcus. in addition, these isolates had a phe- to tyr substitution in the par c, a hitherto unknown mutation. the alterations in the par c gene were seen in these isolates only in the presence of changes in the gyr a gene and comprised amino acid changes at codons , , , , and . the presence of b-lactamase plasmid among the penicillinresistant isolates was determined by their plasmid profiles and further confirmation was carried out by a pcr based protocol. our findings suggest that emergence of penicillin and ciprofloxacin resistance in n. gonorrhoeae isolates from a major std center in india, indicates the need for the increased awareness and prudent use of antimicrobials. in vitro activity of newer antibiotics against methicillin-resistant staphylococcus aureus ps gutierrez zufiaurre mn, sanchez hernandez j, munoz-bellido jl, garcia-rodriguez ja. hospital universitario de salamanca, microbiología, salamanca, spain purpose: mrsa are frequently co-resistant to a number of structurally unrelated antibiotics. more than % mrsa are resistant to gentamycin, ciprofloxacin, macrolides and clindamycin. newer antibiotics active against multi-drug resistant grampositives have been developed. we have tested the in vitro activity of newer antibiotics against genetically-characterized, high level ciprofloxacin resistant mrsa. material and methods: thirty-six ciprofloxacin-resistant, gyra/grla mutant mrsa clinical strains were tested against levofloxacin (lfx), ciprofloxacin (cfx), moxifloxacin (mfx), gatifloxacin (gfx), erythromycin (er), telithromycin (tl), linezolid (lin), synercid (syn) and vancomycin (va). mics were determined by the agar dilution method, according nccls guidelines. results and conclusions: all the strains were resistant to cfx, , % were lfx-susceptible and . % were gfx-susceptible. nevertheless, mics of lfx and gfx for all susceptible strains was in the highest extreme of the susceptibility range. mfx was the most active quinolone. almost all the strains were high-level er-resistant with constitutive mlsb phenotype. tl did not improve significantly its behaviour, although it was -fold as active as er against the only er-susceptible strain. va, lin and syn had excellent activity against all the strains. they showed a very homogeneous behaviour against all the strains that were included in a range of Á/ mg/l of lin and va and . Á/ mg/l. sanchez hernandez j, gutierrez zufiaurre mn, munoz-bellido jl, garcia-rodriguez ja. hospital universitario de salamanca, microbiología, salamanca, spain purpose: corynebacterium urealyticum is the etiologic agent of encrusted cystitis and inespecific utis, and can be also involved in systemic infections. c. urealyticum is frequently multi-drug resistant, so only glycopeptide antibiotics and tetracyclines have high susceptibility rates, while fluoroquinolones resistance rates vary significantly. we have tested the in vitro activity of linezolid, telithromycin, synercid and newer fluoroquinolones against multi-drug resistant c. urealyticum clinical strains. material and methods: sixty-four c. urealyticum clinical strains were tested against levofloxacin (lfx), ciprofloxacin (cfx), moxifloxacin (mfx), erythromycin (er), telithromycin (tl), linezolid (lin), synercid (syn) and vancomycin (va). mics were determined by the agar dilution method according guidelines defined by the nccls for enterococci. results and conclusions: results confirm the high resistance rate to older fluoroquinolones and macrolides, with !/ % cfx resistance and % er-resistance. lfx was more active (mic mg/ml). mfx was the most active fluoroquinolones (mic mg/ml). tl improve its behaviour in respect to er (range . Á/ mg/ml). va, lin and syn had excellent antimicrobial activity. no resistant strains were found. mic were , . and mg/ml, respectively. mics were similar for all the strains independently of their resistance to other antibiotics plasma concentrations, urinary excretion and bactericidal activity of linezolid ( mg) versus ciprofloxacin ( mg) in healthy volunteers after a single oral dose ps wagenlehner fme a , wydra s a , onda h a , kinzig-schippers m b , sö rgel f b , naber kg a . a hospital st. elisabeth, urologic clinic, straubing, germany , b institute for biomedical and pharmaceutical research, (ibmp), nürnberg-heroldsberg, germany purpose of the study: in a randomized cross-over study volunteers received a single oral dose of mg linezolid versus mg ciprofloxacin to assess plasma concentrations (up to h), urinary excretion (by hplc), and urinary bactericidal titers (ubt) up to h. the mean maximum plasma concentration of linezolid was . mg/ ml and of ciprofloxacin . mg/ml. the cumulative renal excretion (mean) of parent drug was %/ % for linezolid/ciprofloxacin. ubts were determined for a reference strain and five gram-positive clinical uropathogens with the following mics (mg/ml) for linezolid/ciprofloxacin: s. aureus atcc ( / . ), s. aureus (mssa) ( / ), s. aureus (mrsa) ( / ), s. saprophyticus (msse) ( / . ), e. faecalis ( / ), e. faecium ( / ). results: median ubts measured within the first h for linezolid were : for enterococcal strains and : to : for the four staphylococal strains. median ubts for ciprofloxacin were : for the two enterococcal strains, : to : for the two ciprofloxacin susceptible, : . for the two resistant staphylococcal strains. areas under the ubt Á/time-curve showed statistically significant differences only for the two ciprofloxacin resistant staphylococcal strains in favour of linezolid. conclusion: linezolid exhibited the same bactericidal activity against ciprofloxacin resistant as susceptible strains. linezolid should be tested for treatment of complicated uti due to gram-positive uropathogens in a clinical trial. whitehouse t a , cepeda ja a , tobin purpose: we performed pharmacokinetics within a double-blind, randomised trial comparing linezolid and teicoplanin in intensive care patients with known or suspected gram-positive infection. they received either mg linezolid intravenously -hourly, or mg teicoplanin -hourly for the first three doses and once daily thereafter (or every rd day if renally impaired). blood samples were collected to create serum pharmacokinetic profiles. linezolid was quantitated by hplc and teicoplanin by fluorescence polarization immunoassay. results: twenty two patients were studied in the linezolid group (m Á/f : , mean age years [range Á/ years]). median treatment duration was . days (range Á/ ). eighteen patients were treated with teicoplanin (m Á/f : , mean age years [range Á/ ]) for median days (range Á/ ). steady state peak concentrations (mean / sd) for linezolid and teicoplanin were . / . and . / . mg/l, respectively. trough concentrations at day were . / . mg/l for linezolid and . / . mg/l for teicoplanin. recommended breakpoints of staphylococcus aureus are mg/l for linezolid and mg/l for teicoplanin. accumulation occurred in one -year-old linezolidtreated patient with impaired renal function. conclusion: current recommended dosing regimens for linezolid and teicoplanin are generally appropriate in the critically ill, though a more detailed analysis is required. stamos g, lebessi e, ioannidou s, paleologou n, kallergi k, foustoukou m, 'p. and a. kyriakou ' children's hospital, microbiology, athens, greece the purpose of the study was to investigate the susceptibility of methicillin resistant staphylococcus aureus (mrsa) isolates from a -bed paediatric hospital to quinupristin/dalfopristin (q/d, streptogramin combination) and linezolid (lzd, oxazolidinone). material: we performed a retrospective analysis of mrsa strains, isolated from patients hospitalized in miscellaneous medical departments [neonatal unit ( ) , surgical wards ( ), orthopaedic wards ( ), oncology unit ( ), other wards ( ) and outpatient clinic ( )], during a -year period ( Á/ ) . the sources of isolation were pus ( ), throat ( ), nasal ( ), bronchial ( ), skin ( ), stool ( ) ear ( ) and other ( ) specimens. all isolates were sensitive to glycopeptides, while . % were resistant to gentamicin and . % to erythromycin. methods: the sensitivity testing was performed by a disk diffusion method (bbl sensitivity disks, becton dickinson), according to nccls guidelines. the breakpoint zone diameters for lzd and q/ d were ]/ and ]/ mm for susceptibility and / and / mm for resistance, respectively. results: all isolates were proved to be susceptible to both antibiotics. the mean inhibition zones were . mm for lzd and mm for q/d. conclusions: lzd and q/d are very promising antimicrobial agents, showing excellent activity against mrsa clinical isolates. the prudent therapeutic use is strongly recommended to avoid the emergence of resistance. in vitro activity of streptogramins and oxazolidinones against streptococcus pneumoniae clinical isolates ps stamos g, lebessi e, paleologou n, psatha m, sanida p, zaphiropoulou a, foustoukou m, 'p. and a. kyriakou ' children's hospital, microbiology, athens, greece the purpose of this study was to evaluate the in vitro activity of linezolid (lzd), a member of oxazolidinones and the streptogramin combination quinupristin/dalfopristin (q/d) against clinical isolates of streptococcus pneumoniae from a tertiary care paediatric hospital. material: a total of pneumococcal isolates exhibiting reduced susceptibility to common antibiotics were included in the study. the strains were isolated from middle ear fluid ( ), eye ( ), nasal ( ) blood ( ) and other ( ) cultures during the last years. the percentages of the isolates that were resistant to penicillin, erythromycin, cotrimoxazole and clindamycin were . , . , . and . %, respectively. methods: the susceptibility testing was performed by a standard disk diffusion method (bbl sensitivity disks, becton dickinson). in case of marginal results or intermediate sensitivity to quinupristin/ dalfopristin, the mic was determined using the e -test method (ab biodisk). results: all isolates were found to be sensitive to lzd. q/d was very active as well, except for two isolates that exhibited intermediate susceptibility, showing cross-resistance to macrolides and clindamycin, as well. conclusions: the new antimicrobial agents show excellent activity against resistant to common antimicrobials pneumococcal isolates, but the clinical use is not suggested, unless no other therapeutic solutions are available. linezolid is a new oxazolidinone with excellent activity against gram-positive organisms including glycopeptide-resistant strains of staphylococci and enterococci. in icu linezolid has been used for the treatment of severe gram-positive infections in a control trial. the susceptibility pattern of all gram-positive isolates from icu patients has been studied. methods: a total of specimens from icu patients were processed, were from patients enrolled in the antibiotic trial. all methicillin-resistant staphylococcus aureus (mrsa), coagulase-negative staphylococci (cons), enterococcus sp and methicillin-sensitive staphylococcus aureus (mssa) were tested. the break point for linezolid was mg/l and for teicoplanin mg/l. isolates were tested for susceptibility by e -test. results: linezolid ( isolates) mic / (mg/l) were as follows: mrsa (n / ) . / , cons (n / ) . / . , enterococcus sp (n / ) . / . , mssa (n / ) . / . teicoplanin ( isolates) mic / (mg/l) mrsa (n / ) . / . , cons (n / ) / . , enterococcus sp (n / ) . / . , mssa (n / ) . / . . all grampositive isolates were inhibited by concentrations of linezolid below the breakpoint including eight strains of staphylococci resistant to teicoplanin. conclusions: linezolid was highly active against grampositive isolates. resistance to teicoplanin was similar to other reported series. there was no emergence of resistance to linezolid. mrsa colonization is often a limiting factor for discharge from icu. clearance of mrsa is seldom achieved with conventional glycopeptide treatment. the oxazolidinone, linezolid, has excellent soft tissue and respiratory tract penetration and might be expected to eradicate carriage in some patients. we recently performed a doubleblind randomized trial in icu patients with known/suspected gram positive infection. received intravenous linezolid, mg b.d., and patients received teicoplanin mg o.d. mrsa clearance was assessed at end of treatment (eot), at -and days follow-up. results: in the linezolid and teicoplanin groups, and were known to be colonized with mrsa at study entry, respectively, while and were not. detection of clearance of mrsa colonization at eot was % for linezolid vs % for teicoplanin group (x b p b/ . ), at days it was % vs % (x b p b/ . ), and at days % vs % (ns). conclusion: short-term mrsa clearance can be achieved in significantly more patients treated with linezolid however this was not maintained at days, either because of incomplete initial eradication or recolonization. further analysis will follow when molecular typing of the isolates is completed. penetration of linezolid into bone, fat and muscle during hip arthroplasty ps lovering am, bannister gc, zhang j, macgowan ap, southmead hospital, bcare, bristol, uk there are limited data describing the concentrations and penetration of linezolid (lzd) into tissues, such as bone, that can be used to guide therapy for non-vascular infections. here we report the concentrations and penetration of lzd for bone, fat and muscle in comparison with cefamandole (cmd). twelve patients received mg lzd as a min infusion and mg cmd as a bolus injection immediately before hip arthroplasty. bone, fat, muscle and blood samples were collected at timed intervals after the infusion and assayed by a validated hplc method. for bone, peak levels of both agents occurred min after administration with mean levels of lzd . mg/kg versus cmd . mg/kg, decreasing to lzd . mg/kg versus cmd . mg/kg at min. correction for blood concentrations gave penetration of lzd % versus cmd % at min and lzd % versus cmd % at min. for fat and muscle, peak levels occurred min after infusion. mean levels were lzd . mg/kg versus cmd . mg/kg (fat) and lzd . mg/kg versus cmd . mg/kg (muscle). correction for blood concentrations gave penetration of lzd % versus cmd % (fat) and lzd % versus cmd % (muscle). we conclude that linezolid exhibits rapid penetration into bone and associated soft tissues achieving levels in excess of the mic for sensitive organisms; with a similar distribution and penetration profile to agents currently used for treatment of infections in these tissues. bacqué e a , barrière jc a , berthaud n b , desmazeau p b , dutruc-rosset g b , dutka-malen s b , ronan b b . a aventis pharma, chemistry, paris, france , b aventis pharma, disease group, paris, france xrp is a new oral streptogramin composed of semi-synthetic synergistic components in a / (w/w) association: rpr ( d-( -morpholino)methyl- d, g-dehydro pristinamycin i e ) from pi a and rpr [( r )- -deoxy- -fluoro pristinamycin ii b ] from pii b by original synthetic routes. the association has antibacterial activity against staphylococci including methicillin */mls b -resistant strains (mic range: . */ mg/ml), streptococci (mic : . mg/ml), pneumococci including multidrug resistant strains (mic : . mg/ ml), enterococci including vancomycin-resistant strains (mic : mg/ ml), m. catarrhalis and neisseria spp. (mic : . mg/ml), h. influenzae (mic : mg/ml), legionella spp. (mic : . mg/ml) and anaerobes (mic range: . Á/ mg/ml). xrp is generally bactericidal at the concentration of Á/ )/the mic against s. aureus , s. pneumoniae , h. influenzae and demonstrates consequent pae ( . Á/ !/ . h at Á/ )/mic, following an exposure of . Á/ h). mutants of s. aureus to xrp were isolated at low frequencies ( . )/ ( Á/ . )/ ( ) at )/ and )/mic while no mutant could be isolated at )/mic. these results suggest that xrp ( / w/w) is a promising compound for the treatment of community-acquired infections. ex vivo evaluation of rpr /rpr (xrp ), a new oral streptogramin ps berthaud n, diallo n. aventis pharma sa, infectious disease group, paris, france the intra cellular activity of xrp , was assessed in j murine macrophages containing ingested staphylococcus aureus (three strains) or l. pneumophila (one strain). at the concentration of )/ the mic, growth of s. aureus was strongly inhibited after a -h period of incubation (d log cfu/ml vs t controls range: (/ . Á/(/ . , according to the strain tested). at the concentration of )/ the mic, growth of intracellular l. pneumophila was inhibited after a Á/ -h period of incubation (d log cfu/ml vs t controls about (/ . and (/ . at and h, respectively). rpr and rpr alone had also inhibiting effect on bacterial growth (d log cfu/ml vs t controls after h of incubation about (/ . and (/ . , respectively). the bactericidal activity of xrp was also assessed against slowly growing s. aureus , under experimental conditions mimicking those observed in patients with an infected indwelling device (first step of infection: adherence to inert support; declared infection: biofilm model). under the experimental conditions, xrp demonstrated a rapid and potent bactericidal effect against s. aureus adherent to an inert support or included in biofilm. this effect was observed at each of the three concentrations tested ( , , and , and )/ mic, respectively). in vivo evaluation of xrp (rpr /rpr ), a new oral streptogramin ps berthaud n, huet y, aventis pharma sa, infectious disease group, paris, france the oral efficacy of xrp , was assessed in staphylococcus and pneumococcal murine infections. mice were challenged ip ( )/ , and )/ times ld ). abscesses were established by intramuscular injection of about bacteria into the right thigh of mice. pneumonia was established by intranasal injection of about bacteria. mice were treated twice a day for (staphylococcus aureus septicaemia and abscesses), (s. pneunomoniae septicaemia) or days (s. pneumoniae pneumonia). six to days post infection, for septicaemia and abscesses the results were expressed as ed , whereas for pneumonia they were expressed as the dose yielding an average survival time (ast) significantly longer than that of the untreated infected controls. xrp was efficacious in the treatment of experimental infections in mice caused by mls b -sensitive and -constitutively resistant s. aureus (ed range: Á/ and Á/ mg/kg/administration in septicaemia and abscess models, respectively). it was also efficacious in the treatment of infections caused by s. pneumoniae whatever the serotype and the resistance profile of the strains tested (ed range: Á/ mg/ kg/administration in septicaemia, ast: mg/kg/administration). these results suggest that xrp might be effective for the treatment of staphylococcal and pneumococcal community-acquired infections. xrp (rpr /rpr ), a new oral streptogramin: bactericidal activity and pharmacokinetics in a model of streptococcus pneumoniae mouse pneumonia ps berthaud n, huet y, diallo n. aventis pharma sa, infectious disease group, paris, france the bactericidal activity against streptococcus pneumoniae of xrp , a new oral streptogramin composed of two semi-synthetic synergistic components in a / (w/w) association (rpr , pristinamycin i derivative and rpr , pristinamycin ii derivative), was assessed in lungs of mice with pneumonia. mice were inoculated intranasally with about cfu of strain - (mls bresistant). eighteen hours later (t ), animals received xrp ( mg/kg p.o). the administration was repeated , , , and h afterwards. to study the influence of varying the ratio of pi to pii component administered on activity and pk parameters, ratios of rpr to rpr ranging from / to / were also administered under the same conditions. after an oral unitary administration at mg/kg, xrp , as well as ratios of rpr to rpr ranging from / to / , demonstrated strong and quick bactericidal activity in lungs. lung levels of rpr and rpr were generally equal or two times higher than blood levels. resulting rpr /rpr ratios in blood and lung, although not in accordance with the initial ratio administered, were synergistic for Á/ h in blood and for Á/ h in lungs explaining the activity observed. conclusion: based on in vitro data, telithromycin is a good candidate for the treatment of rti. in vitro evaluation of abt- , telithromycin and azithromycin against streptococcus pneumoniae , moraxella catarrhalis , haemophilis influenzae and methicillin-resistant staphylococcus aureus ps steele-moore l, berg d, barnes i, couch k, klein j, holloway w. christiana care, infectious disease, wilmington, us macrolide resistant streptococcus pneumoniae (sp) is a worldwide concern predominantly because these isolates tend to be multiply drug resistant. new agents with increased activity against these pathogens are clinically important. the ketolide class of antimicrobial agents demonstrate excellent in vitro activity against sp, even those that are macrolide resistant. the in vitro activities of the ketolides abt- (a) and telithromycin (t) were compared to azithromycin (az) against clinical isolates of sp, moraxella catarrhalis (m.cat), haemophilis influenzae (h.flu) and mrsa. organisms tested: strains of sp (including az resistant), h.flu, mrsa and m.cat. microdilution mic tests were performed following nccls recommendations using freshly prepared plates containing haemophilis test medium for h.flu, cation adjusted mueller-hinton broth (camhb) with laked horse blood for sp and camhb for m.cat and mrsa. the new ketolides, a and t had superior activity against sp including the az resistant strains (mic s: a / . mcg/ml, t / . , az / ). all compounds had excellent activity against m.cat while none demonstrated activity against mrsa. h.flu activity was comparable among a, t and az. these new ketolides are not currently approved by the fda; however t has been approved in europe. ló pez h, vidal gi, salomó n jm, scaglione m, zitto tr. centro de infectología, infectious diseases, buenos aires, argentina objective: we evaluated the impact of the initial treatment failure rate, hospitalisation and costs in outpatient treatment of adult cap in argentina comparing amoxicillin, clarithromycin and telithromycin. method: a probabilistic model was implemented in outpatient treatment of cap. we estimated an initial treatment with amoxicillin, clarithromycin or telithromycin. we assumed expected clinical cure at . , . and . %, respectively. for those patients with failure treatment we evaluated a second-line of antibiotics (amoxicillin followed by clarithromycin and clarithromycin followed by new fluorquinolone) or hospitalisation. patients with telithromycin and failure treatment must be hospitalised without a second line of outpatient treatment. costs of cap included drug's costs by days, medical visits, chest radiographies, analysis and hospitalisation. results: we estimated treatments in patients and first-line drug failure in , and patients with amoxicillin, clarithromycin and telithromycin, respectively. costs in outpatient treatment were: hospitalisation $ , and $ , . ; second-line drug $ and $ ; second-line hospitalised $ . and $ with amoxicillin and clarithromycin, respectively and hospitalisation with telithromycin $ . conclusions: telithromycin showed lower clinical failure, hospitalisation and costs in cap. some studies suggest shortening cap telithromycin treatment to days helping adherence to treatment and decreasing costs even more. the new macrolides */a good alternative of tetracycline in the treatment of mediterranean spotted fever ps popivanova nip a , petrov aip a , boykinova obb a , kazakova zkk a , baltadjiev agb b . a medical university, infectious disease, plovdiv, bulgaria , b department of anatomy, medical university, plovdiv, bulgaria mediterranean spotted fever (msf) caused by rickettsia conorii appears an endemic disease for some regions in bulgaria. frequently the disease has a severe course with multiple organ lesions. the early and adequate treatment is of extreme importance for the outcome of the disease. searching an alternative antibiotic treatment of this disease we considered macrolides for their good cell and tissue penetration and dose-dependent bacteriostatic and bactericide effect. we treated msf patients with doxycycline )/ mg/day, patients with clarithromycin )/ mg/day, as well as patients with midecamycin )/ mg/day and midecamycin acetate mg/kg/day. as a surrogate marker for treatment evaluation the effect on the febrile syndrome was used. our findings showed that by the th day of treatment the fever normalized in . , . and . % of the patients treated with doxycycline, clarithromycin and midecamycin, respectively. for the same period the patient fever decreased below c in , and . %, respectively. the intoxication symptoms were influenced within the same period equally in all treated patients. conclusions: we suggest that the new macrolides appear a good alternative of tetracycline on patients with msf. erythromycin resistance of gram /'// cocci in bulgaria. benefits of the new macrolides in the treatment of respiratory infections ps popivanova ni a , yovtchev ip b , dobreva md c , argirova ta c . a medical university, infectious disease, plovdiv, bulgaria , b medical university, ear-nose-throat disease, plovdiv, bulgaria , c medical university, microbiology, plovdiv, bulgaria in the recent years we tested erythromycin sensitivity of species of gram /'// cocci isolated from throat and nose secretions, ear and eye effusions, sputa, cerebrospinal fluid and blood cultures, vaginal and urethral secretions, urine and fecal samples from patients with inflammatory diseases of the listed organs and systems. staphylococcus aureus , staphylococcus coagulase /(//, streptococcus â-haemolyticus , enterococcus were isolated. of these microorganisms s. aureus was the most abundant. our resistograms revealed sensitivity of the gram /'// cocci in . and . % for and , respectively, and resistance of . and . %, respectively. in the tests with midecamycin and midecamycin acetat the same microorganisms showed sensitivity of . % and resistance of . %. the clinical findings showed excellent effect of the new macrolides including clarithromycin and azalides */azithromycin. we conclude the resistance of gram /'// cocci and especially of s. aureus to erythromycin increases very quickly and has reached dramatical extent. by now, the new , , and -membered ring macrolides and azalides show high antibacterial activity and good clinical effect. pappas ga, liberopoulos e, tsiara s, elisaf m, tsianos e. university hospital, internal medicine, ioannina, greece quinupristin/dalfopristin (q/d) is a novel injectable streptogramin antibiotic which initiation in therapeutics was hailed as an important step towards treatment of vancomycin-resistant enterococcus faecium (vref) species. initial reports concluded in an excellent response of vref to q/d. reports of q/d-resistant strains of e. faecium have emerged, both in usa and europe. we report two cases of e. faecium bacteremia in which the responsible isolate was not sensitive to q/d. the first patient was a woman with acute leukemia and septicemia. e. faecium was cultured from blood samples: the species was resistant to almost all antibiotics, exhibiting sensitivity only to tetracycline (!), while its sensitivity to q/d was indermediate. the second patient was a man with endocarditis, in whom blood cultures isolated e. faecium sensitive to a number of antibiotics, including ciprofloxacin and vancomycin; still the sensitivity to q/d was indermediate. q/d has not been officially introduced to the antibiotic arsenal of greek medicine. moreover, the drug has never been used in our hospital, not even experimentally. other e. faecium species isolated in our hospital have been sensitive to q/d. how much hope can be put on a drug that seems to be partly useless before its initiation? increasing reports of v/ q-resistant strains of e. faecium from all over europe raises fears that the v/d story might well end before it begins. varadinova t a , diakov t a , karagiozova d a , genova p a , pardon p b , baudry c b , quideau s b . a sofia university, virology, sofia, bulgaria , b et institut du pin, centre de recherche en chimie moleculaire, universite bordeaux , bordeaux, france vegetable tannins posses a wide range of biological activities. the aim of the present study was to evaluate the cytotoxicity of five purified vegetable tannins against mdbk cells. the maximal nontoxic concentrations (mnc) and the concentrations required to inhibit cell growth by % (cc ) were evaluated after , and h periods of action. mnc values after h indicated that compounds stimulated cell surveillance when applied in concentrations lower than . mm. cc values indicated: (i) a decrease in cytotoxicity after h as cc were up to times lower than those observed after the h period, and (ii) a re-increase in cytotoxicity when the period of action was prolonged up to h as cc were times lower than those observed after the h period. these data thus appear to reveal the capability of the investigated natural polyphenolic products to stimulate cell surveillance in a time-dependent manner. antibacterial effect survey of enoxolone on periodontopathogenic and capnophilic bacteria isolated from specimens of patients with periodontitis ps salari mh a , kadkhoda z b , sohrabi n a . a tehran university of medical sciences, pathobiology, tehran, islamic republic of iran , b tehran university of medical sciences, dentistry, tehran, islamic republic of iran objectives: most of the microorganisms associated with periodontitis are capnophilic and anaerobic bacteria. purpose of this study was to detection of antibacterial effect of enoxolone on periodontopathogenic and capnophilic bacteria. methods: in this study periodontopathogenic and capnophilic bacteria were isolated from specimens of patients with periodontitis by culture method. an anti-bacterial activity of enoxolone against these microorganisms was investigated by minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) methods. results: based on our findings the mic, mbc and lethal does (ld ) of enoxolone for actinobacillus actinomycetemcomitans , eikenella corrodens and capnocytophaga were ' , , ', ' , , ', and ' , , ' mg/ml, respectively. conclusion: our results show enoxolone has antibacterial effect on a. actinomycetemcomitans , e. corrodens and capnocytophaga spp. sakalova stn. medical university, microbiology, grodno, belarus we have synthesized diamides of dicarboxylic acids, with components such as -nitrothiazole benzolsulphamides and triazol. all the above compounds exhibited bacteriostatic activity towards some microorganisms. for further studies of bacteriostatic activity of amides and diamides of dicarboxylic acids, as well as for determination of 'structure Á/activity' relationship, we have synthesized a range of monoamides. antibacterial activity of the synthesized compounds was studied in vitro by agar dilution methods. for this purpose, approximately various gram-positive and -negative microorganisms including clinical strains of staphylococcus aureus , bacillus subtilis , serratia marcescens , escherichia coli , proteus morganii , micrococcus lisodeicticus , staphylococcus epidermidis , shigella sonnei , salmonella typhimurium , yersinia enterocolitica . minimum inhibitory concentration was expressed in mg/ml. nitazole was used as a comparison substance. analysis that new derivatives of -nitrotiasole have high antibacterial activity relative towards certain microorganisms included strains obtained from infection department's patients. results will be shown. microbial susceptibility to the essential oil of ziziphora clinopodiodes lam. ps purpose of the study: antimicrobial activities of essential oils vary from oil to oil and from one micro organism to another. the antimicrobial and chemical properties of essential oil from ziziphora clinopodiodes lam. has not been studied and hence the present study was planned to evaluate those properties against a series of micro organisms viz, escherichia coli , staphylococcus aureus , pseudomonas aeroginosa , klebsiella pneumonia , bacillus subtilis , bacillus licheniformis , streptococcus faecalis , candida albicans and saccharomyces cerevisiae . results: z. clinopodiodes lam. essential oil was found to have remarkable antimicrobial property against all the microorganisms but p. aeroginosa . the oil exhibited its best antimicrobial activity within a maximum of min. seventeen components were identified by gas chromatography and mass spectrometry (gc and gc/ms) analysis of the oil, out of which pulegone ( . %), neomenthol ( . %), cyclohexene, -methyl- -( -methenyl)trans ( . %), , -cycloheptadien- -one, , , -trimethyl ( . %), piperitone ( . %), and limonene'/ , -cineole ( . %) constitute major parts of the oil. conclusion: monoterpenes seem to have antimicrobial role. it seems necessary to explore antimicrobial properties of new harmless antimicrobial agents from natural sources as substitutes for common chemical drugs. methanol extract of carpobrotus edulis enhances killing of methicillin resistant staphylococcus aureus phagocytosed by thp- human monocyte derived macrophages and promotes the release of modulators of cellular immunity ps although alkaloids from the family mesembryanthemaceae have anti-cancer activity, species of this family have received little attention. because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, we have studied a crude methanol extract of carpabrotus edulis , a common plant found along the portuguese coast, for properties normally associated with plasma membrane active compounds. the results of this preliminary study show that the extract is non-toxic at concentrations that prime thp- human monocytederived macrophages to kill ingested methicillin resistant staphylococcus aureus and promote the release of lymphokines associated with cellular immune functions. the extract also induces the proliferation of thp- cells within day of exposure and days earlier than that induced by phytohemagglutinin. similar results were obtained with monocyte-derived macrophages isolated from human peripheral blood. the active component or components of the plant extract may be exploited as intracellular active anti-bacterials as well as modulators of cellular immunity. enhancing of erythromycin production by saccharopolyspora erythraea nur with common and uncommon oils ps hamedi j a , malekzadeh f a,b saghafi-nia ae b . a department of biology, faculty of science, university of tehran, tehran, islamic republic of iran , b shafe-e-sari co., antibiotic production co., teheran, iran enhancing effect of various oils on the erythromycin production by saccharopolyspora erythraea nur were evaluated in a complex medium consisting soybean flour and dextrin as the main substrates. the biomass, erythromycin, dextrin and oil concentrations, and ph value were measured on a daily basis. also, the kinds and frequencies of fatty acids of the oils used were determined. saturated fatty acids in the shark oil were higher than that of vegetable oils used. erythromycin concentration in the melon (cucumis melo var. inderus cultivar mashhad ) seed oil containing medium was . times higher than that of the control medium (without oil) and . times higher than that of rapeseed oil containing medium. erythromycin concentration in the other oil containing media, including rapeseed, soybean, shark (carcharhinus dussumieri ), and safflower oils was . , . , . , . time higher than that of control medium, respectively. the melon seed oil had the least enhancing effect on the biomass production, and thus decreasing the cost of the biomass separation. can varicella be eliminated by universal childhood vaccination ? */ epidemiological and economic data from germany ps wutzler p a , banz k b , neiss a c , goertz a d , bisanz h d . a institute for antiviral chemotherapy, university of jena, jena, germany , b outcomes international, basle, switzerland , c institute of medical statistics and epidemiology, technical university, munich, germany , d glaxosmithkline, pharma, munich, germany purpose: universal varicella vaccination in childhood is expected to reduce substantially the number of uncomplicated cases of chickenpox and to decrease the number of complicated cases requiring hospitalization. to generate fundamental data for decisions of the health authorities epidemiological and health-economic data were collected in two large studies. using an age-structured decision analytic model the benefits, costs and cost-effectiveness of a varicella immunization program over a period of years were assessed. results: it could be shown, that the vast majority of varicella cases occur in children aged less than years. in . % of the cases a severe course was assessed. overall incidence of complications was estimated to be . %. a routine varicella vaccination program targeting healthy children could prevent . % of varicella cases and over major complications per year provided the coverage rate would be %. under these conditions the elimination of varicella is predicted to be achievable within Á/ years. a combined measles, mumps, rubella and varicella vaccine is expected to provide the required coverage. conclusions: routine childhood varicella vaccination appears to be a highly efficient strategy to significantly reduce the sizeable burden of varicella and leads to significant savings from both societal and payer's perspective. bulgakova va, balabolkin ii, sentsova tb. scientific center of child health, russian academy of medical sciences, scientific research institute of pediatrics, moscow, russian federation objective: to estimate efficacy of vaccine influvac at children with allergic diseases. methods: twenty children aged Á/ years with allergic diseases received vaccine influvac (solvay pharma). for the control group of children, with allergic pathology did not receive this vaccine because of an intolerance of chicken protein ( children). results: all vaccinated children for the observable season of months did not get influenza. general and aboriginal reactions to a vaccine did not occur. in control group for the observable season two children were ill with influenza and four children with acute respiratory virus infection ( %). among vaccinated children there was an increase in titre to a protective level ( : and above) to all to three strains of influenza days after injection. vaccine influvac can be recommended for an immunisation against influenza of children with an allergic pathology because of efficacy and absence of side effects. ló pez h, zitto tr, vidal gi, cánepa mc, salomó n jm, scaglione m. centro de infectología, infectious diseases, buenos aires, argentina objectives: our study examines the possible economic impact of the influenza on health working adults in argentina, and the intervention cost saving with immunization. methods: this is a theoretical study based on a mathematical model. population data was published in s national statistics. the global incidence of influenza infection was estimated at %. we have estimated the direct cost on influenza infection (outpatient visits, drugs and hospitalization) and indirect cost (work absenteeism and productivity loss) and projected net saving for the Á/ year-old vaccinated group. the vaccine effectiveness was estimated at and %. the price of vaccine was $ each. results conclusion: influenza vaccination is effective in diminishing cases of flu and reducing working-day loss. its a safe and cost-effective vaccine. ló pez h, zitto tr, cánepa mc. centro de infectología, infectious diseases, buenos aires, argentina flu infection is a major cause of illness and one of the most common cause of work absenteeism, increasing institution costs, healthcare provider visits, use of drugs, and decreasing work productivity. vaccine against flu has an effectiveness between and %, in health adults. objective: evaluate the impact of flu-like respiratory tract infections in a health institution staff during year, comparing vaccinated with not-vaccinated groups. methods: we evaluated all causes of absenteeism along year ( ), based on the written note made by the professional who has evaluated ill people, selecting flu-like respiratory infection causes. we evaluated age, working days loss related to illness, and cost on vaccinated and not-vaccinated groups. results: one hundred and sixty eight of the total staff ( people) were vaccinated, of them had flu-like infection, resulting in working days lost. for not-vaccinated group, people had flu and lost days. lost cost for vaccinated group was of $ , and for notvaccinated group, $ . conclusion: we observed a decrease in working days loss and money waste related to flu-like infections on the vaccinated group. because of safety and effectiveness of vaccine against flu, the implementation of vaccination will be cost-effective for all institution staff. we studied functioning of the interferon system in children with atopic bronchial asthma (ba) at the age of Á/ years. the control group included healthy children. we investigated the interferon status (method of grigoryan s.) and serum concentrations of ifngamma (ifng) (elisa). there was a decrease in the ifn-producing ability of leukocytes to the synthesis of ifna at % and ifng at % of children with ba. serum level of ifn of children with ba during all period of illness is compared to the children without predisposition to atopy ( . / . and . / . pg/ml accordingly) was significantly decreased. production of ifna increased after using viferon (recombinant a b ifn and antioxidants). decreased ability of gammainterferonogenesis in the most children was not affected by the action of immunomodulators. there was shown interferon system's dysfunction in the development of atopy and increasing predisposition to respiratory infections and to persistent of atypical infections in children with ba. harxhi a, pilaca a, pano k. university hospital center of tirana, infectious diseases, tirana, albania congo-crimean haemorrhagic fever is viral disease with a high rate of mortality that is caused by a nairovirus, bunyaviride species. this is a zoonotic disease, which affects sporadically humans and is geographically distributed even in eastern europe and balkan. during the months of may and june , in northeast of albania were reported eight cases of haemorrhagic fever. serologic tests performed in the laboratory of reference in thesaloniki, greece confirmed the diagnosis of congo-crimean haemorrhagic fever. in the mean time, who reported the outbreak in southwest kosovo of cases suspected for haemorrhagic fever from which were confirmed laboratory as congo-crimean haemorrhagic fever. we are describing here the clinical history of one of eight cases with cchf in albania. from the epidemiological point of view this case was considered peculiar, as it was the only one hospitaly acquired, and due to the gravity of the haemorrhagic syndrome was admitted at the intensive care unit at infectious diseases service, university hospital center of tirana. results: in the study were included patients ]/ years of age with documented hbsag-carrier ]/ months (average age */ . years, male */ %, female */ %). hbv dna in serum was tested by qualitative and quantitative pcr (commercial test-system ampli-sens hbv). hbeag, hbeab, hbsag were detected by elisa (hoffmann la roche). hbv dna by qualitative pcr was detected in % patients, by qualitative pcr was detected in % patients in the concentration ]/ copies/ml, in . % ]/ copies/ml, in . % ]/ copies/ml, in . % ]/ copies/ml ( fig. ). hbv dna level distribution among the hbsag carriers. elevated ( . the upper limit of normal) alt level was determined in . % of the hbv dna negative and . % of the hbv dna positive patients. hbeag was detected in . % of the hbv dna positive patients and had not been determined in the hbv dna negative patient. eleven percent of patient had the combination of the biochemical, serological and virology criteria, which are typical for active chronic hepatitis b (hbsag-carrier !/ months, hbv dna ]/ )/ copies/ml, elevated alt). conclusion: in smolensk % of the hbsag-carriers have viral replication confirmed by qualitative pcr. eleven percent of them have active chronic hepatitis b. kandemir o a , polat a b , kaya a a . a medicine of faculty, mersin university, clinical microbiology and infectious disease, mersin, turkey , b medicine of faculty, mersin university, pathology, mersin, turkey the exact potential of nitric oxide in the pathogenesis of chronic viral hepatitis is not known. the elevated nitric oxide production is assumed to be responsible for the pathological changes in many inflammatory conditions, mainly via peroxynitrite, a potential oxidant that is produced by the reduction of superoxyde anion with nitric oxide. the intensity and the distribution of the immunohistochemical staining of intrahepatic inducible nitric oxide synthase were studied in the biopsy specimens obtained from patients with viral hepatitis and patients with elevated transaminase levels from other etiology. hepatic inducible nitric oxide synthase staining was significantly more intense in the viral hepatitis group (p / . ). inducible nitric oxide synthase staining levels correlated well with the severity of the viral hepatitis using the knodell's liver histological activity index (r / . , p / . ). among the viral hepatitis group, the pathological distribution of the inducible nitric oxide synthase staining favored the periportal regions whereas less staining was observed in the bile duct and parenchyma regions. as nitric oxide mediated nitration of hepatocellular proteins is found to be elevated in the inflamed hepatic tissues and it well correlated with the severity of the disease, we suggest that inducible nitric oxide synthase can possibly have a critical role in the pathogenesis of chronic viral hepatitis. there were patients under observation who were divided into two groups, the first of patients (eight chronic virus hepatitis; three chronic virus hepatitis'/steatosis; four steatohepatitis; three chronic cryptogenic hepatitis; there were men and four women aged from to . the second group consisted of patients, eight with chronic virus hepatitis; four with steatohepatitis; six with chronic cryptogenic hepatitis. there were men and three women aged from to . diagnosis was confirmed with the help of clinical data, biochemical tests, serological markers, psr-diagnostics and ultrasound examination and computer tomography of the abdomen. in the first group of patients the treatment with ursofalk was administered at the dosage of mg/kg of body mass from month to years with improvement in general condition of the patients: heaviness, pain under the right rib, nausea and skin itch have disappeared. in all the cases, improvement in the biochemical blood analysis took place during treatment. the average index of alt activity was before */ . u/l, after */ . u/l and ast */ . and . u/l; alp */ . and . u/l; ggt */ . and . u/l; chol */ . and . mmol/l; tg */ . and . mmol/l. in the second group of patients the treatment was carried out with various hepatoprotectors during the courses from to months. before the average index of alt activity was . u/l, after */ . u/l; ast */ . and . u/l; alp */ . and . u/l; ggt */ . and . u/l; chol */ . and . mmol/l; tg */ . and . mmol/l. treatment of patients suffering from hepatitis of viral and other aetiologies with ursofalk produces a positive effect on both clinical symptomatic and biochemical indices. remission was more stable during a long period of taking the preparation. the hepatoprotective effect of ursofalk during the years was sustained for the whole of the period of the treatment. after stopping, an acute attack of cytolytic syndrome was observed. with other hepatoprotectors we did not get any improvement in clinical scene of the disease or in biochemical indices. shavlov nm, kletsky sk. minsk, belarus hsv- and - have possibility to damage different organs and systems. sometimes they cause damage of the liver, which resemble viral hepatitis. the etiology of such hepatitis may be confirmed only by results of liver biopsy. we have diagnosed cases of herpetic hepatitis: eight children and four adults. clinical course was different. in five cases the acute beginning took place: high temperature, the jaundice at the Á/ day (the level of bilirubin was Á/ mkmol, especially direct), cholestasis, the pain in upper right part of abdomen. the ascites was found in three patients with acute hepatitis during st week from the beginning of disease. in seven cases the beginning was gradual. the temperature was subfebrile, prolonged; malaise and moderate pain in upper part of abdomen were constant complaint. the jaundice was moderate; bilirubin increased until mkmol. the level of alt was moderately increased ( Á/ times). the blood analysis showed moderate leukocytosis with neutrophilia, and increased sre. the serological markers of hepatitis a, b, c, d were negative in all cases. hsv- and - were found in the blood. the diagnosis was defined by the results of hystochemical investigations, when the viruses were found in liver bioptat, and confirmed with the results of specific treatment. specific damage of liver cells was found: protein dystrophy and specific inclusions in cell nucleus. in all cases the treatment with acyclovir were given. the results we have observed during st week: the temperature became normal, the jaundice decreased and bilirubin was normal during Á/ days. in one case the recidive took place weeks later after treatment. the second course of acyclovir with intron a gave good results. nesic z a , delic d b , prostran m a , vuckovic s a , stojanovic r a . a department of pharmacology, school of medicine, university of belgrade, belgrade, yugoslavia , b clinical center of serbia, institute for infection and tropical disease, belgrade, yugoslavia the large number of unsolved cases of acute and chronic hepatitis has most probably the viral etiology. in mid s, two independent groups of authors reported a new human hepatotropic virus, with flavivirus like genomes, hepatitis g virus (hgv). the aim of this pilot study was to determine the prevalence of hepatitis g viral infection among patients at high risk of exposure to blood and blood products, as well as to evaluate if the risk of hgv infection was higher among them than in the general population. immunoenzyme test on microtitration plate for detection antibodies against hgv e antigen in plasma or sera (r&d systems, minneapolis, usa) was used for evidencing anti-hgv igg antibodies in sera. anti-hgv antibodies were detected in the control group (blood donors) in . % ( / ) patients. prevalence of anti-hgv antibodies among i.v. drug users was evidenced in . % ( // ), in hemophiliacs . % ( / ), in patients acquiring multiple blood transfusion . % ( / ), in hemodialyzed patients . % ( / ) and in patients with transplanted organs . % ( / ). our results suggest that patients exposed to blood or blood products have a higher risk of hgv infection than general population. evaluation of ortho total hcv core antigen assay in assessment and follow-up of patients treated for chronic hcv ps lunel f, veillon p, payan c. chu angers, laboratoire de bactério-virologie, angers, france an assay to quantitate 'total' hvc core antigen (hcv ag) in serum or plasma, may reflect viral load, has been developed by ortho-clinical diagnostics. methods: we evaluated hcv ag with two quantitative assays for hcv rna: bdna . (bayer) and monitor . (roche). we studied samples from untreated patients and from patients with chronic hcv treated with ifn or ifn/ribavirin. results: correlation of ag and quantitative assays was high (r / . for bdna . and . for monitor . ). no difference between the levels of rna and ag among hcv genotypes (r / . Á/ . ) was found. ag values, before treatment, were significantly lower in sustained responders (sr) than in other groups ( . log versus log, p b/ . ). in patients treated with ifn or combination therapy, we found very good correlation between decrease and negativation of ag and viral load: log iu/ml decline after m of interferon was significantly correlated with the negativation of hcv ag and sr. thirty-eight/ of sr had a rna load decrease !/ log iu/ml and / had a negativation of hcv ag after m . conclusion: total hcv core ag appears to be a new tool for monitoring patients with hcv infection. hepatitis c virus rna and hcv core antigen kinetics predict the efficiency of interferon-alfa and ribavirin therapy in naive patients infected by hcv genotype or ps fifty-five patients infected by genotype or were treated with a primary dose of (if hepatitis c virus (hcv) rna b/ meq/ml) or million units of interferon alfa- b (ifn) thrice weekly for months. ribavirin was added at month (m), until m if hcv rna was found positive after m of ifn. the viral kinetic was assessed during the follow up by serial measurements of hcv rna (bdna . and monitor . ) and using a new assay from ortho-clinical diagnostics which is able to quantify total hcv core antigen. sustained virologic response was observed in % of the patients ( / ). after month of ifn treatment, sustained responders had a fall of hcv rna and hcv core antigen higher than non-responders ( . / . log ui/ml versus . / . log ui/ml, p b/ . , for hcv rna) and ( . / . log (pg/ml)/ ) versus . / . log (pg/ml)/ ) p b/ . , for hcv core antigen). after month of ifn, the positive and negative predictive values of sustained response were, respectively, and . % for hcv rna negativation and . and . % for hcv antigenemia negativation. these results suggest that both kinetics of viral load and antigenemia are highly predictive of sustained response. theodorou m, petinelli i, pontikaki d, mela c, blana a, papanastasiou a, toliopoulos a, stavrakaki m, sagkana e. microbiology department, western attika general hospital, greece, egaleo, greece greece has accepted a big number of economic immigrants lately. we investigate the prevalence of hepatitis b/c as well as the epidemiological features that might influence the public health. . economic immigrants from: albania , eastern europe and from asiatic-african countries , visited our hospital to be checked in order to get a health certificate to obtain the green card. they where tested for hepatitis b/c. the serological markers were determined by immunoenzymatic method. all hbsag('/) and anti-hcv were further tested for hbv dna and hcv rna by competitive rt pcr. hcv rna('/) were genotyped by strip hybridization immunoassay. in albanians . % were hbsag('/), . % hbv dna('/), . % anti-hcv('/) and . % hcv rna('/). in east europeans . % were hbsag('/), . % hbv dna('/), . % anti-hcv('/) and . % hcv rna. in asians-africans, . % were hbsag('/) and % hbv dna('/). in pakistanis, . % were anti-hcv('/) and . % hcv rna('/). of the rest of asians-africans, . % were anti-hcv('/) and . % hcv rna('/). albanians: higher prevalence of hbv infection ( . %). greek blood donors: % pakistanis: hcv infection is % (predominance of a type), general greek population: %. public health services in greece and europe must take appropriate measures. el zawawy la a , mohamed on b , ali sm a , eissa me a , allam sr a . a faculty of medicine, parasitology, alexandria, egypt , b high institute of public health, microbiology, alexandria, egypt the purpose of the study was to investigate the influence of schistosomal suppression on the antibody response to hepatitis-b vaccine (hbv) and to study if the vaccine has any protective effect on experimental () infection. the results obtained revealed that infection reduced the serum antibody level against hbv. parasitological and histopathological findings showed significant protection against infection. the conclusion reached was; in order to reduce the incidence of virus-b infection especially in schistosomiasis endemic areas, public health officials should evaluate a policy for regulation of hbv booster vaccination to enhance the population immunity against hepatitis-b infection. cooper e, fisher t, shingadia d. newham general hospital, family clinic, london, uk sustained anti-retroviral combination chemotherapy requires excellent adherence to the regimen so as to suppress viral replication sufficiently to delay the emergence of resistance. if chemotherapy were taken to scale, e.g. in africa, erratic adherence might soon lead to multi-resistant circulating virus. we reviewed our experience in a well established london family clinic with a team including community nurses. we reviewed the records of the african immigrant children, aged Á/ , treated with anti-retrovirals exclusively at our centre throughout . whereas had undetectable hiv rna within the year, only four had undetectable rna throughout the year. four failed therapy through proven resistance mutations, but nine were considered through circumstantial evidence to have rising viral loads primarily because of poor adherence. three were known to have stopped taking drugs for extended periods. the three boys over years were unreliable in adherence, but the one girl in this age-group was fully adherent. our preliminary assessment is that for the children in our families, despite a team approach and home visits, nonadherence to haart may be twice as common as selection of a dominant viral mutant as a primary cause of failure to sustain viral suppression. quiros-roldan e, moretti f, castelli f, el-hamad i, carosi g. the prevalence of hiv related lipodystrophy-syndrome depending on the definition and severity of lipodystrophy ranges from to %. we have retrospectively reviewed the medical records of african patients followed. the characteristics are shown in the . % of africans had triglycerides !/ mg/ml and . % had cholesterol !/ mg/ml, none had both metabolic alterations. glycemia !/ mg/ml was observed in . % patients. it is interesting highlight that in any the africans morphological changes were noted and all of them showed weight stable. although the low prevalence of metabolic alterations may be attributed to the different ethnic alimentary behavior if self-body perception by african is not as accurate as by caucasian on the estimation of the body changes have to be investigated. alabaz d a , alhan e a , yaman a b , evliyaoglu n a , kocabas e a , aksaray n a . a division of pediatric infectious diseases, cukurova university, adana, turkey , b department of microbiology, cukurova university, adana, turkey hepatitis a virus (hav) infection is usually asymptomatic in children. however, it may occasionally cause a severe disease with high morbidity and mortality, and loss of school or business days. in a previous study, we have shown that every one of two to three school children from upper social classes living in adana carries high risk of hav infection. it is well known that maternally transmitted anti-hav antibodies interfere with hav vaccination. in an effort to determine the optimal age for hav vaccination, babies ( % girls and . % boys) born in our hospital were prospectively followed up at least months for the presence of maternal antibodies to hepatitis a (anti-hav igg). anti-hav igg titers were measured from the blood specimens obtained at birth from the mothers and from the offsprings at months, , , , , , , , and . the prevalence of positive anti-hav at birth ( %) was similar to those of hav seroprevalance studies carried out in adults in our area. the disappearance of antibodies occurred between the st and st month of life. the prevalence of anti-hav igg among children aged , , , , and months were , , , , and %, respectively. in light of these findings, we suggest that hepatitis a vaccination be given after months of age. earlier vaccination may be ineffective due to interference with maternally transmitted anti-hav antibodies. ghaderi b, alaghebandan r, rastegar lari a. department of microbiology, iran university, tehran, islamic republic of iran diarrhoea is a major public health problem in developing countries. amoebiasis is one of the most common causes of diarrhoea in iran as an endemic area for amoebiasis. little, however, is known about the extent of the condition in our society. the aim of this study is to determine socio-demographic and clinical characteristics of patients with intestinal amoebiasis. during july and august , we collected patients with diarrhoea among patients who visited at a referral hospital in shahriar area (in countryside of tehran), iran. thirty out of patients ( %) had intestinal amoebiasis and were followed up prospectively until the resolution of the illness. nineteen of ( . %) patients were male and the remaining of . % was female. the patients were aged Á/ with mean of . years. most of the patients ( %) were below years of age and the peak of occurrence was between the age of and years. watery diarrhoea with abdominal cramps was the main clinical feature. seventy percent of patients were resident in urban area and the remaining ( %) in rural area. average family income was low and all patients were in low socioeconomic level. water supplying system for all patients was pipeline water. low socioeconomic level associated with poor personal hygiene was the most important factor for highly prevalence of this problem in our society. also it seems that food plays important role in transmission of protozoa then water. the new strategy for allele identification of the genes coding for pertactin and pertussis toxin subunit s in bordetella pertussis ps bordetella pertussis strains demonstrate a significant polymorphism in toxin s subunit and pertactin, which are major protective antigens of the organism. monitoring the changes in prevalence of particular alleles of genes coding for these proteins in local b. pertussis populations is an essential issue in cases of the observed decrease of vaccination effectiveness. we have developed a new method for allele identification of these genes, which eliminates the necessity of dna sequencing. the approach is based on the identification of the number of repeats or the presence of specific nucleotides in the polymorphic regions or residues, respectively, of the genes and utilises products of their full or partial pcr amplification. the nucleotide heterogeneity in each polymorphic site is analysed either by the differential digestion of the amplicons or by the arms (amplification-refractory mutation system) methodology. numbers of repeats in particular regions of the genes are revealed by the size analysis of the adequate pcr products or their restriction fragments. in all cases the presence, size or pattern of dna molecules obtained is visualised by the agarose gel electrophoresis. the preliminary analysis of the recent and archival b. pertussis strains identified in poland was performed using the described approach. the presented strategy provides a much easier, faster and more cost-effective than dna sequencing mean to study the polymorphism of the major b. pertussis antigens. vaccination coverage and history of vaccine preventable infectious diseases among students in second year of medicine and pharmacy of tours university ps borderon jc a , hamed a b , ragot s b . a centre hospitalier universitaire, tours, france , b médecine préventive universitaire, tours, france the purpose of this study was to determine the level of infectious risk in students who will be exposed to patients. information was obtained by a questionnaire for each student, and by checking medical records for immunization coverage and vaccine preventable infectious diseases. answers could be specified for students, of whom females (f) and males (m). the number of non-immunized students was against diphtheria: two, tetanus: three, pertussis: four, poliomyelitis: two, hepatitis b: six, and hepatitis a: , respectively. among the students non-vaccinated against measles, (nine f and five m) had no history of that disease. among ( f and m) non-vaccinated against rubella, ( f and seven m) had no history of that disease, uncertain in seven others (six f and one m). the date of vaccination was often late regarding recommendations. fifteen students had no history of varicella. one student had not received bcg vaccination. fifty-eight students had received two, and three bcg vaccines. post-bcg tuberculin skin testing was missing after first bcg, second and third bcg. the date of the first tuberculin test was often one or several years after bcg vaccination. adverse effects of vaccination were rarely reported: two cases of fever (dt polio, measles); three cases of local reaction (dt polio, dtp polio). one case of contraindication for influenza vaccine: egg allergy. the survey shows failure in immunization coverage actually recommended in health care students. objectives: to present the morbidity of rabies and evaluate the efficiency of our prophylaxis scheme in lasi county. material and method: we made a retrospective study of the rabies cases in the patients admitted in our unit in a th-year period. we have analysed all the clinical, epidemiological and biological aspects. results: in a -year period, cases of rabies were admitted in the clinical infectious diseases hospital lasi. the highest incidence was for Á/ */eight cases ( %); the highest yearly cases were three cases in and . most of the patients were male ( %), came from suburban areas ( patients). eight cases occurred in may Á/june, wild animals were involved in half the cases (fox, wolf). for patients, no prophylaxis was performed and an incomplete course in four cases. the period of time to the appearance of the first symptom was Á/ days. the prophylaxis scheme led to a good protection. conclusions: in lasi county, rabies is a problem with a prevalence of . %/year. trends in the use of antimicrobials in riyadh in were analyzed. data was obtained from a survey of randomly selected families of school children aged Á/ years in a -month period in . one hundred and ninety-nine ( . %) students were on antibiotics in the month preceding the study; ( %) received antibiotics for the diagnosis of pharyngitis; ( %) students antibiotics were prescribed by a physician; and in ( %) the duration of antibiotics was less than week. this study shows a major problem in antibiotics prescription in our community and also the need to establish effective antibiotics policy in general practice to limit the potential emergence of drug resistance bacteria in the community. mir s a , cura a a , erdogan h a , guler s a , sengul gn a , koyu a a , ozinel ma b . a department of pediatrics, ege university medical faculty, izmir, turkey , b department of microbiology, ege university medical faculty, izmir, turkey antibiotic susceptibility spectrum of childhood urinary tract infection agents are geographical variation. the current antibiotic regimens and the selection of antibiotics for prophylaxis should be re-evaluated periodically. the objective of our study was to determine the local resistance rates to antibiotics and to give a direction for the selection of antibiotics in uti treatment. we evaluated urinary culture assays retrospectively, sent from pediatrics and pediatric surgery inpatient and outpatient clinics of our hospital during the last months, and investigated the isolated pathogens and the resistance rates to antibiotics. in addition, the data obtained were compared with of years ago. with respect to the resistance rates to antibiotics of uti pathogens, the resistance rates of e. coli for carbapenems, aminoglycosides and third generation cephalosporins were , and %, respectively as before, but the rates for ampicillin increased from to % and for tmp-smx it increased from to %. we concluded that the resistance profiles to antibiotics should be reviewed every years at least and thus the selection of proper antibiotics would lessen the morbidity as well as the medical expenses. chanturidze tk, tsiklauri r. ministry of labor, health and social affairs, public health, tbilisi, georgia purpose: since infectious diseases (id) are increasing in georgia. this study is aimed to reveal economic barriers of effective id control by assessing financial contribution to id from public and private sources, household's total spending on health and their capacity to pay. sources: ) national household expenditure and revenue survey. ) who fair financial contribution methodology. ) meta-analysis. results: . % of population leaves under the poverty level; % out of total household expenditure (average gel; us$ ) % is spent on food */non-subsistence income covers expenditures on goods and services including health; % of population refuses health services because of inability to pay; public spending on health comprises % of total health expenditures; public spending on id control is below gel per capita; almost all private spending goes to id treatment and equals to . gel per patient. conclusions: insufficient public spending on id control transfers the burden to the population with extremely low capacity to cover health expenses. refusal to utilize health services, and incomplete treatment and increases the threat of id spread and drug resistance. government should increase the allocations to id from public sources for effective id control in georgia. antimicrobial consumption trends in children's university hospital ps ratchina s a , averchenkova l b , jarkova l a . local surveillance of antimicrobial (am) consumption is essential to promote the rational use of this group of drugs. the purpose of this study was to analyze the trends of am use in the children university hospital in and . data on am usage were obtained from the hospital drugstore requests in the -beds multi-ward children university hospital. consumption was expressed as the number of ddd per bed-days (b Á/d). the total am consumption figures were similar in and ( . and . ddds/ b Á/d, respectively) with notable differences in am prescribing patterns. penicillin consumption increased from . to . ddds/ b Á/d mostly due to amoxicillin. the overall aminoglycoside usage remained comparable ( . vs. . ddds/ b Á/d) though amikacin has considerably replaced gentamicin. there was a sevenfold increase of ciprofloxacin ( . vs. . ddds/ b Á/d) along with the evident decrease of tetracycline and co-trimoxazole consumption found ( . vs. . ddds/ b Á/d and . vs. . ddds/ b Á/d, respectively). the tendency to prescribe more effective in respect of the local resistance data and/or more safe am was detected in comparing with that can be explained by the introduction of the local guidelines for infectious diseases management in . clarithromycin in the treatment of chronic prostatitis caused by chlamydia trachomatis */a pilot study ps the aim of this pilot study was to determine the efficacy and tolerability of clarithromycin in the treatment of chronic prostatitis caused by chlamydia trachomatis (ct). fifty-two patients older than years of age with diagnosed chronic chlamydial prostatitis were enrolled. the presence of ct in expressed prostatic secretion or urine specimen voided immediately after prostatic massage was confirmed by isolation on mccoy cells and lugol staining. the majority of patients suffered from suprapubic pain and pain in the groin. twelve patients had no clinical symptoms. according to rectal palpation, prostate gland was normal in patients, tender and soft in and firm in five patients. clarithromycin was administered orally mg twice daily for days. simultaneously the patients' partners received mg orally twice daily for days. clinical efficacy and tolerability of administered clarithromycin were evaluated Á/ days and Á/ weeks after the end of treatment. bactericidal efficacy of administered drug was evaluated Á/ weeks after the end of treatment. the eradication of ct was achieved in out of patients, while patients were clinically cured. two patients had nausea and elevated serum transaminases. in asymptomatic patients, the eradication of ct was achieved in of patients who reported no side effects. this pilot study has shown an excellent efficacy and tolerability of clarithromycin in the treatment of patients with chronic chlamydial prostatitis. women with diagnosis of urinary tract infections (uti) often demonstrate vaginal colonisation with alpha-haemolytic escherichia coli strains. in the present study we decided to evaluate a distribution of virulence genes encoding for cytotoxic necrotizing factor type (cnf ), p-fimbriae, s/f c-fimbriae aerobactin (aer), and afa genes in alpha-haemolytic e. coli strains isolated from gynaecological material in our region and to compare the detected sequences in clinical isolates of other diagnostic groups. of alpha-haemolytic e. coli strains, were isolated from urine, from gynaecological specimen, and were faecal strains. e. coli strains were tested for the production of haemolytic phenotype on blood agar plates. the amplification of virulence factors was performed by pcr according to previously described protocols (le bouguenec et al., ; blanco et al., ; and yamamoto et al., ) . we found that all gynaecological alphahaemolytic strains were positive for cnf , (p b/ . compared to % of urine strains, and p b/ . compared to % of faecal strains). similarly, sfa/foc specific dna sequences were found in % of gynaecological isolates (p / . compared to % of urine strains and p / . compared to % of faecal strains). from this point of view, the female genital tract seems to be a potential reservoir of these uropathogenic e. coli strains. azithromycin in the treatment of pelvic inflammatory disease caused by chlamydia trachomatis ps administered in hospitalized patients with chlamydial pid. the diagnosis was made prior to hospitalization. microbiological analysis of urine, blood and swab specimens collected from endocervix, vagina and urethra confirmed c. trachomatis to be the single suspected causative pathogen of pid. the presence of c. trachomatis in swab specimens from endocervix was examined by dnk/rnk hybridization. azithromycin was administered Á/ days after samples for microbiological analysis were collected in dose of )/ mg iv for days. clinical efficacy and tolerability of therapy were assessed Á/ days after the end of therapy and clinical and microbiological analysis Á/ weeks after completion of therapy. the eradication of c. trachomatis and normalization of gynecological findings were achieved in and disappearance of subjective symptoms in patients. no side effects and deviations from normal values in hematologic and biochemical blood parameters were recorded. this study showed high bactericidal efficacy, rapid clinical effect and good tolerability of once-daily administration of mg azithromycin for days in the treatment of patients with pid caused by c. trachomatis . altindis m a , cevrioglu s b , aktepe oc a , cetinkaya a . a kocatepe university school of medicine, microbiology, afyon, turkey , b kocatepe university school of medicine, obstetrics and gynecology, afyon, turkey diagnosis of the causative organism of the vaginitis is usually based on clinical criteria. a standardized, laboratory based and rapid diagnostic test for the identification of these organisms is desirable. to determine the laboratory method that best predicted the causative organism, we calculated the sensitivity, specificity and predictive value of positive and negative test for clinical criteria, an oligonucleotide probe test (affirm vpiii-bd usa) and compared them with the combination of positive vaginal culture and gram-stained vaginal smear. we evaluated consecutive women aged Á/ years, attending for vaginal discharge. vaginal swab specimens were used for culture of gardnerella vaginalis , trichomonas vaginalis and candida sp, preparation of a vaginal smear for gram-stain interpretation and wet mount evaluation and affirm test. affirm detected g. vaginalis in ( %), candida sp in three ( . %) women and no trichomoniasis case found by any methods. the sensitivity and negative predictive values of affirm test and clinical signs were same ( %) in identifying of bacterial vaginosis. however, affirm test was more specific ( vs %) and also has higher positive predictive value ( vs %) than clinical signs. we did not evaluate the results for patients with candidiasis because of less number. according to these results affirm-microbial identification tests are objective and specific for the rapid diagnosis of the bacterial vaginosis. comparison of efficacy of single dose of tinidazole with standard dose of metronidazole in giardia lamblia infection (preliminary report) ps fallah m a , moshtaghi aa b . a university of medical sciences, parasitology, hamadan, islamic republic of iran , b university of medical sciences, pediatrics, hamadan, islamic republic of iran objectives: giardia lamblia is the most common intestinal protozoa in developing countries. treatment of the infection with metronidazole, the drug of choice, requires a long course of therapy and produced some side effects. the object of this study is to determine efficacy and side effects of tinidazole in g. lamblia infection. this is a preliminary report of an ongoing trial. methods: a randomized controlled clinical trial was carried out and subjects with g. lamblia infection were treated with tinidazole or metronidazole. tinidazole mg/kg single dose and metronidazole mg/kg three times a day for days were given orally to and children, respectively. parasitological cure was documented when there were consecutive negative stool examinations at Á/ weeks after therapy. results: twenty-one of individuals treated with tinidazole and of children treated with metronidazole had parasitological cure. cure rates between two groups were not significant statistically. no major side effects were observed except one case in metronidazole group who had mild headache and abdominal pain for days. conclusions: we concluded, tinidazole at the dose has efficacy equal of metronidazole in the treatment of g. lamblia infection. because of single dose prescription, short course of therapy and good compliance of patients, this preparation is preferred to metronidazole in giardial infection. ebeid fa, seif el-din sh. theodor bilharz research institute, pharmacology, cairo, egypt b-carotein was given in different doses starting from . to mg/kg body weight (b.w.) for different groups of albino mice days before infection with s. mansoni . infection of animals was done by body immersion using egyptian strain of s. mansoni cercariae/mouse. forty-nine days after infection the animals were sacrificed and hepatic and mesenteric worms were extracted and determined. ova count in liver and intestinal tissue and the total number of worms/animals were also determined in experimental groups comparing with infected control animals. the results indicated marked decrease in number of worms and ova count in both liver and intestine comparing with control ones. this reduction increased significantly with increasing dose. it was concluded that b-carotein could be used as a prophylactic agent against s. mansoni infection. barisic z, babic-erceg a, borzic e, zoranic v, carev m, kaliterna v. department of microbiology, public health institute, split, croatia the aim of this study is to determine frequency of pseudomonas aeruginosa urinary tract infection (uti) in outpatient's population in south croatia and to suggest optimal antimicrobial treatment for these patients. during months long observation period, from total number of examined urine specimens, significant bacteriuria was found in specimens. p. aeruginosa was the sixth most common isolate, it was isolated from specimens ( . %). these specimens were taken from different patients. susceptibility testing was performed by disk diffusion method, and the following results were obtained: resistance to cefibuten occurred in . % patients, to norfoxacin in . %, to ciprofloxacin in . %, to gentamicin in . %, to netilmicin in . %, to amikacin in . %, to ceftazidime in . % and to imipenem in . % patients. p. aeruginosa strains showed better susceptibility to tested parenteral antibiotics than to antibiotics for oral use which complicated treatment in outpatients. the best susceptibility was shown to imipenem, but this drug is inappropriate for use in outpatients setting, so the best choice for treatment p. aeruginosa uti in our outpatients is treatment with ceftazidime, and the second choices are aminoglycoside drugs amikacin and netilmicin. silan l a , breza j b , krcmery v jr. c . a department of internal medicine, derer s university hospital, bratislava, slovakia , b department of urology, comenius university school, bratislava, slovakia , c department of pharmacology, st. elisabeth cancer institute, bratislava, slovakia we studied the clinical efficacy of oral treatment with ciprofloxacin/ cpf/ alone and combined with clarithromycin in patients with complicated urinary tract infection/cuti/ with or without an indwelling catherer. patients were randomly allocated to mg cpf/cpf group/ or to mg cpf plus mg cam/combination group/ for days. evaluation was done on day according to the criteria advocated by the japanese urinary tract infection committee. in patients with a urinary catherer, the combination achieved a higher complete bacterial elimination rate / %/ and clinical efficacy rate / %/ than cpf alone / and . %, respectively/. while no significant difference was found in the bacterial elimination rate between the two groups, the clinical effect of the combination / %/ was superior to that of cpf alone / %/ in patients with an indwelling catherer. the better clinical efficacy of the combination may partly be attributed to the antibiofilm effect of cam in the clinical setting. the results also indicated that difficulties still remain in the treatment of cuti in patients with an indwelling catherer. in conclusion, clinical study suggested that cam might have an inhibitory action on biofilm formation in the clinical setting. combination of cam with other appropriate antimicrobial agents may have a favorable effect on the treatment of cuti. vesicoureteral reflux and urinary tract infections */the management of primary vesico-eureteral reflux in children ps the children studied presented with primary vesicoureteral reflux at derer s universitz hospital in bratislava between and . seven hundred and sixty patients, boys and girls, suffering from primary vesicoureteral reflux in age from months to years were tested and systematically analyzed outcomes data for seven treatment alternatives. key outcomes identified were probability of reflux resolution, likelihood of developing pyelonephritis and scarring, and possibility of complications of medical and surgical treatment. available outcomes data on the various treatment alternatives were summarized and the relative probabilities of possible outcomes were compared for each alternative. conclusions: increased of urinary tract infection, vesicoureteral reflux nephropathy includes early diagnosis, appropriate evaluation, effective atb therapy, and surgery indicated. the main determinants of renal damage are bstruction, age, sex, predisposition on renal scarring, reflux grade and laterality, therapeutic delay, individual susceptibility, bacterial virulence and immunogenetic status. ) the one and only absolute indication for surgical management is failure of medical therapy to prevent chronic recurrent pyelonephritis, renal injury or other reflux complications and eliminations of the reflux condition will minimize their likelihood. genetically conditioned immunopathogenic mechanisms are involved in the pathogenesis of the chronic recurrent pyelonephritis in patient suffering from vur. for most children we recommended continuous antibiotic prophylaxis as initial treatment-medical therapy is based on the principle that reflux often resolves with time, and antibiotics maintain urine sterility and prevent infections while the patients awaits spontaneous resolution. ) vur predispose an individual to renal infection, the immunological and inflammatory reaction caused by a pyelonephritic infection may result in renal injury or scarring. silan l a , breza j b . a department of internal medicine, derer s university hospital, bratislava, slovakia , b department of urology, comenius university school of medicine, bratislava, slovakia elderly patients with uti are believed less likely to be cured by antimicrobial therapy than younger patients. the reasons for this poorer outcome have not yet been clarified. we have investigated the efficacy of antimicrobial therapy in elderly patients with complicated uti. five hundred patients, men and women, who had complicated uti/ symptomatic and symptomatic and were Á/ years of age, were treated with one of three different drugs, one was a never quinolone and two were oral cephems. multivariate logistic regression analysis of treatment outcome revealed that the clinical response was significantly related to general underlying diseases and diseases of the urinary tract, but not to age, symptomatic or asymptomatic uti, or infection site such as the kidney or bladder. we concluded that the clinical effectiveness of an antimicrobial agent was not directly related to age, and that urological examination for underlying disease and control of them is quite important for effective treatment and control of complicated utis, especially in elderly patients. the study on the frequency and antimicrobial resistance of escherichia (e ) coli in urine isolates of patients admitted to maribor teaching hospital in and ps rebersek gorisek j a , baklan z a , unuk s a , novak d b . a department for infectious diseases, teaching hospital maribor, maribor, slovenia , b department for microbiology, regional institute of public health maribor, maribor, slovenia purpose: the aim of the study was to determine the frequency and antimicrobial resistance of escherichia coli isolated from urine samples of patients admitted to maribor teaching hospital in and . the frequency and the antimicrobial resistance were compared between years and . methods: in the prospective study going on between and , all urine isolates from patients at maribor teaching hospital were collected and analysed. urine cultures were done using the modified sanford method. the susceptibility testing was performed by disk diffusion method according to nccls. results: in the year , urine isolates and in the year , urine isolates were analysed. e. coli represented . % of urine isolates in and . % of urine isolates in . e. coli resistance rates (%) to amoxycillin was . in the year and . in the year ; to amoxycillin/clavulate was . and . ; to cefalotin was . and . ; to cefaclor . and . ; to trimethoprim sulfamethoxazole was . and . ; to ciprofloxacin was . and . ; to gentamicin was . and . . conclusion: compared to , the frequency of e. coli isolated from urine samples is similar to that in the year . the resistance to amoxycillin, cefaclor and gentamicin is stable. the resistance to trimethoprim sulfamethoxazole and ciprofloxacin is increased and the resistance to amoxycillin/clavulate and cefalotin is decreased. prevalence of the resistance to metronidazole, furazolidone and nitrofurantoin in helicobacter pylori clinical strains ps de la obra sanz p a , roman jl a , lomas e a , villar h b , lopez-brea m a . a hospital de la princesa, microbiology, madrid, spain , b hospital de san agustin, microbiology, aviles, spain the objective of this study was to determine the prevalence of metronidazole, furazolidone and nitrofurantoin resistance in helicobacter pylori clinical isolates. methods: a total of strains of h. pylori were included in this study. all these were tested against metronidazole, and against furazolidone and nitrofurantoin by an agar dilution method. resistance was defined as: metronidazole, mic !/ mg/l; and mic !/ mg/l for furazolidone and nitrofurantoin. results: sixty-eight strains were resistant to metronidazole ( . %). the mic and mic values were and mg/l, respectively. three of strains ( . %) were furazolidone resistant (mic / mg/l), two of these strains were metronidazole resistant (mic / mg/l) and they had mic of mg/l for nitrofurantoin. the mic and the mic were . and . mg/l, respectively for furazolidone. only one of the strains ( . %) was nitrofurantoin resistant (mic mg/l), this strain was metronidazole resistant (mic mg/l) and had a mic / mg/l for furazolidone. the mic and the mic were . and mg/l, respectively for nitrofurantoin. conclusion: the low frequency of furazolidone and nitrofurantoin resistance, compared to metronidazole suggests that the furazolidone and the nitrofurantoin may be good alternatives to metronidazole for treatment of h. pylori infections. antimicrobial resistance of campylobacter isolated from human origins ps zhukhovitsky vg, drabkina iv. department of bacteriology, botkin hospital, moscow, russian federation the purpose of the study was to determine the antimicrobial resistance of thermophilic enteropathogenic campylobacter spp. (tec) isolated from human under acute diarrhoea in in moscow. among tec strains c. jejuni and five c. coli were identified. the antibiotic tested by disk diffusion test on mueller-hinton agar with sheep blood were ampicillin (a), amoxycillin/clavulanate (ac), imipenem (i), meropenem (m), erythromycin (e), clarithromycin (cl), tetracycline (t), doxycycline (d), gentamicin (g), azithromycin (az), chloramphenicol (ch), lincomycin (l), ciprofloxacin (c), nalidixic acid (na). the resistant rate of the tec isolates was highest for na ( %) followed by a ( %), t ( %), d ( %), n ( %) and cl ( %). a moderate resistance rate was obtained for a ( %), ch ( %), az ( %), ac ( %). none of the isolates demonstrated resistance to i, m and g and four of isolates ( %) were sensitive for all the antibiotics tested. mic to na was estimated as mg/l. among na resistant tec strains ( %) were identified as c. jejuni and one ( %) as c. coli . among c. jejuni and c. coli na resistant rate was and %, respectively. one na resistant c. coli and nine na resistant c. jejuni were resistant to ciprofloxacin. ring c, atanassova v. department of food hygiene and microbiology, school of veterinary medicine, hannover, germany aim of the study: poultry meat is known to be often contaminated with salmonella and other foodborne pathogens and thus has to be considered as a possible source for human infections. the aim of the study was to monitor the resistance of salmonella isolates from poultry meat of different european countries to various antibiotics. material and methods: from september to december a total of samples of frozen poultry meat from france, germany, italy, spain, the netherlands and portugal were examined for the prevalence of salmonella using classical cultural detection as well as rflp-pcr. all isolates were tested for their sensitivity towards ampicilline, kanamycine, ciprofloxacine, tetracycline, trimethoprim, sulfamethoxazole, nalidixic acid and erythromycine using standard procedures. results: from . % of all examined samples salmonella spp. were isolated. of these isolates . % were characterized as salmonella , . % as s. hadar and . % as s. typhimurium . nearly % of all isolates were resistant to erythromycin. resistance towards four or more isolates was observed in several cases. discussion: the consumption of poultry meat, if insufficiently prepared, has still to be considered as a major source for human infection with salmonella spp. the question arises whether the resistance of the isolates to various antibiotics is of clinical importance in the treatment of the patients. objective: to provide insight into the epidemiologic situation of salmonellosis for the nis area (the largest area in serbia, with inhabitants */ , ). methods: the material was processed at the institute for public health (epidemiology and microbiology divisions). isolation of microorganisms was performed on apparatus for rapid identification (vitek-biomerieux) and by applying elisa tests and classical microbiological methods. results: in the period Á/ , salmonella laboratory confirmed cases were reported. the greatest number of diseased in the Á/ years group. the most frequent isolated salmonellae were: s. enteritidis ( . %) and s. typhimurium ( . %), s. hadar , s. agona , s. virchow , s. infantis , s. derby , s. enteritidis showed the greatest sensitivity to antibiotics with the infrequent resistance to ampicillin and trimethoprim-sulfamethoxazole. s. typhimurium showed the greater resistance to the wide spectrum of antibiotics and some isolates were resistant to all antibiotics tested. the less common types of salmonella were sensitive to all antibiotics except trimethoprimsulfamethoxazole and ampicillin. conclusion: specific resistance to some antibiotics was related to serotypes. typhoid fever */retrospective study of cases in lebanon ps tohme a, abboud j, ghayad e. hôtel-dieu hospital, internal medicine, beirut, lebanon objectives: to present epidemiological and clinical features of typhoid fever in lebanon. methods: fifty-two patients were seen at hotel-dieu hospital of beirut between and . diagnostic criteria were positive blood culture for s. typhi or paratyphi and/or a somatic o agglutinin titer ]/ / as determined by the widal test with symptoms suggestive of typhoid fever. we also present an epidemiological study of cases registered by the ministry of health during the same period. results: among the cases, % of the patients' ages were between and years and % were less than years. the overall male to female ratio was . and % of cases were seen on january, february and % on august. among the patients, young adults were the most affected. average duration of symptoms before the diagnosis was / days. the main presenting symptoms were: fever ( %), diarrhoea ( %), abdominal pain ( %) and headache ( %). complications were noted in % of cases and digestive complications were the most prevalent. leucopenia was not a helpful diagnostic marker. s. typhi was the most frequent ( %) serotype identified. resistance to ampicilline was %, to cotrimoxazole and chloramphenicol % for each. the mortality rate was %. conclusion: typhoid fever is still an endemic disease in our country and the occurrence of resistant strains of s. typhi will favor ceftriaxone or fluoroquinolones in the treatment. maaloul i a , hammami b a , zambaa f a , elleuch r a , hammami a b , -ben jemaa m a . a chu hedi chaker, service des maladies infectieuses, sfax, tunisia , b chu habib bourguiba, laboratoire de microbiologie, sfax, tunisia although its not very frequent, the psoas abscess is not an exceptional entity. in order to specify its clinical, biological, radiological and evolutionary features, a retrospective study has been led in our service, on a period of years (january Á/december ). on the whole, cases have been listed. they were men and women. the age average was years (extreme Á/ years). the study did not find any underlying diseases, except diabetes mellitus for three patients. the clinical symptoms were dominated by fever with abdomino-lumbar aches ( cases), and psoitis (eight cases). biology showed an inflammatory syndrome in all cases and a hyperleucocytosis in cases. the diagnosis of psoas abscess, evoked on clinical data, has been confirmed by the imagery data: ultra-songraphy ( cases), ct scanning (six cases), magnetic resonance imaging (three cases). the tubercular etiology has been confirmed in six cases, among which two were associated to escherichia coli (one case) and to brucella melitensis (one case). the other etiologic agents were dominated by staphylococcus aureus (eight cases), b. melitensis (two cases), e. coli (one case), bacteroides fragilis (one case), streptococcus anginosus (one case), fusobacterium nucleaticum (one case) and candida glabrata (one case). all patients received an anti-infectious treatment adapted to the micro organism in question. a drainage of the abscess has been realized for patients (percutaneous: nine cases, surgical: six cases). the evolution was favourable for patients. however, two patients had a relapse after stopping the treatment. conclusion: the diagnosis of the psoas abscess, difficult on the clinical data, is based on the imagery techniques (us, ct, rmi). the percutaneous drainage guided by the imagery is recommended (in an etiological and therapeutic aim). associated to an adapted antibiotherapy, it allows to defer a surgical drainage. zezoski mbz, nikolova o, gavriloski pmg. medical center, infectious diseases, prilep, the former yugoslav republic, macedonia purpose: to make a list of the most frequent abdominal changes in patients with human brucellosis. materials and methods: there were new patients with human brucellosis, between and . diagnosis was made using standard clinical, biochemical and serological investigations (bab, wright, coomb's, rvk, -mercaptoethanol, elisa igm and igg), and specially ultrasound examination of the abdomen and retro peritoneum. results: weight loss is the most frequent change, presented in ( . %) patients. follow atypical abdominal pain in ( . %), vomiting in ( . %), diarrhea in ( . %), enlarged liver in ( . %), enlarged spleen in ( . %) and hepatic lesion with increased ast and alt in ( . %). conclusion: although frequent, abdominal changes seldom could be missed in patients with human brucellosis. we recommend routine ultrasound examination with standard biochemical test for liver function, due to avoid unnecessary complications. osteoarticular complications are common in brucellosis. the most common site of involvement is the sacroiliac joint. the osteoarticular complications such as, sacroiliitis and spondylitis are diagnosed with radiologically. in the present study, we aimed to determine the severity (grade) of sacroiliitis by using some laboratory parameters such as esr, crp and tube agg test. seventy-two ( male, female) patients with brucellosis were included in the study. osteoarticular involvement was present in patients. the most common osteoarticular finding was sacroiliitis in the patients ( %). twenty ( ) healthy subjects were formed the control group. there was statistically significant difference between patients and controls regarding esr, crp, and tube agg test (p / . , . , . , respectively). in addition, sacroiliitis has an effect on esr and crp. there was a positive correlation between the grade of sacroiliitis and the value of crp (p / . , r / . ). in conclusion, it has been suggested that, crp may be used as an auxiliary or a secondary parameter in grading sacroiliac joint involvement in brucellosis. magira ee a , papandreoy s a , gounaris t a , spirelis ma a , tasopoulos g a , anagnostopoulou m b , paniara o b , gounari p a , sioulal e a . a evagelismos, internal medicine, athens, greece , b evagelismos, micro- a -year-old greek farmer was admitted to the hospital because of painful scrotal swelling, hepatosplenomegaly, lumbar pain and lowgrade fever accompanied by profuse sweating. his life style included occupational animal exposure ingestion of raw milk and dairy products. the laboratory data were within the normal ranges. focal hypoechoic right testicular lesions, swelling of the concurrent epididimis along with an increase in the vascularity of the right testis were seen on an echo examination. these findings were consisting in unilateral epididimo-orchitis. a ct scan of the lumbar spine area showed a decrease of the signal intensity localized in the anterior aspect of l vertebral body at the diskovertebral junction involving the subchondrial parts of the l and s vertebrae standard tube agglutination test was positive for antibodies to brucella melitensis (titer !/ / ). cultures of blood specimens were positive for b. melitensis . the patient had been given to a combination of antibiotics with doxycycline, streptomycin and rifampin. a remarkable improvement of his clinical condition was showed weeks later. this case illustrates the following point: in areas in which brucellosis is endemic when scrotal abnormalities are seen the possibility of genitourinary tract complications of brucella should be considered. pappas ga a , akritidis nk b , mastora m b , tsianos e a . a university hospital, internal medicine, ioannina, greece , b 'g. hatzikosta ' hospital, internal medicine, ioannina, greece aims and scope: to determine the incidence and forms of complications associated with brucella infection. patients and methods: we studied the most recent patients, in all larger series approaching , diagnosed as suffering from brucellosis, and assessed the presence of signs and symptoms of arthritis and spondylitis, or other forms of bone involvement. the diagnosis of brucellosis was based on serology or isolation of brucella species from blood cultures or cultures from other media. results: osteoarticular complications were noted in patients, presenting with arthritis, and presenting with spondylitis. eight patients presented with genitourinary complications, either orcheoepididymitis (four patients), or hematuria resolving with treatment (four patients). meningitis was present in two patients. gastrointestinal complications (vomit and diarrhea) were present in three patients, while one patient presented with ascites. respiratory tract complications, in the form of pneumonia (four patients) or bronchitis (three patients) were noted in seven patients, while one patient with pneumonia exhibited pleural fluid. skin rashes, of macular type, were present in three patients. no patient presented with complications from the heart. hematologic complications were frequent, in the form of severe (one patient) or moderate (two patients) pancytopenia, isolated thrombocytopenia (three patients), or lymphocytosis (eight patients). akritidis nk a , pappas ga b , mastora m a . a 'g. hatzikosta ' hospital, internal medicine, ioannina, greece , b university hospital, internal medicine, ioannina, greece aims and scope: to determine the incidence and modes of bone and joint involvement in the course of brucellosis. patients and methods: we studied the most recent patients, in all larger series approaching , diagnosed with brucellosis, and assessed the presence of arthritis and spondylitis. the diagnosis of brucellosis was based on serology or isolation of brucella species from blood cultures. results: twenty-three patients exhibited a form of osteoarticular involvement. arthritis was present in patients, most often involving the knees, but also the hips, elbows, even smaller joints as intephalangeal joints of the hand. synovial fluid, when aspirated, was often characterised by an intense mononuclear infiltrate. spondylitis was present in patients, most often involving the lumbar spine, but also the thoracic spine. the characteristic erosion on the upper anterior crest of the vertebral body was visible in plain x-rays in three patients, while mri and bone scan were helpful in other cases. discussion: osteoarticular involvement in the course of brucellosis is the most common focal presentation of the disease. acute brucellosis is often accompanied by bone and joint ache, especially of the lumbar spine, still frank involvement in the form of arthritis and spondylitis is not rare. arthritis usually presents in the acute form of the disease, while spondylitis tends to be characteristic of a chronic form of the disease, often necessitating prolonged use of antibiotics. bosilkovski m, krteva l, caparoska s, grozdanovski k, sajn b. clinic for infectious diseases and febrile conditions, medical faculty, skopje, the former yugoslav republic, macedonia one hundred and twenty-six patients with brucellosis were studied prospectively. seventy-eight ( %) of them had osteoarticular involvement. peripheral arthritis in ( %) patients was the most frequent, followed by spondilitis in ( %), sacroiliitis in ( %), rarely bursitis, tendinitis and osteomyelitis. the overall male to female ratio was : . their average age was (sd ) years. direct contact with animals was the reason for acquisition of the illness in % of patients, in % alimentary or aerogenous route was incriminated, and in % the route of aqisition was unknown. the average duration of the symptoms from the onset to establishing the diagnosis was (sd ) days. the main presenting symptoms were joint pain ( %), sweating ( %), fatigue ( %) and fever ( %). hepatomegaly was present in %. in % of patients, involvement of some other system was evident. comparison with patients, who did not have osteoarticular illness, showed that patients with osteoarticular involvement had significantly more often joint pain, fatigue, weight loss and more prolonged duration of symptoms before the diagnosis was established. doxycycline and chloroquine as combination therapy for chronic q fever endocarditis ps calza l, attard l, manfredi r, chiodo f. division of infectious diseases, university of bologna, s. orsola hospital, bologna, italy introduction: endocarditis is the main clinical manifestation of chronic q fever, occurring in about Á/ % of all reported cases, and it is diagnosed almost exclusively in patients with either cardiovascular abnormalities or an immunocompromised condition. case report: a -year-old caucasian male patient with biological prosthetic aortic valve was first hospitalized because of an interstitial pneumonia. six months later, our patient was re-admitted owing to intermittent fever, chills and weight loss. echocardiographic study showed a small vegetation of mm in diameter on left cusp of aortic valve. serology for coxiella burnetii revealed a complement-fixing igg antibody titer to phase i antigen of more than : , consistent with chronic q fever endocarditis. antimicrobial therapy with i.v. doxycycline and oral chloroquine was started, leading to a clinical and echocardiographical recovery. therapy was continued by oral doxycycline and chloroquine, and the patient remained asymptomatic during a -year follow up. conclusion: the optimal treatment of q fever endocarditis has not been well established: the most effective antimicrobials are fluoroquinolones and rifampin, but chloramphenicol, doxycycline and trimethoprim are also useful. the role of chloroquine in combination with doxycycline seems to be promising, because chloroquine may increase the lysosomal ph, enhancing the doxycycline bactericidal activity. akritidis nk a , pappas ga b , mastora m a , liappis e a , tsianos e b . a 'g. hatzikosta ' hospital, internal medicine, ioannina, greece , b university hospital, internal medicine, ioannina, greece aims and scopes: to review the incidence and the forms of lower respiratory tract infection in patients suffering from q fever, and their clinical and radiological characteristics. patients and methods: twenty-seven patients diagnosed as suffering from q fever, were assessed for the presence of lower respiratory tract infection. the diagnosis was confirmed serologically. results: thirteen patients expressed lower respiratory tract pathology, as confirmed by clinical examination and chest x-ray. in of these patients the main cause of admission was respiratory tract symptoms, ranging from dry cough to hemoptysis. chest x-ray was pathological in patients: patients had lobar pneumonia, two of them multiple nodular opacities, and one of them bronchopneumonia. hepatitis was a common finding. all patients were treated with tetracycline. discussion: although coxiella burnetii infection is acquired via the respiratory tract, it is paradoxical that symptoms attributed to the lung are not invariably positive. q fever pneumonia is an atypical pneumonia that usually follows a benign course. diangostic suspicion is usually raised by the epidemiologic pattern and the accompanying mild hepatitis. pleural effusion is not a common finding. the usual radiologic appearance of q fever pneumonia is that of a lobar or segmental pneumonia. one important aspect of q fever pneumonia is its common presentation in the form of multiple nodular opacities often necessitating the exclusion of malignancy. ( ), culture ( ), and serology'/culture ( ). there were Á/ cases per year, mainly in october ( %). a history of exposition to hare was present in / ( %) and to marmot in / ( %). skinning ( / %), animal contact ( / %), bite ( / ) and wound during bait preparation with frozen meat for hunting ( / ) were noted. the initial clinical presentation was ulceroglandular ( %), glandular ( %) and pneumonic ( %). the involved nodes distribution was axillary ( / ), cubital/axillary ( / ) and cubital ( / ). median incubation period was days (range Á/ ); time to consultation days (range Á/ ), and time for effective treatment days (range Á/ ). an initial diagnosis of tularemia was made presumptively in %. effective antibiotic regimen used was aminoglycosides in % ( / ), and tetracyclines in % ( / ). note that intravenous netilmicin was used in cases. complication rate was % ( / ) with one death ( %), and was associated with delay in effective treatment ( !/ days of illness) (p b/ . ). conclusion: in our area tularemia occured mainly in male population, during autumn, with a short incubation period and history of hare contact. delay before appropriate treatment increased the complication rate. bompolaki i, doukakis s, triantafillidou d, polimili g, kastanakis m, nikiforakis k, vittorakis e, kastanakis s. first medical department, 'saint george ' general hospital, chania, greece a severe frontal and/or retroorbital headache represents the most common neurologic manifestation of murine typhus. other neurologic manifestations as confusion, stupor, nuchal rigidity and in severe cases delirium, extreme agitation or coma appear less commonly. eightyfour patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi, were studied from our team. seventy-four patients ( %) presented headache and nine patients ( %) presented confusion. one patient ( . %) presented nuchal rigidity in combination with severe headache and confusion giving us the suspicion of meningitis. in this case a lumbar puncture was performed emergently and the cerebrospinal fluid (csf) was examined. the findings of csf were proteins: mg/dl, wbc: /ml and glucose: mg/dl and its culture was negative. all patients were treated with a specific anti-rickettsial treatment. the outcome of murine typhus was favorable for all patients ( %). no one patient presented neurologic sequelae. conjunctivitis usually accompany rickettsial diseases such as rocky mountain spotted fever, epidemic typhus and murine typhus. eightythree patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi , were studied from our team, during a period of time between january and the first semester of . the clinical examination of these patients revealed the presence of conjunctivitis in / patients ( . %). in the same time these patients referred retroocular pain and mild photophobia. this study showed that in murine typhus the injection of conjunctivae is rather common. almost a quarter of the patients presented conjunctivitis despite the fact, that this ocular manifestation is less severe than in other typhus and spotted fevers. eighty-three patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi , were studied from our team, during a period of time between january and the first semester of . three blood samples were obtained from each patient for the study of their hematological abnormalities. the first sample was obtained on admission, the second sample weeks after the first, the third sample, month after the second. on admission / patients ( %) presented anemia, / patients ( %) presented leukopenia and / patients ( %) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was . g/dl, . )/ and )/ /ml, respectively. two weeks later anemia was presented in / patients ( %), / patients ( %) presented leukopenia, / patients ( %) presented leucocytosis and / patients ( %) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was . g/dl, . )/ and )/ /ml, respectively. one month later / patients ( %) had anemia and / patients ( %) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was . g/dl, . )/ and )/ /ml, respectively. our study showed that early thrombocytopenia and anemia are frequent in murine typhus and that white blood cells count is usually normal. renal function in murine typhus: a study of cases ps doukakis s, polimili g, triantafillidou d, kastanakis m, vittorakis e, palla k, kastanakis s. first medical department, 'saint george ' general hospital, chania, greece the clinical course of murine typhus is usually uncomplicated and the mortality rate is low ( b/ %). advanced age and prolonged interval before administration of a specific anti-rickettsial treatment are correlated with severity of the disease. renal function in murine typhus is usually unaltered except in elderly patients with prolonged hypotension. eighty-three patients with compatible clinical status of murine typhus and high serological titres of antibodies against rickettsia typhi, were studied from our team, during a period of time between january and the first semester of . three blood samples were obtained from each patient for the study of their renal function. the first sample was obtained on admission, the second sample approximately weeks after the first, the third sample, taken from the half of the patients, was obtained one month after the second. on admission / patients ( . %) presented acute renal failure. the outcome of murine typhus was favourable for all patients ( %). the four patients who presented acute renal failure reversed after the administration of anti-rickettsial treatment and careful administration of fluids. in murine typhus the induction of hypovolaemia insufficiently corrected by normal homeostatic mechanisms may lead to prerenal azotaemia. in these cases the immediate onset of an antirickettsial treatment and the correction of hypovolaemia are essential for the rapid clinical improvement of the patient. experimental ocular toxoplasmosis: clinical, histopathological, immunological and therapeutic studies ps el zawawy lae a , hammoda na a , allam sr a , ali sm a , galal as b . a faculty of medicine, parasitology, alexandria, egypt , b faculty of medicine, ophthalmology, alexandria, egypt the purpose of the study was to investigate clinical, histopathological, immunological and therapeutic features of an experimental model of ocular toxoplasmosis in sensitized and non sensitized rabbits and to assess the influence of treatment by interleukin (il- ) on ocular lesions. the results obtained was that 'toxoplasma' retnochoroiditis developed in both groups of rabbits with more pronounced effect in non sensitized animals. administration of il- improved ocular lesions in both groups with more evident effect in sensitized rabbits. immunological findings were consistent with clinical and histopathological observations. the conclusion reached was that; ocular lesions were manifested in non sensitized rabbits more than in sensitized ones. il- revealed a significant impact on improving the host defense against toxoplasm infection in eye. immunotherapy with il- would open the way for a new range of treatment based on immunomodulation. express-diagnostic of streptococcus antigen for the adequate antibiotic therapy in patients with pharyngitis ps pertseva to, konopkina li, kireeva tv. dsma, internal medicine , dniepropetrovsk, ukraine the purpose of the study: evaluation of effectivness of streptococcus express-diagnostic for the adequate antibiotic therapy in patients with pharyngitis. results: we deal with clinical and microbiological comparison in patients with pharyngitis. using of streptococcus antigen express diagnostic in swabs from the backside of pharynx allowed to get positive results in the seven cases ( . %). following cultural study has confirmed these results. positive test was more probable in patients with pronounced fever (more than c), headache, weakness and in cases associated with chronic tonsillitis. isolated streptococcus pyogenes was susceptible to ampicillin, claritromicin, erytromicin, azytromicin, , clindamicin, ceftriaxon, levafloxacin, oxacillin, cefuroxim, roxytromicin. conclusion: using of the express diagnostic of streptococcus antigen allows to restrict groundless prescription of antibiotic therapy in patients with other types pharyngitis (i.e. viral, candidal etc.). alabaz d a , turgut m a , kocabas e a , tumgor g a , yaman a b , alhan e a . a division of pediatric infectious diseases, cukurova university, adana, turkey , b department of microbiology, cukurova university, adana, turkey chickenpox is a common viral infection that usually follows a benign, self limited course in healthy children. the most common complication in children with varicella is superimposed cutaneous infections with pyogenic bacteria (streptococcus pyogenes and staphylococcus aureus ). varicella gangrenosum, a necrotising soft tissue infection complicating the vesicular eruption of chickenpox, is rare. here we present three cases with necrotising soft tissue infections following chicken pox. these children were admitted because of common crusted lesions and necrotising soft tissue infection over the neck, the back, and the inguinal area. they all had the contact history and ensuing vesiculopapular rush. these infections were caused by group a streptococci in two cases, and s. aureus in one case. after instituting of appropriate antibiotic therapy, each patient underwent a surgical exploration with fasciotomies and debridement. widespread use of varicella vaccine may decrease invasive infections in children, adolescents, and adults, thus decreasing the burden the disease with its complications impose up on the family and the society . cefprozil is a second generation cephalosporin. the aim of this open, multicentre, non-comparative study was to investigate the efficacy and safety of cefprozil in the treatment of streptococcal tonsillopharyngitis. fifty-eight patients were clinically assessed for signs and symptoms of streptococcal infection. laboratory confirma-tion was sought using three tests; culture, rapid strepto test and estimation of antistreptolysin (aso). one or more tests were done in of the patients. treatment was for days, mg/kg per day in children, mg per day in adolescents. patients were again clinically assessed on the th Á/ th day. the results showed clinical success in patients ( . %) and in of the who had laboratory tests ( . %). two patients had treatment withdrawn because of nausea and abdominal pain ( . %). of the patients with laboratory tests at least one test was ositive. the most helpful was the strepto test giving the quickest positive result in % of those tested ( / ). culture was positive in % of those tested ( / ). the aso test was of limited value. in conclusion, cefprozil showed high clinical efficacy and safety in the treatment of streptococcal tonsillopharyngitis. tsiara s, militadou g, milionis c, elisaf m. internal medicine department, university of ioannina, ioannina, greece streptococcus group b agalactiae (gbs) is a rare pathogen for healthy male adults. we present an old man in whom (gbs) was isolated in blood cultures. case report: a -year-old man was admitted to the hospital in order to investigate osteolytic lesions in the lumbar spine. two weeks before, he experienced severe low back pain radiating to the right leg and fever arising to . c with chills and rigors. a gbs was isolated from blood cultures and treatment with penicillin was initiated. a spine ct scan revealed osteolysis in the t and s vertebrae and the patient transferred to us. he was a previously healthy man. he received only antihypertensive therapy. on admission he was afebrile with arthralgias and myalgias. on clinical examination there was tenderness on the right pleurospondylic angle radiating to the right leg. laboratory data on admission: hb: . g/dl, wbc: . )/ /l, esr: mm/h. biochemical values, serum protein electrophoresis, rectal examination and a prostatic specific antigen (psa) were normal. a bone marrow aspiration and biopsy were negative. a transoesophageal ultrasound revealed vegetation on the right cusp of the aortic valve, with low grade regurgitation. an mri of the lumbar spine revealed infectious myositis with concomitant osteomyelitis involving the l , l vertebrae without any evidence of osteolytic lesions. a thorough investigation did not revealed any underlying immunosuppressive disease. treatment with vancomycin and gentamycin iv was initiated and the patient discharged from the hospital in excellent health after weeks. discussion: gbs infections usually occur in neonates and pregnant, or in adults with underlying disease as diabetes mellitus or immunosuppression. the most common site of infection is soft tissue. we present this case because gbs infections are rare in the elderly in the absence of underlying disease. common sites of involvement are soft tissues, while bone, joints, and heart valves account to Á/ % of the involved organs. although our patient had more than one site of involvement he responded well to medical treatment without surgical debriment or heart valve replacement. objective: to evaluate the safety and efficacy of rhugm-csf in combination with broad-spectrum antibiotics for the treatment of ccnf. methods and results: a retrospective review of all patients (pts) with ccnf treated with antibiotics'/rhugm-csf at our hospital from january to december was performed. five patients were identified with the diagnosis of ccnf. ages ranged from to years; there were three women and two men. dental infection was the most common source of ccnf in %. one patient had acute tonsillitis leading to ccnf. all cases studied experienced infection of the neck with spread into the submandibular, submental, sublingual, retropharyngeal, and parapharyngeal spaces. all infections were polymicrobial. diabetes mellitus was a co-morbidity in one case. all pts were treated with dual antibiotic coverage (vancomycin'/meropenem), rhugm-csf ( mcg/kg/daily given s.c.) and aggressive wound care. rhugm-csf was given for Á/ days. in spite of the severity of the infection all pts recovered and do not experienced local or systemic complications. discussion: ccnf is a severe bacterial infection of the cervical fascia resulting in extensive fascial necrosis with widespread undermining of the surrounding tissues. prompt antibiotic therapy combined with rhugm-csf resulted in a % overall survival in our experience. to our knowledge, this is the first report describing the successful treatment of ccnf with use of broad-spectrum antibiotics combined with rhugm-csf. the following case adds to the clinical manifestations and course of meningococcal disease. a previously healthy -year-old girl presented acutely with high fever purpuric rash including conjuctival haemorrhages and hypotension. the child had also neck stiffness. a presumptive diagnosis of meningococcal septicaemia was made and treatment with penicilline, chloramphenicole, fluids and inotropes was initiated. laboratory investigations showed wbc: /ml, hb: . g/dl, hct: . %, esr: mm in the first hour, pt: s, aptt: s. neisseria meningitidis group b was isolated from the blood cultures. csf obtained after antibiotics were started did not grow n. meningitidis . the patient had an adverse outcome. she died after h of hospitalization. this patient developed a fulminating meningococcal septicaemia. shock is a clinical diagnosis arising from the failure of compensatory mechanisms that maintain perfusion of vital organs at the expense of non vital. septic shock results from loss of circulating plasma volume due to increased vascular permeability and maldistribution of intravascular volume. in young children there is a prevalence of serogroup b meningococcal disease which can be explained by the immaturity of the immune system and by the fact the group b capsule synthesis is known to inhibit alternative complement pathway activation. this case emphasizes the need for further protection against n. meningitidis group b. . results: forty-eight patients were included in this study. the etiological agents were: viral (n / , . %, mean ('x ) age / years), streptococcus pneumoniae (n / , . %,'x age / ), neisseria meningitidis (n / , . %, 'x age / ), s. viridans (n / , %, 'x age / ), p. multocida (n / , age ). the peak incidence of bacterial meningitis was in winter (pneumococcal %, meningococcal %, s. viridans %) .the cerebrospinal fluid (csf) findings in viral meningitis were 'x white cells / /mm , 'x pmn / %, 'x glucose csf/ serum . , 'x protein mg/dl and in bacterial meningitis were 'x white cells / /mm , 'x pmn / %, 'x glucose csf/ serum / . , 'x protein / mg/dl, gram stain was positive in %, culture was positive in %. all pneumococcal and meningococcal strains were susceptible to penicillin. the case fatality rates for pneumococcal and meningococcal meningitis were and . %, respectively. conclusions: the cases of bacterial meningitis were according to typical epidemiological features of age and season. the case fatality rate of pneumococcal meningitis appear to be high regardless of susceptibility to penicillin. none had received pneumococcal vaccine prior to becoming ill. diagnosis and therapy of meningococcal meningitis */trend and particularities of a 'romanian model' ps lintmaer i, moroti r, popescu a, popescu c. institute of infectious diseases matei bals , unit , bucharest, romania background: newer diagnosis methods and antimicrobials are expected to change the management of menigococcal meningitis (mm) and to improve its prognosis. objectives: to determine the changes in the diagnosis methods and therapy of mm patients in a infectious diseases hospital. to compare mm management in bucharest with literature data. methods: retrospective rewiew of mm in adult patients hospitalized over a -year period. our results were compared with other studies made in the s, taken from medline. results: there were episodes of mm during the study period ( episodes in Á/ and episodes in Á/ ) . we noticed a defined diagnosis increase and increased blood culture specificity. the antimicrobial monotherapy was maintained but penicilin was replaced by ceftriaxone. hhc was replaced by dexamethasone in pathogenic therapy. we noticed a shorter length of treatment and a reduced lethality. the most important differences between our results and other studies are: monotherapy regimens are less frequent and therapy lengths are longer; however, prognosis is similar. conclusions: the mm management has been modified in the last Á/ years: prognosis is improved, but the changes do not bring clear cost/ effective benefits. tiouiri th, kilani b, amari l, zouiten f, kanoun kf, ghbontini a, ben chaabene t. rabta hospital, infectious diseases, tunis, tunisia objectives: in order to study epidemiological, clinical and therapeutical characteristics of the infective endocarditis (ie). methods: we reviewed all the cases of ie fulfilling the duke criteria. data were collected during a -year period ( Á/ ) in the unit of infectious diseases. results: one hundred and eight cases were identified. the mean age was . years. sex ratio was . . eighty-five ie ( . %) occured in patients with native valve, and ie ( . %) with prosthetic valve. fever was the most common sign, % had a congestive heart failure, . % had cutaneous signs. the most common primary focus of ie was orthodontic. blood cultures were positive in % of cases. in one case, serological test identified rickettsia conori . streptococci and staphylococci were isolated in . and . %, respectively. echocardiography detected abnormalities in . % of cases. rheumatic heart disease was the most predisposing condition. empirical therapy was based on combination of b lactam with aminoglycoside. recovery was obtained for patients. cardiac surgery was performed in cases. overall mortality rate was . %. conclusion: ie affects young persons. prevention needs eradication of acute rheumatic arthritis. a major outbreak of legionnaires' disease in spain: diagnostics aspects ps guerrero c a , toldos cm a , yagü e g b , ramírez c a , rodríguez t a , -segovia m a . a hospital morales meseguer, servicio de microbiología, murcia, spain , b departamento de microbiología, facultad de medicina, universidad de murcia, murcia, spain objective: to evaluate the value of different methods (serological tests, culture of respiratory secretions, blood cultures and urinary antigen testing) for the diagnosis of legionella pneumophila pneumonia during an outbreak in spain. results: we have studied patients from a recent outbreak of legionellosis in murcia (spain). the diagnosis was achieved in patients. urinary antigens were positive in patients. in the patients with urinary antigen negative the serological response was demonstrated by indirect immunofluorescence (ifa) in patients. all blood cultures processed were negative. sputum samples were obtained from patients, of these l. pneumophila was isolated only in six patients. in all of them direct immunofluorescence test (dfa) was positive. conclusions: although the serological diagnosis was the most sensitive method the urinary antigen testing was of great value in the rapid diagnosis of the legionella's outbreak in murcia. the isolation of l. pneumophila by culture showed a poor sensitivity probably because of the low severity of the illness. purpose: to evaluate chloramphenicol for an initial empiric antibiotic treatment of purulent meningitis in adults. study group: one hundred and twenty patients hospitalized for the diagnosis purulent meningitis in the department in years Á/ , males and females, age range Á/ years, mean age . years. children up to years were not included. method: a retrospective analysis of the study group focused on antibiotic treatment both initial and changes during treatment. results: chloramphenicol was used as an initial antibiotic in ( %), rd generation cephalosporin in ( %), penicillin in ( %), ampicillin in five ( %) and other antibiotic in five ( %), respectively. during treatment chloramphenicol was switched for rd gen cephalosporin in seven of patients with streptococcus pneumoniae meningitis and in five of patients with meningitis of unknown etiology. the reason for the change was non-improving csf formula in three, persisting csf culture positivity in two and persisting coma in seven patients. conclusion: because of repeated necessity to switch chloramphenicol for rd gen cephalosporin during treatment of purulent meningitis of pneumococcal and unknown etiology the initial treatment strategy was changed in . third gen cephalosporin is now used as a first choice antibiotic, what is in consent with international recommendation of treatment. to evaluate and compare groups treated initially with chloramphenicol and with rd gen cephalosporin will need several more years. low prevalence of multi-drug resistant mycobacterium tuberculosis in jerez de la frontera-cadiz (spain) ps alados jc, aller ai, de miguel c, de francisco jl, calbo l. hospital del sas-jerez , microbiologia, jerez de la frontera, cadiz, spain introduction and aim: previous reports indicate that multi-drug resistance mycobacterium tuberculosis (mtb) is an worldwide problem. the aim of this study was to review the resistance of mtb to the first-line antimycobacterial agents in our area. material and methods: over a period of years ( Á/ ), strains of mtb isolated from non-treated patients with tuberculosis ( strain in , in , in and in ) were studied. these isolates were tested for in vitro drugs susceptibility to isoniacid-i, rifampicin-r, streptomycin-s and ethambutol-e using the bact/ alert method (organon teknica) as described by the manufacturer. results: our results showed that . % ( / ) strains were resistant to one or more drugs. single drug resistances were detected on nine strains to i ( . %), one to r ( . %), two to s ( . %), one to e ( . %). three mtb strains were resistant to more than one drug but only one was multi-drug resistant (i'/r).the incidence of i-resistant strains over the period fell from % in to . % in . conclusions: ( ) multi-drug resistance is not an important problem in our area. ( ) isoniacid resistance was declined to an admissible level. to investigate the anti-tuberculosis drug resistance pattern of pulmonary tuberculosis isolates in southern taiwan, an area with higher tuberculosis incidence and mortality than other regions of the island, we performed a hospital-based surveillance at a southern taiwan medical center from to . the combined drug resistance rates to at least one of five first-line agents was . %, and to both isoniazid and rifampin (multi-drug resistance, mdr) was . %, indicating high resistance rates compared with those reported in the who/iuatld global project and in northern taiwan. the resistance rates to two second-line drugs, cycloserine, and kanamycin, were . and . %, respectively. a significant decreasing trend in resistance rates to all drugs except streptomycin was observed during the -year period. though combined drug resistance rate may not be the most accurate tool as it includes previously treated cases which inflates the resistance rate, the observation of trends in the susceptibility of pulmonary tuberculosis in accompany with the increasing percentages of tuberculosis patients receiving complete treatment course and the decreasing percentages of cases lost of follow-up in kaohsiung after the institution of new governmental regulations for case management in suggest the usefulness of intervention programs. lipid profile in patients with multidrug resistant pulmonary tuberculosis ps extrapulmonary tuberculosis may sometimes present with confusing clinical manifestations. a -year-old female patient was admitted with a history of recurrent supra-sternal abscess for year. mri confirmed the presence of sternal osteomyelitis and an anterior mediastinal mass. the diagnosis of tuberculosis was proved by histologic examination and acid-fast stain. the patient was treated with first-line agents, which isoniazid, rifampin, pyrazinamide, and ethambutol. tobacco smoking as a factor of the decrease of chemotherapy effectiveness and of the development of the drug resistance in patients with pulmonary tuberculosis ps shprykov as a , zhadnov vz a , shprykova on b . a medical academy, department of tuberculosis and lung diseases, nyzhny novgorod, russian federation , b medical clinic for infectious # , laboratory of bacteriology, nyzhny novgorod, russian federation studies of the effect of smoking on the results of chemotherapy of patients with tuberculosis of lungs. intensive tobacco smoking slowed down clearance of positive sputum and of lung tissue destruction (in smokers . % and . vs. . % and . % in nonsmokers, p b/ . ). drug-resistant mtb strains have been found to be isolated more often in smokers */ . vs. . % in non-smokers, p b/ . . resistance to streptomycin and isoniazid prevailed, reaching in heavy smokers . and . %, respectively. resistance to rifampicin increased . times. the concentration of rifampicin in the blood serum of heavy smokers decreased in . times. clinical data are in complete correlation with the findings of our experiments: % of experimental cultures developed resistance to streptomycin, isoniazid and less to rifampicin in the study of drug sensitivity under the effect of tobacco smoke condensate. thus, our findings show the development of drug resistance in mtb under the effect of components of tobacco smoke and also showed less effectiveness in therapy. kilani kb, ammari la, tiouiri ht , ben chaabène tbc. rabta hospital, infectious diseases, tunis, tunisia guerrero c a actinomycosis is a chronic disease characterized by abcess formation, tissue fibrosis and draining sinuses. it is caused by anaerobic bacteria belonging to the genus actinomyces. thoracic actinomycosis may involve the lungs, pleura, mediastinum or chest wall. the authors present a case of pulmonary actinomycosis complicating a cervicofacial site. a -year-old man with a history of cervicofacial actinomycosis treated by penicillin g years ago was admitted because of right-sided chest pain for months before presentation, cough and fever. physical examination shows a painless indurated mass in the neck with multiple fistula of the sternum. chest radiograph and ct scan revealed a mass in the upper lobe of the right lung with an infiltrate of the upper lobe of the left one. magnetic resonance imaging confirms the previous lesions, with extending process to the sternum and right collar bone. bronchoscopy was performed while patient was on antimicrobial therapy. culture of bronchoalveolar lavage fluid was negative. transbronchial biopsy was not conclusive. fungal serologies were negative for aspergillosis, histoplasmosis, blastomycosis. bacterial examination of purulent drainage from sternal wound shows inclusion bodies identified as actinomyces. he was treated then with penicillin iv for months, than switched to doxycycline. after months of treatment, he is asymptomatic with radiological improvement. kanellopoulou m a , skarmoutsou n a , iglezos i b , mylona e a , martsoukou m a , apostolopoulou f b , papafrangas e a . a laboratory of clinical microbiology, sismanoglio general district hospital of attica, athens, greece , b nd department of pneumology, sismanoglio general district hospital of attica, athens, greece introduction: achromobacter xylosoxidans is a rare human pathogen. it is an important cause of bacteremia in patients with cardiac diseases, malignancies and immunosuppression. it has been recently recognized as an emerging microorganism in cystic fibrosis (cf), whose its pathogenic role is unknown. aim: to investigate the sensitivity to eleven different antibiotics of a. xylosoxidans strains isolated from adults with cf, during . methods: the susceptibility was tested by kirby bauer and microdilution methods (wider i, fransisco soria melguizo, s.a.), according to nccls recomendations. results: the resistance to antibiotics was as follows : gentamicin, tobramycin, aztreonam %, amikacin %, ceftazidime %, ticarcillin %, carbapenems, cotrimoxazole %, colistin % and piperacillin %. conclusions: ( ) a. xylosoxidans isolated from cf patients appeared resistant to the most usually tested antibacterial agents. ( ) colistin which is used as aerolized antibiotic for cf patients seems to be effective in the half of the isolated strains. ( ) piperacillin was the most active antibiotic against a. xylosoxidans . morris ka, perry jd, jain s, gould fk. microbiology department, freeman hospital, newcastle upon tyne, uk alafosfalin, l-alanyl-l- -aminoethylphosphonic acid, is an antibacterial peptide mimetic which inhibits peptidoglycan biosynthesis. we report the in-vitro activity of this compound in combination with ceftazidime, cefsulodin, fosfomycin, piperacillin/tazobactam, aztreonam, ciprofloxacin and timentin. drug combinations were evaluated against burkholderia cepacia strains, and pseudomonas aeruginosa strains isolated from patients with cystic fibrosis. for this purpose a chequerboard technique was adopted using doubling dilutions of each antibiotic incorporated into a highly defined agar medium free of antagonists. the minimum inhibitory concentrations (mics) and fractional inhibitory concentrations (fics) of all the antibiotic combinations were determined which revealed the antibiotic interaction occurring. alafosfalin in combination with ceftazidime, meropenem, piperacillin/tazobactam and timentin demonstrated the highest percentages of synergy in both b. cepacia and p. aeruginosa . synergy was shown to occur in , , and % of b. cepacia strains respectively, and in , , and % of p. aeruginosa strains. these four combinations were re-tested with all isolates and the results were shown to be reproducible. alafosfalin shows potential as a treatment for cystic fibrosis patients colonised with p. aeruginosa and/or b. cepacia , when applied in combination with these agents. community-acquired pneumonia */does its aetiology matter? ps lintmaer i, popescu a, popescu c. institute of infectious diseases matei bals, unit , bucharest, romania the aetiology of a pneumonia is not one of the criteria used to determine pneumonia's severity. however, it is accepted that identified based Á/based therapy is less expensive (and possibly more effective). objectives: our study aims were to: ( ) to evaluate the role of aetiology identification in pneumonia; ( ) to evaluate the first-line therapy in pneumonia. methods: we conducted a retrospective study in an infectious diseases hospital on patients with pneumonia. we excluded all the cases with nosocomial pneumonia. primary end-point was the day clinical failure (deaths, icu admission), secondary end-points were the average time of fever and length of stay and the antimicrobial regimen changes. results: causative agent identification rate was . %. the evolution was different for patients with identified aetiology compared with other patients in terms of: -day failures, length-of-stay and changes of the antimicrobial regimen. the patients with inadequate first-line therapy had a more severe course of illness with a greater rate of day clinical failure, longer fever and length-of-stay. conclusions: pneumonia's treatment was better for the patients with identified causative agent. that is why we should include aetiology among the pneumonia severity criteria, especially at an 'after -day therapy' re-evaluation. results: pneumomococcal aom was detected in children ( . %) and s.pn. was the only pathogen in . %. the resistance rates of the organism to antibiotics were as follows: penicillin . % (micb/ mg/ml; intermediately resistant . %, mic . mg/ml . %, and mic!/ mg/ml; highly resistant . %), erythromycin . %, clindamycin . %, cotrimoxazole . % and chloramphenicol . %. all isolates were uniformly susceptible to rifambicin and vancomycin. the large majority of pneumococcal isolates ( . %) had the mphenotype and the remaining strains ( . %) the constitutive mls phenotype. a various of serogroups were detected; the serogroup was the most predominant one ( . %), followed by serogroups ( . %), ( . %) and ( . %). the non-typable s.pn. strains compromised the . % of the strains. conclusions: high prevalence of resistance to penicillin, macrolides and cotrimoxazole in pneumococcal aom of childhood was recognized. a strategy for preventing aom caused by drug-resistant pneumococci is mandatory to start. material and methods: a total number of strains were examined. the sensitivity test was performed by kirby bauer, microdilution method (pasco, difco) according to nccls guidelines and by e -test. results: a percentage of . % of s. pneumoniae strains revealed high level resistance to penicillin (mic]/ mg/ml), while the % showed intermediate resistance (mic . Á/ mg/ml). the resistance to erythromycin and cotrimoxazole was . % (mic ]/ mg/ml) and . % (mic]/ / mg/ml), respectively. all strains were sensitive to cefotaxime (mic . mg/ml), vancomycin (mic / . mg/ml), meropenem (mic / . mg/ml) and levofloxacin (mic / mg/ml). conclusions: ( ) the prevalence of high resistance s. pneumoniae to penicillin seems to be low in examined strains ( . %). ( ) intermediate resistance to penicillin of s. pneumoniae isolates was high as expected ( %). ( ) most of the strains were sensitive to erythromycin ( . %) and cotrimozaxole ( . %). ( ) s. pneumoniae isolates were completely ( %) sensitive to levofloxacin, vancomycin and meropenem. beghi g, aiolfi s, maghini l, patruno v, aiolfi e. s marta hospital, pulmonary rehabilitation unit, a.o., rivolta d'adda, italy aim: of this study was a retrospective ( Á/ ) evaluation of the more effective and practical antibiotic treatment in ae-copd patients (pts) admitted to our unit. methods: before introducing any antimocrobial drug, sputum specimens were collected for microbiological purposes, while blood analysis, to monitor adverse systemic effects, were performed at the beginning and the end of treatment. antibiotic treatment ranged from to days according to four regimens: regimen a ( pts) /oral therapy only: . % with amc g b.i.d.; . % with cip mg b.i.d.; . % with dox mg u.i.d.; . % with lev mg u.i.d.; . % with cla mg b.i.d.; . % miscellaneous. regimen b ( pts) /sequential therapy (e.v. for days /oral): . % with amc g b.i.d.; . % with cla mg b.i.d. regimen c ( pts) /e.v. therapy only: same drugs. regimen d ( pts) /an association of two antibiotics. results: of evaluated pts, only ( . %) required a second regimen of treatment because of failure of the previous one: . % of regimen a; . % of regimen b; . % of regimen c, and % of regimen d. mild adverse effects were detected only in four pts. our results confirm that oral antibiotic treatment is practical, safe, and effective, and can be considered as the first line regimen also in hospitalized patients with ae-copd. becher g a , gillissen a a , rothe m b . a st. george medical center, robert-koch-hospital, leipzig, germany , b filt, lung and chest diagnostics ltd., berlin, germany patients with severe form of chronic obstructive pulmonary disease (copd) are prone by frequent exacerbations. bacterial infections are judged to cause at least half of exacerbations. haemophilus influenzae and streptococcus pneumoniae are the most frequent isolates, gramnegative bacilli account for the severe cases, aggravating the inflammatory process in the airways eventually leading to respiratory insufficiency. the aim of this ongoing placebo controlled, parallel group, mono center study trial is to evaluate beneficial effect of cefixim to reduce bacterial load and pulmonary inflammation in patients (n / ) with acute bacterial exacerbation of severe copd. thus, patients received in randomized fashion either cefixim ( mg/day) or placebo ( days). on days , , and breath condensate is collected using 'ecoscreen' (jaeger germany) for ltb -, il- -, nitrite-and ph-analysis. in parallel sputum gathered for detection of bacterial strains, and for ltb -, and il- quantification purposes. these data are compared to clinical outcome parameters such as lung function tests, radiographic findings, serum inflammatory markers and length of hospital stay. the preliminary data obtained confirm successful antibiotic therapy with oral cefixim in bacterial related acute exacerbations of copd is a useful approach to reduce bacterial load, and concomitantly lower inflammatory indices of the central and peripheral airways leading to clinical improvement of the patients. soriano garcia f a , fenoll a b , fernandez-roblas r a , coronel p c , gimeno m c , rodenas e c , garcia m a , granizo jj d . a fundacion jimenez diaz, microbiology, madrid, spain , b instituto de salud carlos iii, centro nacional microbiologia, majadahonda, spain , c tedec meiji farma, scientific, alcala de henares, spain , d fundacion jimenez diaz, epidemiology, madrid, spain purpose: to describe the susceptibility of streptococcus pneumoniae against cefditoren and other antimicrobials by serotype a multicenter study in south europe was carried out. a total of strains were collected between september and march from adult patients (more than y.o.) with respiratory tract infection (respiratory tract samples and blood cultures). all the isolates were sent to a central laboratory (fundació n jiménez díaz, madrid, spain) where susceptibility test was performed by broth microdilution (sensititre) following nccls recommendations. serotype was determined by quellung reaction and dot assay in carlos iii institute in strains. results: a total of strains ( . %) were not typable. the most prevalent serotypes were ( . %), ( . %), ( . %), ( . %), ( . %) and ( . %). two hundred and sixty-four strains were grouped in different serotypes. the proportion of susceptible strains by serotype to penicillin, erythromycin and levofloxacin were: serotype ( . , . , %); ( . , . , . %); ( . , . , . %); ( . , . , . %); ( . , . , . %); ( . , . , . %). the mic to cefditoren was / . (serotype ); . (serotype , and ) and mg/l (serotype and ). conclusions: the most prevalent serotype was . the susceptibility was higher in serotype than in serotypes and . community acquired pneumonia */a study among closed military community of young people ps martynova av, turkutyukov vb, vostrikova aa, andryukov bg. vladivostok state medical university, epidemiology, vladivostok, russian federation purpose: the etiology of pneumonia is still partly unknown. we should like to clear up an etiological role of respiratory pathogens in community-acquired pneumonia among youth. and we had chosen for it a model of a closed community both investigation of etiology of disease and for further investigation of mechanisms of transmission drug-resistant mechanisms. methods: we studied adults in age of Á/ from closed military collectives who presented to two public hospitals (one urban and one rural) with acute radiologically confirmed pneumonia during winter Á/ . we did blood and lung-aspirate cultures, mycobacterial cultures, serotype-specific pneumococcal antigen detection, and serology for viral and atypical agents. results: streptococcus pneumoniae is recognized as an important cause of community-acquired pneumonia, it probably accounts for % of cases of community-acquired pneumonia among youth. chlamydia pneumoniae and mycoplasma pneumoniae responsible for approximately % of cases. haemophilus influenzae caused . % sever cases of disease, % of all cases were due to moraxella catharralis . conclusion: pneumococcial infection accounted for % of the cases diagnosed. s. pneumoniae was the most common bacterial infective agent, with a low incidence of both m. pneumoniae and s. pneumoniae . other causative pathogens occurred only within groups of individuals with deficiency of immunological status. berezin en a , cardenuto md a , nobuko e b , guerra ml c , brandileone mc d . a santa casa, pediatrics, s. paulo, brazil , b santa casa, microbiology, s. paulo, brazil , c adolfo lutz, microbiology, s. paulo, brazil , d adolfo lutz, bacteriology, s. paulo, brazil to determine antimicrobial susceptibility of sp isolated from the upper respiratory tract, we collected np swab specimens from children, between months and years old. those children attended the outpatient clinic in s. paulo city, with diagnosis of bacterial infection requiring antibiotic therapy between march , to may , . penicillin susceptibility of isolates was determined by screening with oxacillin mcg disk and performing the minimal inhibitory concentration by the e -test. we performed also susceptibility test for amoxicillin and cefaclor. results: sp was recovered from np of children ( . %). the carriage of sp was more prevalent in children attending day care centers, children with young siblings at home, and children with tobacco users at home. the prevalence of penicillin non-susceptible strains was . % all of them with intermediate resistance. all the strains were susceptible to amoxicillin and . % were resistant to cefaclor. serotypes . b, f, n, , a and were the most common. these findings suggest that nasopharyngeal isolates of streptococcus pneumoniae from children with upper respiratory infections can be used to conduct surveillance for antimicrobial resistance in a defined geographic area. we were able to conclude also that penicillin intermediate resistent strains can be susceptible to amoxicillin. hinojosa rm a , saenz a a , collazo m a , echaniz g b . a universidad autonoma de nuevo leon, infectologia, monterrey, mexico , b instituto nacional de salud publica, epidemiologia, cuernavaca, mexico the emergence of penicillin-and multidrug-resistant pneumococcal strains has become a global concern, necessitating the identification of the epidemiological spread of such strains. material: ninety streptococcus pneumoniae clinical isolates were collected from march through march . typing was done by the capsular reaction with pooled, type, or group antisera. susceptibility testing to antimicrobials was done by the e -test and the disk diffusion method. results: only ( %) s. pneumoniae strains were classified by serotyping; the most frequent types were a/b, f, v, f and . the oxacillin screening test detected . % penicillin-resistant s. pneumoniae strains isolated from children and . % from adults. the susceptibility percentage of s. pneumoniae to ceftriaxone was % in both children and adults. s. pneumoniae isolates from children exhibit a susceptibility of % to azithromycin, while in adults % of the isolates were susceptible. for the rest of the antimicrobial agents, the susceptibility ranged from to %. s. pneumoniae had a lower susceptibility to ceftazidime and ciprofloxacin. conclusions: ceftriaxone and azithromycin had a good in-vitro activity against s. pneumoniae strains. but the percentage of penicillinresistant s. pneumoniae detected in this study is alarming, therefore we conclude that a continuous surveillance system is necessary in mexico. vertkine al, prokhorovitch ea, alexanyan la. a retrospective analyses of cases of ambulant pneumonia with fatal outcome was made. among the patients who died from ambulant pneumonia the prevailing age was over ( . %) and the prevailing sex was male ( . %). . % had pneumonia accompanied with some pathology: chronic lung disease ( . %), alcoholism ( . %), diabetes mellitus ( . %). . % of the patients had a big volume of lungs lesion */ . % of the patients suffered from bilobular pneumonia and . % */from trilobate pneumonia. in . % of the cases pneumonia was complicated with abscess formation and/or exudative pleurisy. we studied the antibiotics therapy used for the patients treatment. the change of antibiotics was made only in cases ( . %) whereas in the other cases no change of preparations was made though the signs of the therapy non-effectiveness were obvious. thus, the rational antibiotics therapy with the timely change of non-effective antibacterial drug is significantly important. while choosing the antibiotics, the patient's age, the accompanying diseases, the volume of the lungs lesion and complications which define pneumonia seriousness are to be taken into consideration. perronne c a , rouveix b b , guillemot d c , zuck p d , reitz c e , tsatsaris a e . a hôpital raymond poincaré, service de maladies infectieuses, garches, france , b hôpital cochin, paris, france , c institut pasteur, paris, france , d hôpital de metz, metz, france , e laboratoires abbott, rungis, france objectives: to describe the management of lower respiratory tract infections (lrti) in healthy adults, by general practitioners (gp). methods: a questionnaire was sent to a representative national sample of gps. this questionnaire assessed their perception and management of lrti, the indication for antibiotics (ab) in a case of lrti in a healthy adult with no focal signs and no signs of severity, knowledge of the micro-organisms responsible for acute bronchitis and knowledge of the afssaps (french agency for the safety of health products) recommendations. results: three thousand seven hundred and thirty-eight gps, who reported seeing an average of patients per week, including . / . patients with lrti, returned the questionnaire. the main results are presented in the following for the majority of gps, the main objective of the visit is to determine the indication for antibiotics. according to gps, alp and whooping cough are rare, while atypical pneumonia is frequent. gps also declare that the diagnosis of alp is often easy right from the first visit, in contrast with that of atypical pneumonia. complementary investigations are not often requested. gps consider that they often delay prescription of antibiotics ( %) and declare that they tend to prescribe a macrolide as first-line treatment. finally, gps have a poor knowledge concerning the micro-organisms responsible for acute bronchitis and the majority of gps declare to be familiar with afssaps recommendations. perronne c a , rouveix b b , guillemot d c , zuck p d , reitz c e , tsatsaris a e . a hôpital raymond poincaré, service de maladies infectieuses, garches, france , b hôpital cochin, paris, france , c institut pasteur, paris, france , d hôpital de metz, metz, france , e laboratoires abbott, rungis, france objectives: to study the management of one case of lower respiratory tract infection (lrti) in adults by general practitioners (gps). methods: prospective study conducted on a representative national sample of gps. each gp had to include the first healthy adult patient seen during the -week data collection period, either on a home visit or in the office for recent cough and acute fever !/ . c. clinical data, the diagnostic perception and the therapeutic approach to the patient were collected by means of a standardised questionnaire, distributed by abbott laboratories. results: three thousand seven hundred and thirty-eight general practitioners included patients. conclusions: during lrti in adults, gps observe few focal signs, confirming the marked predominance of bronchitis compared to pneumonia. in view of the frequency of the signs, the diagnosis of pneumonia appears to be overestimated. one-third of clinical situations were diagnosed as 'bacterial superinfection of acute bronchitis', despite it is not a recognised diagnostic entity. antibiotic prescription was immediate in % of cases and delayed in % of cases. this last point shows that clinical practice differs from the gp's perception of their described prescribing practice shown in a simultaneous survey ( % of gps declared that they prescribed antibiotics immediately, while % delayed this prescription). results: thirty-three patients ( men, mean age b years, mean apache ii score . &bdqup;b . ) during the years Á/ were prospectively studied. thirteen patients ( %) had no identifiable risk factor for severe cap. an etiologic factor was revealed in patients ( %). in of them this was achieved with noninvasive methods. psb cultures were taken from eight patients and were positive in only . the offending organisms included: streptococcus pneumoniae in six cases, gnb as the sole pathogen in six cases, haemophilus influenzae (with s. pneumoniae or klebsialla pneumoniae ) in two cases, s. aureus in two and legionella pneumophila in one patient. initial antibiotic regimen a combination of marcodile '/ rd gen cephalosporin /aminoglycoside was successful in patients who all survived and had to be changed empirically or according to culture results in patients who had a mortality of %. the overall mortality rate was %. the identification of the causative factor did not seem to have any impact on survival. conclusion: severe cap in our icu was most often caused by s. pneumoniae and gbn. the high mortality of this entity seems to be influenced by the immediate use of the appropriate antibiotic combination and not by the identification of the causative organism. this underscores the need for knowledge of topical microbiology which helps in designing an effective empirical initial antibiotic regimen. mily mn a , golubev sa b , lugovoy vy a , voronov gg b . a vitebsk emergency hospital, pharmacotherapy unit, vitebsk, belarus , b basic and clinical pharmacology department, vitebsk state medical university, vitebsk, belarus purpose: the study aims were to assess the spectrum and predictors of the antibiotic use during pneumonia management in a regional emergency hospital in belarus. patients, treatment and physicians characteristics of cases ( Á/ ) were collected and possible associations were examined with using defined daily doses methodology (ddd) and american thoracic society (ats) guidelines. results: the mean treatment duration was . / . days, the total antibiotic ddd/ bed-days was . . the ddd/ bed-days of the most used antibiotics were: penicillins . ; aminoglycosides . ; macrolides . ; cephalosporins . ; tetracyclines . . in manova certain ats categories were associated with ddd/day, but not with frequency of definite antibiotic use. ddd/day was higher in case of multilobar infiltrates ( conclusion: our study indicated the low rate of macrolides and cephalosporins using, and the high one of aminoglycosides. antibiotic prescriptions were associated with disease severity and physician personality rather then with empirical choice rules recommended. acute exacerbation of copd: most frequent infecting agents and their suspectability to the different types of penicillins. analysis of medical documentation ps pertseva to, bogatska ke, gashynova ky. dsma, internal medicine , dniepropetrovsk, ukraine number of copd cases has been increased in ukraine. treatment of acute exacerbation (ae) of copd is not always successful because of inadequate antibiotic therapy. the aim of study was to reveal most frequent infecting agents and their susceptibility to the different types of penicillins in patients with ae of copd. medical documentation of patients with ae of copd (type i) was studied. data of sputum analysis and susceptibility of isolated agents to penicillin, ampicillin, oxacillin and amoxicillin/clavulone acid were evaluated. there were patients with haemophilus influencae/parainfluencae ( . %), klebsiella pneumoniae ( %), staphylococcus aureus ( . %), pseudomonas fluorescens , pseudomonas putida , serratia marcescens , serratia liquefaciens ( . % each), streptococcus agalactiae , acinetobacter baumanii ( . % each) in samples of sputum. in . % cases there was mixt infection. . % had no any bacterial agents. only % of agents were susceptible to penicillin, % */to ampicillin, % */to oxacillin. however, % of microorganisms were susceptible to amoxicillin combined with clavulone acid. this study has shown that most frequent infecting agents caused ae of copd were gram-negative microorganisms and s. aureus . according to antibiogram the prescription of amoxicillin/clavulone acid is most expedient in this case. pertseva to, bogatska ke, konopkina li, kireeva tv, gashynova ky. dsma, internal medicine department, dniepropetrovsk, ukraine we examined patients ( men, mean age . / . years) with acute exacerbation of cob (type i). the most frequently isolated agents were haemophilus influenzae and parainfluenzae */eight patients, gram-negative rods in , staphylococcus aureus in two and mixed in six and one patient had no bacterial agents isolated in their sputum. high susceptibility to azithromycin was found in all cases of gram-positive agents and in h. influenzae and parainfluenzae . other gram-negative agents were resistant to this drug in vitro. however, treatment with mg/day during days was clinically effective in . % of cases. only . % of patients had a further acute exacerbation of cob. there were peculiarities of the infecting agents causing acute exacerbation of cob in this study: klebsiella pneumoniae and s. aureus were found more frequently than in other studies. high efficacy of surnamed in the treatment of acute exacerbation of cob was established in . %. . % had partial positive clinical effect after this therapy. there were no patients with adverse events. efficacy and tolerance of amoxicillin mg/kg bid versus amoxicillin mg/kg tid in the treatment of acute otitis media (aom) in children / years ps borek m a , guggenbichler jp b . a biochemie gmbh, international medical department, kundl, austria , b department of pediatrics, university of erlangen, erlangen, germany five hundred and sixteen patients (mean age / . years) with clinical and otoscopic diagnosis of aom were included in a randomized, double blind, multicentre study, and were treated days either with amox mg/kg bid or amox mg/kg tid. assessments were made during therapy (day Á/ ), after end of therapy (eot, day Á/ ) and follow up (fu, day Á/ ). the primary efficacy endpoint was the clinical response at eot defined as success (cure/improvement) or failure. both regimens were well tolerated; one or more drug-related adverse events (aes) were reported in . % ( / ) of bid patients and in . % ( / ) of tid patients. the most frequently reported drugrelated aes in each group were gastrointestinal symptoms (bid . % vs. tid . %), which were mainly of mild or moderate severity. both regimens were clinically equivalent. the higher cure rates in the bid group suggest a possible higher benefit from bid therapy in children / years. children attending family day-care (fdc) should be less exposed to upper respiratory tract infections than those in group day-care (gdc) and therefore to antibiotic treatment; fewer should thus carry resistant bacteria. to test this hypothesis, np carriage of sp and hi with reduced susceptibility to penicillin (pdsp and hi bl'/, respectively) was investigated among children in fdc (maximum three children) and in gdc ( Á/ children) in the alpes maritimes (france) between november and march . a two stage cluster sample of children attending gdc or fdc was selected. np samples were cultured for sp and hi. penicillin susceptibility was tested by disk diffusion and e -test, and b-lactamase production by api-nh † tests (biomerieux, lyon). two hundred and thirty-five children in fdc and in gdc aged Á/ months were sampled. age and sex distribution were similar in both groups. sp was isolated in children in fdc ( %), and in ( . %) children in gdc (p b/ Á/ ). proportions of pdsp were . and . %, respectively (p / . ). hi was present in . % of children in gdc vs. . % in fdc (p b/ . ). proportions of hi bl'/ were . % vs. . %, respectively (p / . ). antibiotic exposure during the previous months concerned . % of children in gdc vs. . % in fdc (p / . ). there was no correlation between antibiotic use and carriage of pdsp or hi b'/ strains. sp and hi carriage rates are significantly lower among children in fdc than in gdc. advising alternative types of daycare for children attending gdc should reduce exposure and thus limit the spread of resistant bacteria. however, the proportion of pdsp and hi bl'/ is similar in both groups and comparable patterns of antibiotic use are observed. continued efforts must concentrate on parental education and enforcement of recommendations for management of pediatric upper respiratory tract infections. during a period between january and august , invasive haemophilus influenzae (hi) isolates had been collected at the children's hospital of tunis. we used haemophilus test medium to test antibiotic susceptibility. the mic of beta-lactams was measured by e -test. beta-lactamase production was determined by using the cefinase test and biotyping by apinh. presence of capsular antigen was determined by using hi typing anti sera. hi strains were isolated from meningitidis ( ), bacteremia ( ) and arthritis ( ). all strains were serotype b and . % of them belonged to biotypes i and ii. amoxicillin resistance with beta-lactamase producing mechanism occured in . %. mic of beta-lactamase producing strains was vs . mg/l in non-producing one. there is no betalactamasenegative amoxicillin resistant among these invasive isolates. antibiotic resistance concerned chloramphenicol: . %, trimethoprim-sulfamethoxazole: . %, tetracycline: . % and kanamycin: . %. invasive hi infections in tunisian children's were always associated with type b strains. introduction of a hib vaccine programme in tunisia is recommended. the aim of this study was to analyse the clinical picture and treatment of neurological manifestations of neuroborreliosis in children. the study included children ( Á/ years) with neuroborreliosis diagnosed on the basis of the clinical and serologic criteria. symptoms of facial palsy occurred in six children symptoms of iii Á/vi cranial nerves palsy in three children, meningitis in four and paresthesias in three. symptoms of v or viii nerve palsy, mental disturbances, radiculoneuritis or cerebellitis were found in singular cases. all children received ceftriaxone intravenously Á/ weeks. total recovery was obtained in children following the first course of therapy. recovery following the second course of therapy (amoxicillin) occurred in one child with mental disorders and one with vi nerve palsy. improvement was achieved after the second course in the patient with radiculitis, however, muscular atrophy persisted. irreversible, unilateral deafness was found in a child with viii nerve palsy in spite of three courses of therapy applied. infection with borrelia burgdorferi in children causes a wide spectrum of neurological manifestations. facial palsy was the most common sign in our study. applying ceftriaxone in the treatment of neuroborreliosis is characterised by a good effectiveness. double infection by c. pneumoniae and m. pneumoniae as a cause of cystic changes in the lungs ps streharova a, moravcikova d. department of infectious diseases, university of trnava, trnava, slovakia chlamydia pneumoniae and mycoplasma pneumoniae are human respiratory pathogens manifested in early childhood. immunological disbalance could trigger autoaggressive diseases. the authors describe the case of a -year-old girl with development of multiorgan failure and septic state, which followed multiple cystic changes in the lungs. the girl did not have a diagnosis of cystic fibrosis. the authors consider that cystic changes are a consequence of double infection by c. and m. pneumoniae. dzarlieva m, momeva l, temelkovska g, balevska p, pejkovska m. medical centre, neonatology, bitola, the former yugoslav republic, macedonia unicef skopje has supported a nationwide safe motherhood needs assessment in representative samples of hospitals. eighteen of maternity wards and facilities renovated and certified as 'babyfriendly'. all mature newborns with successful adaptation to extra uterine life and satisfactory vital parameters are h during the day with their mothers at rooming-in system. aim: with rooming-in system we reached decreasing of incidence of neonatal infections. material and methods: history records of newborns from our department. for the period of months, neonates have been borne. with suspection of infection there */ babies ( . %). newborns borne through meconium stained liquid */ ( . %). results: microbiological findings: from blood culture */staphylococcus coagulaza negative from swabs */staphylococcus aureus , escherichia coli , staphylococcus epidermidis. conclusion: in , from all babies who had risk factors for infection in newborns ( %) we had positive findings and in year (before rooming-in sistem), in ( . %). after that period (with rooming-in) newborns ( . %). with rooming-in system we reached decreasing in incidence of neonatal infections by breaking the chain of infection */only mothers take care of their babies with help of the staff. newborns are in their micro environment, the same they will have at their home. with this practice we have also reduction of nosocomial infections. a study on antibiotic susceptibility and resistance factor transmissibility among antibiotic resistant salmonellae isolated from children affected to diarrhea ps sharifzadeh a. azad university of shahrekord, microbiology, shahrekord, islamic republic of iran in spite of happened drug resistance, antibacterial therapy still is the best route of treatment of salmonellosis in man and animals. in order to detection of dominants serotypes of salmonellae in children and detection of antibiotic susceptibility and r-factor transmissibility among those isolated salmonellae. this study was conducted on diarrheic stool samples were collected from children affected by diarrhea in ayatollah kashany hospital of shahrekord, during spring of to autumn of . after isolation and identification of salmonellae, seven serotypes were detected. one of those was s. typhi and another six serotypes were s. paratyphi b . in order to detection of antibiotic different antibiotic disks were used in disk diffusion method. best results were taken from ceftizoxim, cephtriaxon, cephazolin and chloramphenichol. the r-factor were transferred from isolated salmonellae to escherichia coli k in all of cases of resistance to penicillin and ampicillin. pharyngeal colonization by streptococcus pneumoniae and group a bhs were evaluated in randomly selected school children aged Á/ years in riyadh, saudi arabia. fifty-six children ( . %) had positive culture for either organisms of the isolates from school children, ( %) were s. pneumoniae , of them ( %) were penicillin-sensitive, three ( %) were penicillin-resistant, and two ( . %) were resistant to two antimicrobials. forty isolates of bhs ( %) were group a bhs. all isolates were penicillin and erythromycin sensitive. the carrier rate among school children for penicillin-resistance s. pneumoniae and resistance to two antimicrobials were ( . %) and ( %), respectively. the carrier rate of group a bhs was ( . %). riyadh has a low rate of antibiotic-resistant s. pneumoniae and a similar rate of group a bhs carriers among school children as that seen in temperate areas. boukadida j a , boukadida n b , hannechi n a , said h b , erraii s a , elmhabrech h a . a chu f. hached, laboratoire de microbiologie, sousse, tunisia , b c s base, sousse, tunisia the acute pharyngitis is a very frequent pathology in which group a streptococcus is the most incriminated bacteria. however, other non a b-haemolytic streptococcus (sbna) could be responsible. the aim of this work is to determine the part of each non a b hemolytic streptococci (sbna) in acute pharyngitis and the related antibiotics susceptibility pattern. the study was realized in sousse-tunisia (north africa) during months from may . the origin materials of isolates are throat swab (transystem venturi, copan, bovezzo). the mean age of patients is years with extremes Á/ years. the samples are cultured on blood agar plates in a delay of h maximum. identification was done to samples that have over than b-hemolytic colonies, groupage with pastorex strep. sanofi pasteur france, susceptibility pattern according to nccls norms, mic is determined by e -test. the control strain is s. pneumoniae atcc . twentyone clinical isolates of sbna are distinguished from clinical isolates of b-hemolytic streptococci recovered from patients with acute pharyngitis without symptoms of viruses' infections (tearing, corysa, sneeze). all b-hemolytic streptococcus represents . % of all collected samples. sbna were . % of the isolates. sbna were strains group g streptococci, seven strains group c streptococci and three strains group f streptococci. susceptibility pattern of each sbna to antimicrobial agents is as follow: group g streptococci: peni g / %, amoxicillin / %, pristinamycin %, clindamycin and erythromycin / . %, tetracyclin / . %, telithromycin . % and levofloxacin / . %. group c streptococci: peni g / %, amoxicillin / %, pristinamycin %, clindamycin / . %, erythromycin / . %, tetracyclin / . %, tel-ithromycin / . %, levofloxacin / %. group f streptococci-peni g / %, amoxicillin / %, clindamycin / . %, erythro / . %, tetracyclin / . %, telithromycin / %, levofloxacin / . %. all sbna have mic to penicillin g under . mg/l. according to available data, penicillin g and amoxicillin still the reference treatment of acute bacterial pharyngitis in spite of the new antibiotics introduction. berezin en a , quevedo sg b , nicolla l b , viegas d c , eizenberg b d , pedrosa f b , santos ag a . a santa casa, pediatrics, s. paulo, brazil , b elly lilly, scientific, s. paulo, brazil , c fac. abc, pediatrics, santo andre, brazil , d university hospital, pediatrics, s. paulo, brazil a multicenter, open label, prospective, randomized trial in which patients Á/ years of age with proven gabhs pharyngitis were randomized to receive either -day course of the broad spectrum oral cephalosporin, cefaclor or a -day course of amoxicillin. patients were included if they have signs and symptoms of streptococcal tonsillopharyngitis and a rapid streptococcal rapid test positive . patients were evaluated at days Á/ , Á/ and Á/ posttherapy. pharyngeal cultures were conducted at baseline and at follow-up visit ( Á/ days). we considered for bacteriologic eradication analysis only patients with positive culture to gabhs. there were patients with a rapid streptococcal rapid test. clinical success were achieved in around % of the patients. for evaluate eradication of the initial pathogen we considered patients of the amoxil arm and patients of the cefaclor arm. thirty-four of patients of the amoxil arm ( . %) and of ( %) patients of the cefaclor arm were considered bacteriological cured at the second culture performed at day Á/ . ten days of a penicillin or amoxicillin therapy may not be the best therapeutic choice for all pediatric patients. in developing countries where rheumatic fever is still an important problem to evaluate the bacterial eradication achieved with the different antibiotics may be important. prophylactic affect of saccharomyces boulardii for antibiotic-associated diarrhea in a paediatric age group ps erdeve o, tiras u, camurdan mo, tanyer g, dallar y. ankara education and research hospital, pediatrics, ankara, turkey the process resulting in antibiotic associated diarrhea is the alteration of enteric flora due to bacteria. saccharomyces boulardii is a yeast, isolated from cover of a kind of hazelnut and its usage became widespread recently. there is no enough study about s. boulardii activity at pediatric ages. we aimed to define s. boulardii activity on azithromycin and sulbactam-ampiciline, and associated diarrhea at pediatric age group. the . % of cases only with antibiotic usage developed diarrhea, whereas rate was . % for probiotic using group (p b/ . ). all of the clostridium difficile toxin a defined cases were from sulbactam-ampiciline using group. the rate of diarrhea for sulbactam-ampiciline using group was . %, while it was . % for group used s. boulardii as probiotic beside sulbactam-ampiciline (p b/ . ). it was observed that probiotic usage decreases diarrhea rate four times (p b/ . ). when age groups considered, the rate of sulbactamampiciline associated diarrhea increased at Á/ ages and s. boulardii effect on preventing diarrhea was significant at Á/ ages (p b/ . ). antibiotic associated diarrhea is a common clinical problem at pediatric age group. s. boulardii is a hope giving probiotic especially for sulbactam-ampiciline associated diarrhea. grey e a bilikova e a , hafed bm a , kovacicova g a , chovancova d b , huttova m b , krcmery v a . a school of health, trnava university, trnava, slovakia , b postgraduate academy of medicine, neonatal clinic, bratislava, slovakia the purpose of the study: the aim of the study was to find out, whether artificial abortion of a mother has impact on neonatal infection of her future baby. the results obtained: therefore, we compared neonates with infection, who were born to mothers with past history of abortion ( Á/ artificial abortions within years), with the babies of mothers, who have not experienced abortion before. control group consisted of neonates hospitalized at the same clinic, at the same time period. according to the analysis of risk factors for neonatal infections, it was found, that prematurity ( Á/ weeks of gestation) ( . vs. . %, p . ) and low birth weight- Á/ g ( . vs. . %, p . ) were significantly more frequently observed in babies of mothers with past history of abortion. drug abuse (heroin) ( . vs. . %, p . ) and nicotine use ( . vs. . %, p . ), were more significantly related to neonatal infections in babies of mothers with past history of abortion. etiological analysis showed that only candida albicans ( . vs. %, p . ) was significantly related to neonatal infections in mothers with past history of abortion. the conclusion reached: in conclusion, artificial abortion has not only direct impact to the health of the mother, but also on her next pregnancy. bacteraemia and patient mortality in a peadiatric intensive care unit ps armenian sh, singh j, arrieta ac. children's hospital of orange county, infectious disease, orange, usa purpose: this study will identify the factors that significantly contribute to mortality in patients with bloodstream infections (bsi) at a pediatric intensive care unit (picu). results: medical records of patients who were admitted to the picu and had a documented bsi were reviewed. there were separate episodes of bsi's, with nine patients having multiple bsi's during their hospital stay. a casecontrol model was used. cases were bsi's with eventual mortality (n / ; . %) and controls were those who survived bsi (n / ; . %). patients who died were older ( . vs. . years; pb/ . ), more likely to have a nosocomial bsi ( . vs. . %; pb/ . ), longer hospitalization prior to bsi ( . vs. . days; pb/ . ), and have a polymicrobial bsi ( . vs. . %; pb/ . ). infection related mortality (irm)-defined as death within days of bsi */was significantly higher in those receiving inadequate antibiotic treatment at the time of diagnosis of bsi ( . vs. . %; p b/ . ), as well as in those with gram negative bacteremia and/or fungemia ( vs. %; p b/ . ). logistic regression was used to adjust for potential confounding variables. conclusions: we found that being older, multiple organisms, and a longer hospitalization prior to the bsi were significantly associated with overall patient mortality. irm was significantly higher for those with inadequate initial antibiotic coverage and in those with gram negative bacteremia and/or fungemia. somer a a , gü r d a , diri s a , yalcýn i a , salman n a , ongen b b , gü rler n b . a department of pediatric infectious diseases, istanbul university istanbul medical faculty, istanbul, turkey , b department of microbiology and clinical microbiology, istanbul university istanbul medical faculty, istanbul, turkey teicoplanin is a glycopeptide antibiotic active against a broad range of gram-positive pathogens including methicillin-resistant staphylococci and offers the advantages of once daily administration, choice of administration route, lack of requirement for routine therapeutic drug monitoring and lower propensity to cause nephrotoxiticy and anaphylactoid-like reactions. in this study the efficacy and safety of teicoplanin were evaluated retrospectively in children with serious bacterial infection. sixty-three children ( girls, boys) aged between month and years were treated with teicoplanin (three loading dosages of mg/kg at h intervals, followed by a maintenance dosage of mg/kg/day). the infections treated were pleural empyema (n / ), joint and bone infections (n / ), septicemia (n / ), skin and soft tissue infections (n / ), and lung abscess (n / ). the pathogens isolated were staphylococcus aureus (n / , of which were methicillin resistant), coagulase-negative staphylococci (n / ), s. pneumoniae (n / ), and group a hemolytic streptococci (n / ). the duration of therapy ranged from to days (median days). clinical success (cure plus improvement) was achieved in . % of cases. no side effects attributable to teicoplanin therapy were encountered. teicoplanin appears to be an effective and well-tolerated treatment for serious gram-positive infections in children. the risk of infection is present in all children with acute leukemia. two hundred and sixty episodes of febrile neutropenia were analyzed in children (aged . Á/ years) with aml */ cases and all */ cases over years during cytostatic therapy (protocols: mbfm */ aml and mbfm */ all). a degree of fn occurred in % of cases in aml, in % */in all. the sites of infection were: blood'/ central venous catheter ( %) and respiratory tract ( %). pathogens isolated from blood were: gram-positive */cns */ %, streptococcus spp. */ . %, gram-negative */enterobacteria */ %, pseudomonas aeruginosa */ %, candida spp. */ . %, aspergillus spp. */ . %, other */ . %. for the treatment of fn we used empirical antibiotics regimens. clinical response was noticed: in st line of therapy */ cephalosporins Á/ generation used */ %, carbapenems used */ % as nd Á/ rd lines */vancomycin */ %, amphotericin b */ %. thirtytwo percent of children with aml and . % children with all died because of sepsis. conclusion: carbapenems are more active in the st line of antibiotics therapy both for patients with aml and all. vancomycin is useful in the nd line for patients with all. amphotericin b and vancomycin are useful in combination in the nd line for patients with aml. pavlenishvili ivp. medical academy, pediatrics, tbilisi, georgia our drug of choice in cases of complicated neonatal sepsis i. pavlenishvili, g, iobashvili tbilisi, medical academy georgian society of pediatrics chemotherapy (gspc) with collaborations of drug and therapeutic committee (dtc) of childers hospital 'republic' finished the observation study about nosocomial infections of neonatal icu in above mentioned hospital. study begins from november . during this period we observe cases of neonatal sepsis, most of them were due gram-negative bacteria. thirty-four cases of neonatal sepsis were cause different stains of escherichia coli , proteus spp. and acinetobacter . we notice that during last years the role of acinetobacter in etiology of neonatal sepsis and nosocomial complications of neonatal sepsis in our hospital is gradually increased. as our observation reveals most optimal in the case of complicated neonatal sepsis was (according criteria of dts) use of ceftasidime. almost in all cases we used ceftasidime with success. we have only one case of mortality. since the macrolide resistance (mr) in streptococcus pyogenes has been increasing in europe, we studied the incidence and genetic basis of mr in s. pyogenes in russia. s. pyogenes isolated at baseline and during follow-up visits from children with acute pharyngitis receiving penicillin or midecamycin ( -membered macrolide) were studied. mr was evaluated by agar dilution (nccls), resistance mechanisms */by pcr. a total of s. pyogenes were obtained at the baseline, ( %) of which were erythromycin-resistant: strains ( %) were erm (a)-positive, one */mef (a)-positive, and one was negative for all primers used. all erm (a)-strains were inducibly resistant to clindamycin and represented seven pfge profiles with one profile found in / strains. in three midecamycin treated patients mr was selected during the therapy (one strain had erm (a), one */mef (a), one */ unknown resistance determinant). mef (a)-strain obtained during follow-up visit had different pfge pattern with mef (a) strain isolated at baseline, while both the baseline and follow-up strains with unknown mechanism of resistance had unique pfge pattern. these data showed moderate incidence of erythromycin-resistant s. pyogenes in smolensk. ribosomal methylation (erm (a)) was the most common mechanism and though the polyclonal nature of mr was established, most erm (a)-strains belonged to only a few clones. bacillus cereus infections in traumatology-orthopaedy department: retrospective study and re-evaluation of healthcare practices ps bacillus cereus was cultured for patients from traumatology department who had developed postoperative wound infections between august and march . all patients presented inferior members open fractures, frequently contaminated with telluric material and requiring external fixators. genomic study of clinical isolates by pulsed field gel electrophoresis and random analysis polymorphic dna, allowed us to eliminate an outbreak. furthermore, the reduced delay in which patients developed the infection ( days'//- ) led us to re-evaluate the procotols used in our institution. indeed, all patients had received amoxycillin '/ clavulanate iv g for antibioprophylaxis during anesthetical induction then relayed per os for hours. because of the production of a potent beta-lactamase by the bacteria, this association could not be efficient. furthermore, accordingly to the afnor en norm, we have tested clinical isolates' sensitivity to the principal antiseptics used for antisepsis and disinfection (iodophors, chloride derivated and biguanidines) and observed a major resistance of all strains tested. even if these postoperative wound infections are considered as nosocomial because of the delay in appearance, we actually think that bacillus cereus were initially present in telluric material. this fact led us to propose a systematic screening for bacillus at admission for this type of wound and to administrate quinolones such as ciprofloxacin for prophylaxis. internal thiols and reactive oxygen species in the candidacidal activity exerted by a n-terminal peptide of human lactoferrin ps the purpose of the study: the emergence of candida albicans strains resistant to current antifungals points to the need for new antifungal agents, e. g. antimicrobial peptides. the results obtained : we report that hlf( - ), a synthetic nterminal peptide of human lactoferrin, displays excellent killing effect against fluconazole-resistant c. albicans and that sub-optimal concentrations of this peptide combined with fluconazole act synergistically. previous investigations revealed that hlf( - ) required an energized mitochondrion, atp release by candida , and ligation of atp receptors for its killing effect. we now report that reactive oxygen species (ros) are involved in the killing effect of hlf( - ). since internal thiols protect cells from oxidative damage, our observation that hlf( - ) caused a % reduction of internal thiols in candida is of interest. as expected, n-acetyl-l-cysteine (nac), which is a precursor of glutathione and a ros scavenger, inhibited the killing effect of hlf( - ). diamide, which oxidizes internal thiols, was candidacidal and hlf( - ) and diamide acted synergistically in killing c. albicans and ros production. moreover, the hlf( - )-induced activation of mitochondria was inhibited by nac, indicating that internal thiols/ros affect mitochondrial activity. the conclusion reached : the candidacidal activity of hlf( - ) involves ros production and reduction of internal thiols. objective : an audit was conducted to explore the observation of increasing resistance to ciprofloxacin in enterobacteriacea isolated from specimens from such patients in the hematology unit. results : enterobacteriacea isolates in specimens from such patients processed over the years to were examined. number of specimens per year was */ , , , and respectively and the annual percent ciprofloxacin resistant enterobacteriacea from these was %, %, %, % and %. conclusion : conclusion drawn from the audit was that rapid rise in ciprofloxacin resistance may possibly be attributed to the use of this fluoroquinolone as a single agent in dose of / mg [cut off patient weight kg] twice a day in neutropenic patients till neutrophils exceed /c.mm. subsequent to the audit, prophylaxis protocol was modified to use of oral colistin -mega units/twice a day in combination with ciprofloxacin / mg twice a day. a prospective audit is proposed to test the benefits of this combination. rhinocerebral zygomycosis: diagnostic dilemma for emergency physician: can the associated morbidity and mortality in this rare but deadly disease be reduced? ps samavedam s, guleri as. western infirmary, clinical microbiology, glasgow, united kingdom rhinocerebral zygomycosis [rcz] is a rare, invasive, rapidly progressive opportunistic infection caused by ubiquitous fungi of the order mucorales. it usually occurs in diabetics or immunocompromised patients. the emergency physician will typically see patients with rcz in its earliest stages masquerading as a variety of other less serious diseases. the key markers like necrotic patch on hard palate, nasal septum or turbinate, marked facial pain, and cellulites with marked eye and neurological signs may present late in disease. we report a case of rcz caused by rhizopus arrhizus [oryzae] in an year old woman with poorly controlled diabetes. she presented with a right-sided facial droop of short origin and being generally unwell. ct scan was non-conclusive and delayed presentation of key markers of rcz permitted disease to rapidly progress. despite an intensive antifungal therapy with ambisome and insulin sliding scale, patient rapidly succumbed within days. fine needle aspiration cytology is less invasive, easier and equally effective alternative to pre op biopsy. the key to successful reduction in morbidity and mortality associated with this rapidly fatal disease is -increasing awareness of the disease, an early diagnosis, correction of underlying metabolic derangement, prompt intensive antifungal therapy with amphotericin b and radical surgical debridement of the necrotic tissue. an 'optimal dosage' of ambisome requires discussion. clinical audit in the haematology ward of a tertiary care hospital: study of degree of correlation between bacteraemia and oro-pharyngeal screens in immunocompromised patients over five years and role of antibiotic prophylaxis ps guleri as, butcher i. western infirmary, clinical microbiology, glasgow, united kingdom introduction : a clinical audit was carried out over -years [ ] [ ] [ ] [ ] [ ] in the immunocompromised patients including neutropenic patients and bone marrow [autograft] transplant recipients in hematology ward of gartnaval general hospital, glasgow, a tertiary care center. objective : it was aimed to establish the degree of correlation between bacterial isolates in oro-pharyngeal screen during bacteraemia episodes and role of antibiotic prophylaxis. methods purpose : to assess whether antiretroviral therapy (art) intensification, gm-csf use and remune initiation before stopping art lead to viremia containment, and long periods off art. methods : ten adults with chronic hiv disease, hiv- rna levels (vl) b/ . log copies/ml and median cd '/ -t cell count of /ml were enrolled. after art intensification with ddi ( months) '/ hydroxyurea [hu]( mo.) '/ gm-csf ( mo.) and a remune dose, art was stopped but remune continued. art was resumed if rebound vl did not decrease to b/ . log in months or if cd '/ counts decreased to b/ . results : vl rebounded in all patients after stopping art, and developed an acute retroviral syndrome (ars). cd '/-t cells decreased, and cd '/ '/ increased ( -fold). after a median stoppage of weeks, art was resumed in patients and vl decreased to b/ . log, cd '/ counts were regained, il- and il- levels rose. at the nd interruption, / patients had a rebound, and / had a nd ars, but peak vl and loss of cd '/ were lower (p / . ). after . weeks off art, patients resumed therapy. the breadth and magnitude of hiv-specific activity increased and thymus size grew. the patients were off art for a median of . out of weeks. two of them are off art for and weeks respectively ( vlb/ log). conclusions : this approach led to a high ars incidence, long periods off art, increases in hiv-specific responses, il- and il- levels, and thymus size. urinary tract infection in hospitalized population is a significant problem. this is a study of catherized patients urinary infection in corfu hospital. in Á/ , urine cultures were sent to our bacteriology laboratory. of them were collected from the catheters. clinical data of age and type of disease were analysed. the samples were cultured on mc conkey agar-urotube and vitek cards. the organisms identified with vitek automatic system. the sensitivity was tested with vitek and kirby-bauer method. results: no growth of organisms in . %. positive cultures %. . % of the bacteria were gram((/) rods ( . % e. coli , pseudomonas spp. %), % were gram('/) cocci (enterococcus spp.) and % was candida spp. the resistance of e. coli was: % to ampicillin , % to co-trimoxazol and % to quinolons. e. faecalis was resistant % to vancomycin . conclusions : ( ) the possible interference of acetaminophen in the amoxicillin/ clavulanic acid (a/c) or erythromycin (ery) efficacy in the treatment of acute otitis media (aom), and its possible role in the evolution to otitis media with effusion (ome), were determined in a gerbil model. a f streptococcus pneumoniae strain exhibiting a mic of a/c and ery of / . and . mg/l, respectively, was used. both antibiotics were tested at . and mg/kg. acetaminophen at mg/kg was administered min before each antibiotic dose. antibiotic concentrations in serum and middle ear exudate were determined. both antibiotics significantly reduced the number of culture-positive ears and colony counts, with serum concentrations over the mic of the microorganism for ]/ % of the dosing interval. antibiotic concentrations in middle ear exudate were almost identical in animals receiving and not receiving acetaminophen. clinical and microbiological efficacy was correlated with antibiotic concentrations in middle ear exudate ]/ . times the mic of the microorganism, for both antibiotics. both antibiotics demonstrated efficacy in the treatment of pneumococcal aom, with the same rate of ome. acetaminophen, concomitantly administered, did not interfere the efficacy of the two antibiotics tested and did not prevent the evolution of aom to ome. parra a a , ponte c a , cenjor c b , garcía-olmos m a , giménez mj c , aguilar l c , soriano f a . a fundación jiménez díaz, medical microbiology, madrid, spain , b fundación jiménez díaz, otorrinolaryngology, madrid, spain , c glaxosmithkline, medical department, madrid, spain a gerbil model of otitis media with effusion (ome) induced by haemophilus influenzae (amoxicillin/clavulanate-a/c-and erythromycin-ery-mics of / . and mg/l, respectively) was used to evaluate the efficacy of a/c ( / and / mg/kg) and ery ( and mg/kg). antibiotics were administered subcutaneously h post-middle ear inoculation, and continued t.i.d for h, with or without acetaminophen (ap), at mg/kg, administered min before each antibiotic dose. antibiotic concentrations in serum and middle ear (me) were measured by bioassay. me samples for colony counting were collected on day . a/c reduced (p / . ) positive me samples and colony counts versus untreated controls or ery: me positive cultures of % for controls, % for a/c , % for a/c , % for a/c '/ap, % for ery , % for ery and % for ery '/ap. this was due to a/c (but not ery) concentrations in me exceeding . times the mic despite the higher percentage of antibiotic penetration of ery versus a/c ( versus / %). animals receiving ap showed less polymorphonuclear cells and more bacteria in me than those receiving only antibiotics, suggesting that the anti-inflamatory drug diminish the phagocytes and therefore, the efficiency in bacterial clearance. amoxycillin treatment for acute otitis media caused by penicillinresistant streptococcus pneumoniae . a pharmacodynamic analysis pm parra a a , ponte c a , cenjor c b , garcía-calvo g a , giménez mj c , aguilar l c , soriano methods: a serotype f streptococcus pneumoniae strain exhibiting a mic of amoxycillin of mg/l was used in an experimental model performed in gerbils (meriones unguiculatus ) following previously described procedures. amoxycillin was tested at the following doses: . , . , . , . , . and mg/kg. amoxycillin concentrations in serum and middle ear exudate were determined after drug administration. results: doses of ]/ . mg/kg significantly reduced the number of culture-positive ears, colony counts and otorrhoea (p / . ) as compared with untreated controls or animals treated with doses lower than . mg/kg. doses of ]/ . mg/kg achieved antibiotic concentrations in the middle ear . Á/ . times higher than the mic of the infecting strain and serum concentrations over the mic for Á/ % of the dosing interval. conclusions: amoxycillin at doses achieving serum concentrations similar to those obtained in children after standard doses, obtained therapeutic and microbiological efficacy regardless the susceptibility of the infecting strain. better correlation was found between antibiotic efficacy and antibiotic concentrations in middle ear exudate than between efficacy and serum concentrations, which were suboptimal from the pharmacodynamic perspective. increasing prevalence of amoxycillin Á/clavulanate-resistance among e. coli strains in a hungarian university hospital pm veréb i, vígh a, urbán e, hajdú e, nagy e. department of clinical microbiology, university of szeged, szeged, hungary background: amoxicillin Á/clavulanate resistance (acr) is an emerging problem in escherichia coli as reported from different parts of europe. the aims of the present study were to evaluate statistically the prevalence of acr among e. coli isolates and to investigate the genetic background of the resistance. methods: all e. coli strains isolated between and were screened for acr by kirby Á/bauer disc diffusion method. the resistance to other beta-lactam Á/beta-lactamase inhibitor combinations and to different beta-lactam antibiotics were also tested. selected strains underwent determination of beta-lactamase activity. confirmatory tests for suspected extended spectrum beta-lactamase were performed. pcr testing for tem and shv genes were carried out on plasmids isolated from selected strains. results: in out of e. coli strains ( . %) were found to be resistant to amoxicillin clavulanate (amc). most of the resistant strains ( %) were obtained from the genitourinary tract and no acr isolate was found in blood cultures. in out of isolates ( %) proved to be acr and . % were isolated from blood cultures and . % from the genitourinary tract. thirty-five selected strains were further analysed. thirty-two were also resistant to (sam) and six were further resistant to tzp. quantitative beta-lactamase determination showed increased activity in strains which were partially susceptible to amc. the presence of esbl could be proved only in three acr isolates. this open parallel-group study compared the efficacy and tolerability of cef with cfix mg once daily in the treatment of community acquired uncomplicated uti. seventy-eight female patients were randomized to receive either oral cef or cfix for or days. the efficacy of treatment was evaluated by clinical response (by symptoms of uti dysuria frequency urgency suprapubian pain and by clinical signs) by bacteriologic response and health status measures at baseline and posttherapy. results: the clinical cure (complete resolution of symptoms and signs) rate for patients receiving cef was . % of the evaluable patients and . % of the patients receiving cfix. bacteriologic response (based on the results of urine cultures obtained posttherapy) the pathogen was eradicated in . % for cef . % for cfix. no drug related side effects have been reported in cef and side effects were experienced by . % of the patients receiving cfix. improvement in health status comparing visual scale scores baseline and poststudy to have detected a higher change in average score from to in cef, from to in cfix. wilcoxon improvement value was significant on the rd day of therapy in case of cef and on the th day of therapy in cfix group. in conclusion, the results of this study indicate that cef course is more effective than cfix in producing a favourable clinical outcome and achieving higher bacteriologic eradication rate, furthermore cef was better tolerated. cefepime is a fourth generation cephalosporine that has a broader spectrum of antibacterial activity than the third generation cepfalosporines and is more active in vitro against gram-positive aerobic bacteria. the purpose of this study was to measure cefepime concentrations in plasma, bile fluid and gall bladder tissue in patients undergoing cholecystectomy. thirty patients male, female, mean age: years had data acceptable for analysis and were included in this study. all patients received iv g of cefepime. several hours after administration and at different time intervals, during surgery, samples were obtained from plasma, bile fluid and gall bladder tissue concomitantly. antibiotic levels were measured by an agar diffusion method. the mean delta time was / min. the values for plasma, bile fluid and gall bladder tissue, were . / . , . / . and . / . mg/ml, respectively. the plasma/bile fluid ratio was . / . . there was a significant correlation between plasma and gall bladder tissue concentration (r / . , p / b/ . ). a correlation between bile fluid and plasma cefepime concentration was not observed. the minimum inhibitory concentration (mic) data from previous in vitro studies indicate that the cefepime concentration observed in plasma bile and tissue samples of this study would be adequate against typical biliary tract pathogens. furthermore, these cefepime concentrations correlated well with the favorable clinical outcome reported in previous clinical studies in biliary tract infections. there was also good correlation between delta time and plasma and tissue concentrations and if the dose were given closer to the time of surgery, cefepime concentration would be higher reducing the possibility of an infection. objectives: the use of antibiotics may lead to decreased colonization resistance and increased formation of resistant bacteria. present concept was developed to overcome these untoward effects. methods: b-lactamase of bacillus licheniformis was overproduced in bacillus subtilis . this targeted recombinant b-lactamase enzyme (trbl) was released in the small bowel from a controlled-release formulation. beagles (n / ) were treated bid with either mg/kg ampicillin (i.v.)'/placebo (p.o.), mg/kg ampicillin (i.v.)'/trbl (p.o.) or only placebo (i.v.'/p.o.). stool was collected at days and . samples were cultured for total and main groups of aerobic and anaerobic bacteria and yeast. temperature gradient gel electrophoresis (tgge) was used to separate the ribosomal rna genes. results: ampicillin'/placebo group had clearly decreased counts of both aerobic and anaerobic bacteria during the treatment, whereas those receiving trbl had only minor overall changes and some occasional changes by single species. intravenous ampicillin decreased the fecal similarity percentage to %. the similarity percentage during treatment with ampicillin'/trbl did not differ from that of placebo ( vs. %). conclusions: according to our results the trbl can maintain the large intestinal microflora almost unchanged. these results indicate that trbl is a promising novel approach for overcoming the ecological adverse effects on gut flora caused by b-lactam antibiotic agents. adamis since broad-spectrum â-lactams combined with amikacin are often applied for nosocomial infections, their pharmacokinetic interactions might be interesting. one gram of aztreonam and . g of amikacin were administered intravenously single and in combination in six healthy volunteers. blood samples were collected at regular time intervals and concentrations of antimicrobials were determined by a microbiological assay applying a strain developing resistance to single agent after serial passages. mean concentrations of amikacin in serum when administered alone and in combination with aztreonam were . and . , . and . , . and . , . and . , . and . and and . mg/ml immediately after and . , , , and h after infusion of antimicrobials. respective concentrations of aztreonam were . and . , . and . , . and . , . and . , . and . and . and . mg/ml. aucs for amikacin when administered alone and in combination with aztreonam were . / . and . / . mg h/l, respectively. respective auc for aztreonam were . / . and . / . mg h/l. it is concluded that the co-administration of aztreonam and amikacin results in earlier clearance of aztreonam and in higher levels of amikacin compared to the administration of each single antimicrobial. molecular modelling of b-lactams reveals the structural basis for their inhibition of penicillin-binding proteins, susceptibility to b-lactamases and oral bioavailability pm grail bm, gupta s, payne jw. school of biological sciences, university of wales, bangor, uk b-lactam antibiotics are peptide mimetics that act as suicide substrates for transpeptidase enzymes that cross link bacterial cellwall peptides. for the first time, the structural and electronic features needed for their recognition by transpeptidase have been fully described, using innovative molecular modelling techniques to compare the conformational forms adopted by cell-wall peptides and blactams. comparison of features in the backbone and c-terminal regions of conformers of active b-lactam antibiotics and model cellwall peptides, has allowed definition of the molecular recognition template required for substrate recognition by transpeptidase. these shared structural features allow both to act as substrates and to acylate the active-site serine. however, a significant difference in a critical backbone torsion between the two substrates, provides an explanation for the inability of the enzyme Á/antibiotic complex to undergo the deacylation step that causes inhibition of transpeptidase. on the other hand, b-lactamases appear to have evolved molecular mechanisms that facilitate the deacylation reaction through compensating for the altered structural orientations in b-lactams caused by the different backbone torsion. finally, analysis of the conformer repertoires of blactams for structural features required for substrate uptake by peptide transporters, provides insights into how their structures can be tailored for optimal oral absorption. antimicrobial susceptibility of proteus mirabilis clinical isolates producing extended spectrum beta-lactamases (esbls) pm objectives: the aim of the present study was to determine in vitro susceptibility to antimicrobials of proteus mirabilis isolated from urinary tract infections. methods: we studied the susceptibility profile of esbl positive p. mirabilis strains in three adopted children from india with age range from months to years. esbl was identified using the synergic effect of clavulanate with betalactams (ceftazidime and cefotaxime the in vivo efficacy of amoxicillin (amx) sub-therapeutic doses ( . mg/kg, t.i.d for h, achieving serum levels over the mic of only % of the dosing interval) and concomitant specific serotherapy (single intraperitoneal dose of / diluted hyperimmune serum (hs) obtained from mice immunized with the heat-inactivated strain) was assessed in a pneumococcal sepsis balb/c mouse model. mice (five mice/ treatment group) were intraperitoneally infected with . )/ cfu/ ml of a serotype b penicillin-resistant strain (mic of and mg/l for penicillin and amx, respectively). treatments started h after bacterial inoculation. study groups were: control (k; receiving nonimmune serum (nhs)), amx'/nhs, hs, and amx'/hs. survival rates (%) over time were: purpose of the study: the study was performed to determine the consumption of imipenem and resistance of gram-negative pathogens (pseudomonas aeruginosa , acinetobacter sp., klebsiella sp., escherichia coli , proteus mirabilis , serratia marcescens , enterobacter sp. ) to imipenem. gram-negative pathogens were isolated at the sestre milosrdnice university hospital from zagreb, croatia, in and . the imipenem sensitivity testing was performed by disk diffusion and e -test methods. the consumption of imipenem was expressed in ddd/ hospital days in the same periods. results obtained: imipenem resistance of acinetobacter sp. decreased significantly in the year (p / . ), especially in the first months (p / . ) when the lowest consumption of imipenem was recorded. imipenem resistance of other gram-negative pathogens did not decrease significantly. conclusion reached: comsumption of imipenem might lead to changes in resistance to imipenem among acinetobacter strains. vacheva-dobrevski rs, savov ez. military medical academy, clinical microbiology, sofia, bulgaria purpose: acinetobacter baumanii is becoming increasingly frequent nosocomial pathogen at our hospital, and beta-lactam resistant strains are on the increase, especially among icu isolates. to study the susceptibility of a. baumanii clinical isolates to beta-lactams and to determine the esbl-producing strains during , year. a total gram-negative nonfermenters (gnnf) isolates was investigated by semiautomated mini api system (bio merieux, france). eighty-four a. baumanii non-repeated isolates was studied for esbl-producing by double-disk synergy test (ddt) and atb-blse test (bio merieux, france). mics for beta-lactams were determined by e -test (ab biodisk, sweden). results: a. baumanii (n / ) showed a multidrug resistance. the isolates were resistant to cefotaxime ( %), cefoxitin ( %), ceftazidime ( %), amoxicillin/clavulanate ( %), piperacillin ( %), aztreonam ( %), imipenem ( %). the ( %) of investigated a. baumanii expressed esbl activity and originated more frequently from icu ( %). esbls producing strains were isolated from endotracheal aspirate ( %), surgery wounds ( %), blood culture ( . %). conclusions: in general resistance levels were higher in clinical isolates a. baumanii to beta-lactams. the ddt seems to be a practical method for esbl-screening; atb-blse method is more sensitive. our study display to be the first report of esbl-producing a. baumanii strains from our country. carbapenems seems to be the most active agents against a. baumanii . salmonella infantis , strain , was isolated from a newborn baby at wassila bourguiba maternity in tunis. it exhibited high resistance to penicillins, extended-spectrum cephalosporines (cefotaxime, ceftriaxone, ceftazidime, cefpirome) and aztreonam but remained susceptible to cefoxitine and imipenem. involvement and characterization of enzymatic mechanism in b-lactam resistance were investigated in strain . isoelectricfocusing revealed that this strain produced a b-lactamase of pi . this enzyme had a broad-substrate profile, hydrolyzing amoxicillin, ampicillin, ticarcillin, cephaloridine, cefuroxime, cefotaxime, ceftriaxone, cefpirome and ceftazidime. the highest specific activity was observed with ampicillin. cefotaxime was hydrolyzed the most efficiently of the extended-spectum cephalosporines. the pi extended-spectrum b-lactamase (esbl) was inhibited by clavulanic acid and sulbactam. no inhibition of the esbl was observed with mm edta. thus, no metal ion is involved in hydrolysis for this b-lactamase. resistance due to the production of the pi esbl was transferred with dna plasmid into escherichia coli . on the basis of substrate and inhibition profiles and isoelectric point, the pi esbl was not previously described in s. infantis in tunisia. the presence of such a resistance on a plasmid raises concer for rapid dissemination among bacteria and loss of effectiveness of blactams. poizot-martin i a , enel p b , benhaïm s c , vion-dury f c , dinh t c , drogoul mp c , gastaut ja c . a assistance publique hôpitaux de marseille, cisih sud, pr ja gastaut, marseille, france , b assistance publique hôpitaux de marseille, cellule santé publique dmi , marseille, france , c assistance publique hôpitaux de marseille, cisih sud, marseille, france objective: to assess liver biopsy (lb) practices in a cohort of co-infected hcv and hiv patients followed up in an hiv specialized medical unit. method: transversal study with questionnaire among patients in pre-therapeutic's evaluation with pcr'/ and without lb at months. results: among the patients, ( %) are lost of follow up, ( . %) have had lb, ( . %) have no lb. the characteristics of these patients are: median age / . / years; sex ratio / . , cdc-stage a / . % b / . % c / . %, undetectable viral load / . %, median cd / / , anti-retroviral therapy / . %, hcv-genotype / . %; a / . %; / . %. causes of non-made lb are: ( ) refusal from patients because of biopsy's fear / . %; ( ) contraindications because of hiv infection / . % (clinical events / . % which contraindicate anti-hcv treatment, grade iii thrombocytopenia / . % which contraindicate biopsy, non-adherence to previous hiv follow up / . %); ( ) other / . % (alcoholism / . %, psychiatric/depressive disorders / . %, decompensated cirrhosis / . %). drug use or methadone/buprenorphine treatment are not considered as contraindication. conclusion: one-third of patients are afraid of lb. alcoholism and psychiatric/depressive disorders are the principal contraindications to anti-hcv treatment. it seems important to improve information of patients about lb and to focus on alcohol and psychiatric/depressive disorders management in such population. kashiwagi kk a , furusyo nf a , nakashima hn a , kashiwagi sk b , hayashi jh a . a department of environmental medicine and infectious disease, kyushu university, fukuoka, japan , b national kyushu medical center, fukuoka, japan the purpose of the study: the aim of this prospective study was to explore the effect of htlv-i co-infection on the development of hcc among patients with chronic hcv viremia. a total of consecutive patients with chronic hcv viremia were studied and followed-up over a mean period of . years: ( . %) were infected with htlv-i infection and ( . %) were not. the results obtained the annual hcc development rate was . % in patients co-infected with hcv and htlv-i and . % in patients infected with hcv alone. hcc was significantly higher in ( . %) of the patients co-infected patients than in ( . %) of the patients infected with hcv alone (p b/ . , logrank test). in patients under the age of years, hcc development was significantly higher in seven ( . %) of patients co-infected with hcv and htlv-i than in eight ( . %) of patients with hcv alone (pb/ . , logrank test), whereas there was no significant difference in hcc development between patients over age with or without htlv-i infection ( ( . %) of and ( . %) of , respectively). the conclusion reached htlv-i infection accelerates the development of hcc in chronic hcv patients, especially among patients under the age of years. to analyze hbv genotype-related clinical differences among patients with chronic hbv infection, all patients were serially tested for serum alanine aminotransferase (alt) and hepatitis b e antigen (hbeag) and followed up for a mean . ( . ) year period. genotypes b and c were found in ( . %) and ( . %) of the patients, respectively. hbeag positivity and alt abnormality rates at the start of the observation period were significantly higher in genotype c patients ( . and . %) than in genotype b patients ( . and . %). the annual rate of spontaneous hbeag disappearance in genotype b patients was much higher than in genotype c patients ( . versus . %, respectively). patients with genotype c who were continuously hbeag negative from entry had significantly higher alt abnormality ( . %) than those with genotype b ( . %). interestingly, patients with genotype c who became hbeag negative by interferon treatment had high alt abnormality ( . %). all patients with alt abnormality were serum hbv dna positive. these findings indicate that hbv genotype c patients are more severe liver deterioration because of the delay of hbeag disappearance and continued hbv replication after hbeag disappearance. nossik nn a , nebolsin ve b , zheltukhina ga c , yevstigneeva rp c . a the d.i. ivanovsky institute of virology, viral reproduction, moscow, russian federation , b pparminterprisis co., chemistry, mocow, russian federation , c moscow state academy of fine chemical technology, piptide chemistry, moscow, russian federation objective: to study the effects of 'gamma'-l-glutamylhistamine (glu-ha) derivates on non-specific immunity ('alfa'-ifn, 'gamma'-ifn and nk cell activity) and antiviral activity on the experimental influenza and herpes virus infections in mice. the glu-ya and its derivate glu-ii were synthesized by peptide chemistry techniques. the glu-ha and glu-ii was administered i.p. . and . mg/kg before and after influenza virus (type a/aichi) and showed a protective effect even at the high infective dose ( ld ) */the rate of protection / Á/ / % in the positive/control group. they were not very effective in the protection of herpes simplex virus encephalitis in mice. the model of the physico-emotional stress in mice was used to investigate the ifn system and nk cell activity. the production of ifns and nk cell activity of splenocytes decreased in h after the stress and back to normal level in Á/ days. it was shown that glu-ha and glu-ii can protect or substantially prevent the decrease in nk cell activity and ifns synthesis in post-stress period (so normally did not induce the ifns' synthesis). conclusions: the glu-ha and glu-ii showed antiviral effect against influenza virus infection in mice. the immunomodulating activity and ability to normalize the ifn synthesis and nk cell activity depressed the post-stress period and probably play an essential role in the antiviral activity. no dose adjustment of an anti-influenza prodrug oseltamivir is required in patients with hepatic impairment pm oo c a , snell pr b , liu b a , martin d b , simkins t b , small i b , ward p b . a hoffmann-la roche inc., global development, nutley, usa , b roche products ltd., global development, welwyn garden city, uk background: oseltamivir (ose; ro - , tamiflu † ) is an oral ethyl ester prodrug of its active metabolite oseltamivir carboxylate (oc: ro - ), a potent and selective neuraminidase inhibitor of the influenza virus. the purpose of the study is to evaluate the need for ose dosage adjustment in hepatic impaired patients (hi). method: healthy volunteers (hv) versus hi (child-pugh score Á/ ) [matched on the basis of age ( / years), gender and weight ( / %)] were compared. each subject received mg ose. results: based on c max (ng/ml) and auc inf (ng h/ml) analysed using nominal times, ls mean ratios and % ci between hi and hv were similar. ose* values in hi were marginally elevated but not sufficiently to require dose adjustment. the aim of this study is to investigate the influence of molecular structure of macrocyclic pyridinophanes and their analogs on antiinfluenza and antiherpetic activity of these compounds. we used d-qsar approaches on the basis of simple representation of molecular structure. such representation for biologically active substances allows the description of the spatial structure of compounds with the complete stereochemical information. it determines spatial structures either promoting or interfering of the concrete biological activity. it is easy to realize the molecular design of compounds with the given level of activity with the help of the combinations of simplexes. statistic characteristics for qsar of partial least-squares models are satisfactory (r / . Á/ . ; cvr / . Á/ . ). the molecular fragments that increase the antiviral activity were defined and will be demonstrated. this information was used for design and directed synthesis of several novel antiviral agents with predicted high anti-influenza or antiherpetic activities. predicted activities were confirmed experimentally. d-qsar approaches are useful for development of antiviral compounds. this work was partially supported by intas foundation (grant intas - ). lozitsky vp. ukrainian mechnikov research anti-plague institute, chemotherapy, odessa, ukraine the purpose of this study was to research the anti-influenza activity of proteolytic inhibitor e-aca. it prevents the enhancement of proteolysis during the interaction of virions with cell membranes and decreases penetration of virions into cells. e-aca brings down proteolytic cleavage of ha-precursor to ha- and ha-polypeptides and reduces the infectious virus harvest. it shows the prophylactic and therapeutic action during the experimental influenza reducing the enhancement of alkaline proteases activity in lungs after infection. e-aca promotes the intensification of specific antibodies production and cell immunity, prevents vessels' permeability and hemorrhagic phenomena, decreases the destruction of bronchi's epithelium. it reduces the duration of intoxication, catarrhal instances and hyperthermia in sick children. e-aca improves the indexes of immunity, non-specific resistance and decreases the rate of bacterial complications. application of e-aca for treatment influenza and other arvi in children is recommended in ukraine on the base of results of our researches. the higher effects demonstrated as a result of combine usage of e-aca with specific ig, or deitiforin, or unithyol, or ribavirin. in our opinion, the study of effectiveness of e-aca combine application with inhibitors of influenza na is the perspective direction of anti-influenza researches development. we should we treat immediately all varicella patients with acyclovir if the patient is older than years pm during past years in institute for infectious and tropical diseases in belgrade, immunocompetent varicella patients were treated and cured. among them were older than years ( . %). x-rays were performed in all patients. diagnosis of pneumonia was made in patients ( . %), but in ( . %) patients older than years. varicella is a benign, self-limited disease, if it strikes early, i.e. preschool, school children and teenagers. at that time there is no need for specific therapy. but in neonates, immunocompetent adults and in all immunocompromised patients it can be difficult and life-threatening disease. in immunocompetent adult population pneumonia is a very serious, sometimes fatal complication. knowing the pathophysiology of primary varicella Á/zoster infection, specific therapy with acyclovir should be started immediately after making the diagnosis in patients older than years, without waiting for x-ray proof of pneumonia. brivudin compared to acyclovir for the treatment of herpes zoster: effects on acute disease and posttherapeutic pain pm objective: comparison of efficacy and safety of brivudin )/ mg and acyclovir )/ mg, both for days, in the treatment of herpes zoster. methods: randomised, double-blind study on immunocompetent patients ]/ years (brivudin: n / , acyclovir: n / ). a subgroup of patients ]/ years (brivudin: n / , acyclovir: n / ) was examined for the occurrence of posttherapeutic pain in a poststudy survey. posttherapeutic pain was defined as any zoster-associated pain, regardless of intensity, after the end of acute zoster. results: brivudin was superior to acyclovir in reducing time to last occurrence of new vesicles (rr(itt): . [ . Á/ . ], p / . ). the advantage of brivudin was more pronounced in patients ]/ years (rr(itt): . , [ . Á/ . ], p / . ). incidence of posttherapeutic pain was significantly lower with brivudin ( . %) than with acyclovir ( . %, p / . ). duration of pain was comparable in both treatment groups (rr: . , [ . Á/ . ], p / . ). potentially treatment-related adverse events occurred in . % of the brivudin recipients and in % of the acyclovir recipients. conclusions: brivudin mg once daily for days is superior to standard acyclovir in stopping viral replication in acute herpes zoster. in patients ]/ years, brivudin is more effective than acyclovir in reducing the risk of developing posttherapeutic pain. brivudin is as well tolerated as acyclovir. varadinova t a , genova p a , garcia-raso a b , terron a b , fiol j b , badenas f b . a laboratory of virology, sofia university, sofia, bulgaria , b chemistry, universitat de les balears, palma de mallorca, spain we have published that cu(ii) complexes of acyclovir (acv) are active against hsv infection (mbd, ) . here we present data on the activity of acv complexes of ni(ii), cd(ii), co(ii) and ag(i) against resistant to acv hsv strain r- in comparison with the effect against acv sensitive strain victoria. selectivity indexes (si) compared to that of acv were indicative for activity. the following data were obtained: (i) was times less selective inhibitor of strain r- than of strain victoria; (ii) under the action of [cd(acv)cl ], [ni(acv) (h o) ]cl ×/ acv and [ni(acv)(no )] ×/ h o was up to % higher than that in the control; (ii) was times less selective inhibitor than acv; (iv) si of was two times higher for strain r- and five times lower for strain victoria then that of acv. these data show that the selectivity of acv against resistant hsv strains can increase when acv is bond to a proper metal ion. methods: an antiviral activity against herpes simplex virus of the type i (hsv-i/leningrad/ / ) and variant hsv- (vvt/ / r) resistant to acyclovir was determined using commonly accepted method. viruses were grown on a continuous culture. maximal toxic dose was determined by the administration of compounds orally ( mg/kg) or intraabdominally ( mg/kg) to white mice that had mass Á/ g. condition of the animals was controlled during h. mice pneumonia model was used for the testing activity in vivo. results: derivatives tested have activity against hsv- and hsv- resistant to acyclovir. maximum protection of the cells up to % was reached at concentration of compounds Á/ mg/kg. tested compounds have low toxicity and animals did not die after intraabdominal and after per oral administration of the substances. using these compounds led to essential relief of diseases in animals. the number and square of virus specific areas of inflammation in lung was decreased to compare with control untreated group. tested compounds protected animals similar to acyclovir that was used as control. conclusion: derivatives of carboalkoxysulfanilic acids are active against hsv in vitro and in vivo and act on the acyclovir resistant variant viruses. markiewicz r a , szepietowski jc b . a jelfa s.a., medical department, jelenia gora, poland , b department of dermatology, university of medicine, wroclaw, poland background: denotivir is a -benzoamino- ?-chloro- -methyl- isothiazolecarboxanilide anti-inflammatory agent with antiviral and immunomodulatory activities. it possesses also mild antibacterial and antifungal action. the purpose of the study: the aim of this presentation is to give an overview of recent studies demonstrating denotivir efficacy in herpetic infections. the results obtained: in vitro studies revealed that denotivir in the doses below its cytotoxicity (about um) significantly inhibited (by Á/ %). herpes simplex virus (hsv)- and hsv- replication in fibroblast and kidney cell cultures. moreover, it was showed that denotivir in the dose of mg/ml markedly inactivated hsv- after min incubation in c. in giunea pigs research, % denotivir in % dmso appeared to be superior to % dmso alone and untreated groups in the therapy of animal skin infected by hsv- . there was no huge difference in eythema and oedema scorings between studied groups, however in the group treated with denotivir, in contrast to others, no vesicles developed. several clinical studies showed usefulness of denotivir in controlling herpetic infections in dermatology, ophthalmology and otolaryngology. in the majority of studies within few hours after the drug application itch and pain relief was noted and within Á/ days the vesicular lesions were dried up. the conclusion reached: in conclusion, denotivir is an effective antiherpetic agent. this study was conducted on cows affected with teat papillomatosis. in the first step, each cow was located on one of three groups. the first group contained four cows from to years that were treated with fig tree (ficus carica) latex. the second group contained four cows from . to years that were treated with a % solution of salicylic acid, and the third group contained four cows as control. in group one and two following treatment with fig tree latex and salicylic acid, superficial necrosis begun from day and all of the warts disappeared by day . in the control group, after day , there were no changes in number of lesions, but some of them were larger than first observation. on day , one of the marked warts disappeared and on day another wart was disappeared but six were present until day . comparison of effects of salicylic acid and fig latex showed similar effects in treatment of udder papillomatosis in cow. laboratory markers of skeletal muscle toxicity in hiv-infected patients: a cross-sectional case-control survey of frequency, potential correlation with antiretroviral therapy, clinical significance, and outcome pm manfredi r, motta r, patrono d, calza l, chiodo f, boni p. to assess skeletal muscle toxicity among Â/ hiv-infected outpatients (p), the p who had ]/ altered cpk assay ( !/ u/l) between may and november , were compared with p randomly selected among those who had ]/ laboratory exams in this -month interval, in a : case-control study. among the p with altered cpk levels only six were females, and received antiretrovirals. the overall frequency of altered cpk among all p who underwent ]/ laboratory workouts in months was . %. cpk alteration was transient in p, with values ranging from to (mean . / . ) u/l, but was recognized ]/ times in Á/ months in p ( %), of them showing concomitant high aldolase levels ( . Á/ . u/l). a myopathy or a rhabdomyolisis were recognized in four p only; a myositis was confirmed in one p by histopathology. in a multivariate logistic regression analysis, when excluding the unexpected prevalence of the male gender (p b/ . ), no significant difference emerged between p and controls as to age, risk for hiv infection, iv drug use, duration of hiv infection, prior anti-hiv therapy and its length, selected drug combinations, administered nucleoside analogues,hiv disease stage, mean cd '/ count and hiv viremia, signs and duration of lipodystrophy, increased glucose, triglyceride and cholesterol levels, and other therapies. muscle abnormalities, though frequently asymptomatic, are underestimated hiv disease complications, and the role of metabolic (i.e. mitochondrial) alterations, deserves investigation. poor efficacy of non-nucleoside reverse transcriptase inhibitor (nnrti)based salvage haart in hiv-infected patients heavily pre-treated with all other classes of antiretroviral compounds pm manfredi r, calza l, chiodo f. infectious diseases, university of bologna, bologna, italy poorly comparable literature series show conflicting results of nnrti-based rescue haart: Á/ % rate of virologic success. to assess the response to a Á/ -drug rescue haart including a nnrti, patients (p) treated with nucleoside analogues (na) and protease inhibitors (pi) for !/ and !/ months, respectively but naïve to nnrti, with a viremia !/ copies/ml, were prospectively followed during Á/ years, provided that they ensured a !/ % adherence. efavirenz was used in p, nevirapine in , and delavirdine in two. most p ( . %) had an early laboratory improvement, but mean peak viral load decrease was . log, and a significant reduction vs baseline (p b/ . ) lasted months only. a mean % increase of zenith cd count was obtained (p b/ . ), but only . % of p remained !/ cells/ml year after switch to nnrti. in a multivariate analysis, the concurrent introduction of novel pi(s) ( p) and/or different na(s) ( p) acted favorably until the th month of follow-up (p b/ . ), while genotypic mutations conferring nnrti cross-resistance, usually associated with a broad resistance profile, predicted failure in all p (p b/ . ), and the response did not vary according to duration and type of prior therapy, and selected nnrti. a deep salvage nnrtibased haart has a poor and transient virologic outcome also in nnrti-naïve p, while a more evident and sustained immunologic response is expected. p who can introduce novel pi/na and have no mutations impairing nnrti activity are entitled to a better outcome. fatal lactic acidosis without elevation of liver-enzymes during the treatment with stavudine, didanosine and efavirenz: a case report pm winzer r, langmann p, väth t, zilly m, klinker h. medizinische poliklinik der universität würzburg, schwerpunkt hepatologie/infektiologie, ürzburg, germany nucleoside reverse transcriptase inhibitors (nrtis) cause various side effects, many of which are thought to be due to their effects on mitochondria. a -year-old hiv positive (hiv rna: copies/ml, cd cell count: /ml), obese (body-mass-index: . ), therapy-naïve female patient, who after months of well tolerated and effective antiretroviral therapy (stavudine, didanosine, efavirenz), had slight gastrointestinal discomfort and suddenly developed a lactic acidosis (arterial-ph . [ . Á/ . she died days later despite intensive care (continuous venovenous haemodiafiltration, sodium-bicarbonate infusion, high doses of vitamins, respiration). the pathologic examination showed an enlarged liver ( g) with yellowish appearance and pasty consistency, which microscopically appeared as a massive macro-and microvesicular fatty degeneration, and only slight signs of terminal pancreatitis. this reported case gives evidence that a massive lactate acidosis may develop without previously disarranged laboratory parameters for liver or pancreatic function. a fatal outcome may evolve without further accompanying-illnesses. efficacy and tolerability of atorvastatin in the treatment of hypercholesterolemia in hiv-infected patients receiving haart pm calza l, manfredi r, chiodo f. division of infectious diseases, university of bologna, bologna, italy introduction: significant increases in plasma triglyceride and cholesterol levels have been reported in patients treated with haart, and prolonged metabolic imbalances could significantly act on the longterm prognosis and outcome of hiv-infected persons. patients and methods: fourteen hiv-infected patients on pi-based haart since at least months and presenting hypercholesterolemia ( !/ mg/dl) of at least -month duration and unresponsive to a hypolipidemic diet and physical exercise, have been treated with a single daily dose of atorvastatin ( mg) for months. results: one patient was ecluded from evaluation due to early dropout. ongoing antiretroviral treatment included ritonavir in four cases, indinavir in four, nelfinavir in three, and saquinavir hard-gel in two. at the close of -month follow-up of atorvastatin therapy, a decrease of total cholesterol level of . % versus respective baseline value was observed; eight out of patients reached normal values for cholesterol. mild gastroenteric symptoms were found in only one of the treated patients, while no skeletal muscle and liver toxicity has been observed. discussion: in our study, pharmacological treatment with atorvastatin proved certainly effective in the management of diet-resistant hypercholesterolemia, and was associated with a favourable tolerability and adherence profile. the effect of combination antiretroviral therapy regimens on hiv- proviral dna level in peripheral blood mononuclear cells (pbmcs) was examined in hiv- -positive patients, using endpoint dilution pcr and serially cloning and sequencing of the gag region of hiv- . the major clone was defined as the most numerous of analyzed clones, and observation periods ranged from to months (mean, . / . months). in five patients (one with primary-stage hiv- infection) receiving three antiretroviral drugs, hiv- rna levels reduced to undetectable (i.e. b/ copies/ml). hiv- proviral dna levels and the number of major clones reduced in four of these patients. hiv- rna levels reduced, but remained detectable, in five other patients. in the two remaining patients (both receiving two rather than three antiretroviral drugs) hiv- rna levels increased. these results suggested that the population of the major clones may be affected when hiv- rna levels reduce following combination regimens of antiretroviral therapy. saquinavir hard gel (shg) as a part of a spontaneous Á/ -month deintensification anti-hiv regimen following successful highly active antiretroviral therapy (haart) pm manfredi r, calza l, chiodo f. infectious diseases, university of bologna, bologna, italy the induction-maintenance concept was poorly studied in hiv'/ patients (p), and shg was never assessed after prolonged response to potent protease inhibitors (pi)-based haart. shg-naïve p who refused indinavir, ritonavir, or nelfinavir-based haart after achieving long-term viral suppression, and resorted to shg'/ nucleoside analogues (na), were followed prospectively. in . % of the p assessed for Á/ months, ]/ na was changed. prior haart was interrupted after . / . months, due to adverse events ( p), or p's request ( p), while a viremia b/ copies/ml was present since . / . months. a viremia of Á/ hiv-rna copies/ml was maintained in p ( . %), while a higher viral load occurred in p after . / . months, and was related to a pre-haart viremia !/ / ml, a more frequent !/ % recovery of cd count, mutations of codons Á/ , and failure to change na (p b/ . Á/ . ). a cd drop !/ %/ cells/ml was found after . / . months in only eight p, who also had virologic failure: immunologic deterioration was earlier and deeper when na were not changed (p b/ . ). all the p who introduced shg'/novel na after a successful !/ -month induction with a potent pi-based haart had a stable Á/ -month outcome. a suboptimal haart including the less effective but better tolerated shg may be effective for !/ year, especially when novel na are introduced, and specific mutations are absent. despite a lower potency, drugs with a good safety and compliance profile may be recovered for simplified regimens. objective: to evaluate efficacy of antiretroviral therapy (art) with two or three drugs in the nervous 'reservoir'. patients: thirteen acute neurological and art naive aids patients underwent a paired and simultaneous sample from plasma and cerebrospinal fluid (csf) for a quantitative detection of hiv- rna (amplicor roche) before art. all patients underwent a ct and/or mr of the brain to perform a diagnosis. all of them had an hiv related neurological acute inflammatory disease. after diagnosis all patients received art: / received two nrti and / received haart including two nrti and one protease inhibitor. all patients underwent a paired and simultaneous follow-up from plasma and csf during the nd month of treatment. results: in all patients baseline levels of hiv-rna were higher (p b/ . ) in the plasma (log . '/ . ) than in the csf (log . '/ . ). the / patients who received dual therapy had undetectable levels (cut-off copies/ml) of viral rna at the follow-up in csf, but not in plasma: three of these seven patients had a detectable plasma hiv- rna. all / patients with haart had undetectable hiv- rna both in plasma and in csf at the follow up. conclusions: dual nrti therapy is rapidly effective in csf (because of an high penetration of drugs through a more permeable blood Á/brain barrier and lower hiv rna baseline levels) but not in plasma. haart is rapidly and equally effective both in csf and plasma. there are no reports of fulminant and fatal hepatic failure after the start of highly active antiretroviral therapy (haart) in an hiv subject without chronic viral hepatitis. case report: a -year-old naive aids woman with clinical symptoms due by a pcp was observed. baseline alt was increased ( . m.n.v.) because of a mild hepatosteatosis and a silent cholelithiasis. serology for hbv, hdv and hcv was negative; igg anti-hav, anti-ebv, anti-cmv and anti-hsv were present. hiv- rna was . log , cd '/ count was /ml. during the pcp treatment with cotrimoxazole alt values increased ( !/ m.n.v.); nevertheless, she completed the treatment. liver enzymes returned to the pre-treatment values over several days. then she started haart with stavudine, lamivudine and efavirenz. after days the patient showed an efavirenz-related skin rush that resolved within days, without treatment discontinuation. fourteen days after the start of haart jaundice appeared. laboratory revealed severe alt increase ( !/ m.n.v.) and hyperbilirubinemia ( mg/dl) and she died because of an acute liver failure syndrome within few days. an haart efavirenzbased regimen can result highly hepatotoxic when given in presence of a hepatosteatosis, of a recent hepatotoxicity caused by a nonantiretroviral treatment and of a previous idiosyncratic reaction to efavirenz. our experience with bulgarian herbal extracts for improving the general condition of hiv-positive patients pm methods: we used a combination of bulgarian herbal extracts and treated six patients, divided in two groups: three with symptomatic and three with asymptomatic hiv-infection. the all three patients with asymptomatic hiv-infection were treated only with herbal extracts, another three patients with symptomatic hiv-infection were treated with combination of herbal extracts and anti-retroviral therapy. the general status of patients has been evaluated by both subjective and objective surveillance. the immunologic monitoring has been performed by absolute count of cd '/ lymphocytes. results: all patients have shown an obvious improvement in their general condition: high spirit and working capacity, good appetite and sleep, a restoration of body weight. the number of cd '/ lymphocytes has been lightly increased or constant. conclusion: the combination of bulgarian herbal extracts has shown significant positive effect on the general condition and improve the quality of life. antifungal activity of in vitro and in vivo combinations of voriconazole with -fluorocytosine and amphotericin b against candida and cryptococcus spp pm hitchcock ca, andrews rj, lewis bgh, pye gw, oliver gp, troke pf. pfizer global research and development, department of discovery biology, sandwich, uk purpose: the present study was designed to determine whether the activity of voriconazole (vor), a novel triazole, was reduced against candidal and cryptococcal infections by the addition of standard antifungal agents, amphotericin b (amb) and -fluorocytosine ( -fc). vor was tested in combination with standard antifungal agents both in vitro, using a checkerboard mic determination test, and in vivo in immune normal guinea pig models of fungal infections. the results indicate that the efficacy of vor against candida albicans and c. neoformans was not antagonised by amb or -fc in vitro. furthermore, in guinea pig models of systemic candidiasis and intracranial cryptococcosis, no antagonism was observed between the lower doses of vor and either amb or -fc on the basis of reductions in tissue fungal loads. at the highest doses of vor, both amb and -fc showed some antagonism, but the combinations were still effective in significantly reducing fungal tissue loads compared with vehicle-treated control animals. conclusion: these results suggest that vor may be used in combination with standard antifungal agents, and future studies to elucidate the clinical potential of vor combination therapies in the management of candida and cryptococcus infections are warranted. itraconazole in the treatment of pityriasis versicolor pm tiodorovic j, jovanovic d, binic i, nikolic lj. faculty of medicine, clinic of dermatovenerology, nis, serbia, yugoslavia a comparison of two short-term dose schedules with itraconazole was carried out in patients with pityriasis versicolor. the patients were divided in two groups. each group consisted of patients who completed the therapy and controls. the clinical diagnosis was confirmed mycologically, by direct microscopic examination. the first group received mg of itraconazole daily for days. the second group received mg daily for days. the patients were controlled clinically and mycologically and days after the initiation of treatment. erythema, scaling and pruritus was evaluated clinically. clinically and mycologically cured patients accepted as cured. the cure rate were % in the first group and % in the second group at day . the effects of these two groups are similar. none of the patients reported side-effects. fungal urinary infections: emerging species, antifungal susceptibility trends and antibody response pm badawi he a , kamel ai b , fam ns a , el-sayed me a , elian sae c . a theodor bilharz medical research institute (tbmri ), microbiology, giza, egypt , b theodor bilharz medical research institute (tbmri ), urosurgery, giza, egypt , c faculty of medicine, medical microbiology and immunology, cairo university, cairo, egypt objectives: to assess the role of candida species in patients with urinary tract infections (utis) with or without schistosomiasis and/or cancer bladder, to compare chromogenic; chromagar (cma), biggy agar; morphologic (corn meal, rice agar-tween ) media and biochemical candifast test for identification of candida species. susceptibility to antifungal agents using e -test and candifast and the performance of elisa test for detection of anticandida antibodies (igm and igg) in serum were evaluated. results: c. albicans was the most frequent ( . %) species responsible for fungal utis. however, non-albicans species, c. glabrata ( . %), c. tropicalis ( %) and c. krusei ( . %) were also isolated. rice agar-tween was found to be cheap, available and sufficient to make a final identification ( %). cma could not identify c. glabrata . biggy agar could not adequately differentiate candida species. candifast biochemical identification showed low sensitivity of . %. e -test on sabouraud dextrose agar (sda) is simple method for mics determination and could detect s-dd strains in case of azoles. conclusion: the emergence of non-albicans species such as c. glabrata , c. tropicalis and c. krusei have contributed to complicated utis. this necessitates accurate isolation and identification of candida to the species level. morphology on rice agar-tween and antifungal susceptibility using e -test on sda is a simle rapid scheme for routine identification of clinically important yeasts. purpose: classification of allergic fungal rhinosinusitis (afr) is based on the immunologic relationship of the host to the fungus. afr must be differentiated from other fungal rhinosinusitis infections, which include acute invasive, chronic invasive, fungal balls and saprophytic colonization. although many cases of fungal rhinosinusitis is caused by species of aspergillis , dermatiaceous moulds have become an emerging pathogen in immunocompetent individuals. results: our case study involved a year male suffering from facial pain, headache, postnasal drip and loss of smell. he was hiv negative and a nonsmoker. the following laboratory tests were performed: ige- . ( . Á/ . ) iu/ml iga- . ( . Á/ . ) g/l igm- . ( . Á/ . ) g/l allergen specific ige for alternaria */ . ( . Á/ . ) ku/l fbc-normal except eosinophils slightly raised . ( . Á/ . ))/ / l. ct scans indicated fungus proliferation, bone erosion and extension of disease into adjacent anatomic area. sinus tissue following debridement was sent for microscopy and culture. hyphae was microscopically observed and cultures yielded two dermatiaceous fungi, bipolaris spp and alternaria spp . conclusion: it is important to differentiate these two species from curvularia , helminthosporum , drechelria and exserohilum . knowledge of these dermatiaceous fungi is important in directing appropriate antifungal therapy and selecting the correct antigens for postsurgical immunotherapy after initial debridement and irrigation. antifungal activity of in vitro and in vivo combinations of voriconazole with -fluorocytosine and amphotericin b against aspergillus fumigatus pm hitchcock ca, andrews rj, lewis bgh, pye gw, oliver gp, troke pf. pfizer global research and development, department of discovery biology, sandwich, uk purpose: a key requisite for a new antifungal drug is to demonstrate that it is devoid of significant antagonism in combination with other agents. combinations of the new triazole, voriconazole (vor), and standard antifungal agents ( -fluorocytosine or amphotericin b; -fc or amb) were tested against aspergillus fumigatus in vitro and in guinea pig models of infections to confirm that antifungal activity was not antagonised by using combination therapies. vor was studied in combination with amb or -fc in vitro, using a checkerboard mic determination test, and in vivo, using immune normal and immunocompromised guinea pig models of systemic aspergillosis. results: the results indicate that the potency of vor was not antagonised by amb or -fc in vivo; indeed, at lower concentrations of vor, significant improvements in reducing fungal burden in both in vivo models were achieved by the addition of amb. in vitro, no antagonism was found between vor and amb, although -fc had a significant antagonistic effect on vor activity. conclusion: these results from in vitro and in vivo models of aspergillosis suggest that vor may be used in combination with standard antifungal agents and, therefore, justify further examinations of vor combination therapies in a clinical setting. in vitro activity of caspofungin compared to that of amphotericin b, fluconazole, and itraconazole against candida species pm arikan s, sancak b, hascelik g. department of microbiology and clinical microbiology, hacettepe university medical school, ankara, turkey purpose: to evaluate the in vitro activity of caspofungin against various candida spp. and particularly against isolates with decreased amphotericin b, fluconazole, and itraconazole susceptibilities. methods: susceptibility tests were done by nccls m a microdilution guidelines for clinical candida strains. the mics (mg/ml) were read at and h. results: caspofungin mics at h are shown in the table. mics at h were similar to h readings. expectedly, no evidence of crossresistance was detected between caspofungin and other drugs tested. caspofungin was similarly active against fluconazole-or itraconazolesusceptible and resistant isolates. conclusions: ( ) caspofungin is active in vitro against all candida spp. tested. ( ) caspofungin mics are slightly higher for c. parapsilosis compared to other species. ( ) its activity against fluconazole-and itraconazole-resistant isolates is noteworthy. ( ) validation of these data require clinical investigations. oropharyngeal microbiological samples of bmt patients were evaluated. weekly cultures (days (/ , and '/ ) revealed presence of fungi in patients ( . %): in four ( %) patients before bmt only, in ( %) after bmt only, and in ( . %) both before and after bmt. in one patient candida norvegensis was isolated from the throat, buccal and palatal surfaces. three c. albicans , two c. krusei , and three c. norvegensis from four patients were chosen to comper their antifungal sensitivities and extracellular virulence factors. using fungitest method we determined the sensitivities of these isolates to flucytosine, amphotericin b, miconazole, ketoconazole and fluconazole. the fluconazole sensitivities were also determined by the e -test. on the basis of the fungitest data the three c. norvegensis isolates were sensitive to flucytosine, amphotericin b, and also to ketoconazole. in case of fluconazole and miconazole they proved to be susceptible dependent upon dose. the mic fluconazole values determined by the e -test for the three c. norvegensis isolates were , ]/ and ]/ mg/ml, respectively. the oropharyngeal isolates of c. norvegensis produced high amounts of extracellular aspartic protease and phospholipase similarly to c. albicans strains. these enzymes may contribute to the pathogenesis of this new emerging candida species. in vitro activities of antifungal and antiseptic agents against rhodotorula sp pm preney l, théraud m, guiguen c, gangneux jp. laboratory parasitologie-mycologie, chu de rennes, france purpose of the study: rhodotorula species are common saprophyte yeasts widespread in nature. since the last years, they have been implicated in several severe infections, especially in immunocompromised patients, and various antifungal therapies were used. however, only limited data are available on the susceptibility of rhodotorula sp. to antifungal and antiseptic agents. material and methods: in this work, we evaluated the in vitro activities of eight antifungal agents against strains of rhodotorula ( strains of r. rubra and nine strains of r. glutinis using atb fungus system (biomerieux) and etest strips (ab-biodisk). beside, the effect of eight antiseptic agents was assessed on a suspension of r. rubra . the quantification of yeasts after exposure to antiseptic agents was performed by subculturings using a microtitration method in well plates. results and discussion: all strains tested were susceptible to amphotericin b, fc, and nystatin. twenty-nine out of strains were susceptible to ketoconazole, out of were intermediate to econazole. all strains were resistant to fluconazole (cmi!/ mg/ml) and itraconazole (cmi!/ mg/ml), and out of were resistant to miconazole, suggesting that antifungal therapy must be adapted when rhodotorula yeasts are implicated in invasive infection. beside, min exposure to sodium hypochlorite , chlorhexidine . % or ecodiol (isopropyl alcohol'/alkylamin) showed fungicidal activities. susceptibility testing of aspergillus fumigatus and emerging aspergillus pathogens by a modification of the nccls m -p method pm logotheti m a , kapsanaki-gotsi e b , velegraki a a , zagoura d b . a department of microbiology, mycology reference laboratory, medical school, university of athens, athens, greece , b biology department, section ecology and systematics, university of athens, athens, greece aspergillosis in high risk groups of patients is still associated with high mortality rate ( Á/ %). aspergillus fumigatus is the primary pathogen, while other opportunistic aspergillus species are emerging. amphotericin b (ab), itraconazole (it), voriconazole (vo) and terbinafine (te) minimum inhibitory concentrations (mic) were determined by modifying the nccls m -p microdilution method. stock drug solutions were prepared in rpmi , dimethyl sulfoxide (dmso), and polyethylene glycol (peg ). inocula, of the a. fumigatus group ( ), a. flavus group ( ) ( ) and the m -p quality control strains were prepared according to, and by modifying, the nccls guidelines. plates were incubated at and c and read at and h. peg effectively dissolved it and vo, while either dmso or peg dissolved te. low and c Á/ h ab, it, vo and te mics ( . Á/ . mg/l) were recorded. a. terreus ( ) and a. parasiticus ( ) were resistant to ab. certain clinical isolates demonstrate clinical and in vitro resistance. standardization of susceptibility testing would offer reliable assistance in selecting and monitoring antifungal therapy. otag f, aslan, g, ozturk c. microbiology department, faculty of medicine, mersin university, mersin, turkey rates of opportunistic fungal infections have risen markedly. because some of these species have potential resistance to antifungal agents, rapid presumptive species level identification is crucial in allowing for directed antifungal therapy. in this study, isolated yeasts from the clinical specimens were identified by atb id c (biomerieux, france). the number of identified yeasts were, respectively; ( %) candida albicans , six ( %) c. glabrata , five ( . %) c. tropicalis , three ( . %) c. parapsilosis , two ( . %) c. krusei , two ( . %) c. kefyr , one ( . %) c. guillermondii , one ( . %) c. dubliniensis . twenty-one of strains were investigated for antifungal sensitivities by atb fungus kit (biomerieux, france). the results are as follows: % sensitivity was detected to myconasol, % to flusitozin, nystatin and econasol, % to amphtericin b and ketokonazol. it is important to achieve empirik treatment of the opportunistic candida infections and the following of resistance to antifungals. shakhmatov da, strelchenco, ov. novosibirsk state medical academy, dermatovenerology, novosibirsk, russian federation at the present stage in russia with a background of a high case rate of syphilis, it becomes necessary to exclude biological false positive serological tests. because the serodiagnosis of syphilis has significant limitations, the direct detection of t. pallidum in suspect blood may serve as an alternate diagnostic strategy. polymerase chain reaction (pcr) has been the most widely used amplification method. the study of patients receiving examination related and treatment for syphilis in std clinic and persons directed from other hospitals where routine serologic examination revealed doubtful results. pcr reaction was carried out with nested primer pairs based on the dna sequence of the Á/ and Á/ kda gene of t. pallidum . pcr was utilized with whole blood. a complex of serological tests: fta-abs and tit was used as the &rdqup; gold standard''. as a result the sensitivity of pcr was . % and specificity was . %. selective comparison of pcr results with vdrl, the fta-abs and treponemal immobilisation test (tit) has shown concurrence . %. in conclusion, the preliminary results of pcr in whole blood in syphilis detection revealed its high sensitivity and specificity; possibility to obtain rapid results in unclear cases. chlamydia pneumoniae (cp) is an atypical pathogen whit intracellular location, whose eradication is very difficult. in the past years it has been objects of many studies that lead to the demonstration of a relationship between its presence and the development of widespread multifactorial pathologies such as atherosclerosis and asthma. the lack of its eradication can become an important clinical and social problem. the study objective is the comprehension of pathogen Á/host interaction mechanism, to characterize therapeutics protocols that cold lead to complete eradication of cp from organism. the research had been principally made on the studying the molecular mechanisms that are at the root of pathogen permanence inside host cell. using proliferation and apoptosis tests we underlined a different behaviour of infected cells towards control cells. in presence of p ( mg/ml), i.e. a peptide that can inhibit the proliferation and induce apoptosis in vitro inhibiting nf-kb, uninfected cells proliferation decreased of % in comparison whit the controls, while the one of infected decreased only of about %. moreover, using various apoptosis-inducers, the infected cells showing apoptosis were about % while the uninfected were about %. the caspace iii activity increased significantly in uninfected cells. in conclusion, cp could delay its elimination from the host inhibiting the apoptosis via nf-kb activation. hryniewiecki t a , gzyl a b , rawczynska-englert i a , a department of acquired valvular heart disease, national institute of cardiology, warsaw, poland , b department of sera and vaccines, national institute of hygiene, warsaw, poland infective endocarditis (ie) frequently causes problems in diagnosis, especially where blood cultures are negative and with fungal etiology (also as a fungal superinfection in bacterial ie). the purpose of the study: the purpose of the study was to evaluate the usefulness of broad-range fungal pcr in diagnosis of fungal superinfection of bacterial ie. twenty-five blood samples were taken for analysis from patients with infective endocarditis. ie was diagnosed according to duke criteria including positive blood cultures. suspicion of fungal superinfection was established on serological investigation in five patients, confirmed by blood culture in two patients. control group consisted of patients without infection. dna was isolated using the commercially available s.n.a.p. kit. amplification products were analyzed by gel electrophoresis stained with ethidium bromide. the results obtained: fungal dna was found in two patients with fungal superinfection of bacterial ie confirmed by culture. in the remaining patients with ie and controls no fungal dna was found. the conclusion reached: broad-range fungal pcr is a fast and inexpensive tool for the detection of fungal dna, but it is more prone to contamination than species-specific pcr. the method may be valuable in the identification of fungal superinfection of bacterial ie or diagnosis of fungal ie. rivanera d, lilli d, lozzi ma, piunno m, mancini c. microbiology, science and public health, rome, italy aim: the aim of this study was to evaluate the eia method for detection of antibody to ttv virus (ttv) and to investigate the anti-tt virus prevalence in patients with hepatitis b (hbv) virus, hepatitis c (hcv) virus, in group of 'high risk'subjects to hepatitis and in healthy subjects. the elisa methods (nuclear laser vienna lab) using ttv s and ns antigens: orf ( aa) and orf ( aa) was applied to detect anti-ttv; the serological screening was performed from samples to italian subjects. results: the positive rates of anti-ttv antibodies were . % in patients with hepatitis b Á/c and . % in 'high risk' hepatitis patients. the anti-ttv was also found in . % in healthy people. conclusions: the anti-ttv were detected in all groups studied, however, its positive rate was similar in patients with hepatitis b Á/c and in 'high risk' hepatitis respect to heathly people. our results shown that tt virus is frequent in italy both in patients infected by others transmitted viruses and in general population. the positivity found in healthy adults included in our studies suggests that the virus might be transmitted non-parenterally. the study of pattern of antibody to ttv may be an infectious marker of ttv similar to that of anti-hcv. a stress test on a miniaturized identification system designed for neisseria and haemophilus pm rich m a , bannatyne rm a , memish za b . a king fahad national guard hospital, division of microbiology, riyadh, saudi arabia , b king fahad national guard hospital, infection prevention and control, riyadh, saudi arabia we report an incident that occurred in our laboratory when the bbl crystal identification system for neisseria and haemophilus was used to identify a haemophilus-like-organism. the numerical profile generated was not in the system database. conventional biochemical tests subsequently revealed an identification of brucella melitensis , a common isolate in our area. as a result of this revelation we subjected this system to a mini 'stress-test' with a collection of isolates of b. melitensis . two numerical profiles were obtained, and , neither of which are listed in the system database. brucella species have been misidentified as moraxella species, moraxella phenylpyruvica , and as haemophilus influenzae biotype iv in various identification systems. two cases of laboratory-acquired brucellosis have been attributed to misidentification. to its credit the bbl crystal identification system for neisseria and haemophilus neither generates a profile number with a misidentified organism nor assigns a confidence level. instead it properly directs the user to resort to conventional methods to secure an identification. if further studies on additional brucella isolates and strains from different geographical sources confirm the unique biochemical profiles identified here, it may be worthwhile to incorporate these into the database where they would be of considerable assistance in areas where brucellosis is widespread. cloning and characterization of aflmp in aspergillus flavus pm chong tk, woo pcy, leung asp, yuen ky. the university of hong kong, microbiology, hong kong, hong kong purpose of the study: to clone and characterize an antigenic protein for serodiagnosis of infection caused by aspergillus flavus which is the commonest aspergillus species causing aspergilloma (ao) and invasive aspergillosis (ia) in asia. result obtained: we cloned the aflmp gene, which encodes the first antigenic cell wall protein in a. flavus . aflmp codes for a protein, aflmp p, of amino acid residues, with sequence features that are present in mp p and afmp p, the antigenic cell wall mannoprotein in penicillium marneffei and aspergillus fumigatus that we described previously. it contains a serine-and threonine-rich region for o glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. specific anti-aflmp p antibody was generated with recombinant aflmp p protein purified from escherichia coli to allow further characterization of aflmp p. indirect immunofluorescent staining indicated that aflmp p is present in the cell walls of the hyphae and conidia of a. flavus . furthermore, it was observed that patients with ao and ia due to a. flavus develop a specific antibody response against aflmp p. conclusion reached: this suggested that the recombinant protein and its antibody may be useful for serodiagnosis in patients with ao or ia, and the protein may represent a good cell surface target for host humoral immunitiy. grape purpose: to investigate the basis for increasing resistance to trimethoprim and sulphamethoxazole. methods: pcr screening for integrons of clinical urinary tract isolates was performed. isolates were tested for resistance to antibiotics. integrons in isolates were sequenced. results: integrons of class were found in isolates and class integrons were found in . eight isolates in the study were resistant to five antibiotics or more and not shown to carry any integron. nineteen of isolates resistant to trimethoprim did not carry integrons. only one of these isolates was shown to carry sul and is thus probably also carrying an integron. none of the isolates were shown to carry dfr , one of five trimethoprim resistance genes known to exist outside integrons. three isolates were resistant to sulphonamides but were not shown to carry neither sul nor sul . only dfr and aad gene cassettes were found in the sequenced integrons. conclusions: resistance to trimethoprim in of trimethoprim resistant isolates is mediated by genes not detectable, as in the case with three sulphonamide resistant isolates. sequenced integrons that contain dfr genes do not carry any gene cassettes mediating resistance to modern antibiotics. unusual diagnosis tool for an unusual presentation of alveolar echinococcosis: report of two cases of local progression after an animal bite pm bardonnet k, bart jm, loiseau j, gérard a, estavoyer jm, heyd b, badet jm, dubiez a, piarroux r, bresson-hadni s. who collaborating centre for prevention and treatment of human echinococcosis, university of franche-comté, besançon, france introduction: the classical human contamination route for alveolar echinococcosis (ae) is ingestion of eggs. two exceptional human cases are reported with extensive local evolution of ae after a bite. case no. : between and , a patient underwent surgery seven times for a muscle growing tumour which developed after a bite. the diagnosis of muscle ae was assessed on histopathological examination. in , serological tests were in accordance with echinococcus sp infection. case no. : in , a man presented 'cat-scratch fever' with a right supraclavicular tumefaction following a cat bite. between and , five recurrences occurred. different surgical explorations indicated multiple abscesses of the cervical muscles. in , serological tests were in favour of echinococcus sp infection and the pathologist described a parasitic wall suggesting hydatidosis, but specific pcr from histological samples prompted the diagnosis of ae. conclusion: in these exceptional observations, the liver which is the most usual location of ae was lesion-free. the chronic inflammatory ae lesions have developed in the local lymphatic chain area of the bite site. to perform diagnosis in these very unusual forms of ae, it is necessary to add unusual tests such as specific pcr to classical tests. antibiotic resistance in foodborne salmonella is an emerging public health concern. integrons are now recognized as the main genetic vehicles of antibiotic resistance in gram-negative bacteria, including in salmonella . the purpose of the present study was to investigate the presence of class i integrons in resistant isolates of several serotypes of salmonella isolated from poultry products and to determine their association with multidrug-resistance phenotypes. a total of isolates of salmonella belonging to seven different serotypes were tested. the most frequent multiresistant phenotype, found alone or together with other resistances, was to streptomycin and tetracycline. all but seven were resistant to three or more antimicrobial agents, including quinolones and amoxicillin. pcr analysis with the ?cs and ?cs primers detected the presence of class i integrons of . kb in one isolate, with the multiresistant phenotype: amoxicillin, chloramphenicol, streptomycin, trimethoprim-sulphametoxazol and tetracycline. our findings suggest that the uncontrolled use of the antimicrobial agents in food animals may have contributed to the development of the pattern of resistance observed in salmonella isolates. also the presence of integrons in low prevalent human salmonella serotypes but associated with food animals underscores the public health problem of antibiotic resistance acquisition and spread. prevalence and antimicrobial resistance of campylobacter jejuni and c. coli isolated from broilers and pigs in france pm avrain l a , humbert f b , sanders p c , kempf i a . a afssa, umb, ploufragan, france , b afssa, hqpap, ploufragan, france , c afssa, lermvd, fougères, france in , caeca from standard, export or free-range broilers and in , fecal samples from pigs, were collected in french slaughterhouses. prevalence of campylobacter jejuni and c. coli strains was . % in standard, . % in export and % in free-range broilers. in standard and export productions, the most often isolated species was c. jejuni , whereas c. coli was predominant in free-range production. . % samples collected from pigs contained c. coli . the sensitivity of strains to ampicillin, nalidixic acid, enrofloxacin (broilers) or ciprofloxacin (pigs), tetracycline, erythromycin and gentamicin was tested by an agar dilution method. in broilers, the percentages of resistant strains were, respectively , , , , . and % for c. jejuni and , , , , and % for c. coli . in pigs the percentages of resistant c. coli were, respectively , , , , and %. in broiler production, significant differences between distributions of species or percentages of resistant strains were observed according to type of production or administrated antimicrobials. the enzyme dhps (dihydropteroate synthase) participates in the folate synthesis pathway, and is well recognized as the target for sulphonamides. the enzyme preceding dhps in this pathway, pppk (dihydropterin pyrophosphokinase), is another interesting candidate drug target. the metabolic role of pppk is to provide one of the substrates for dhps. earlier studies have suggested that pppk and dhps enzymes need to have physical contact with each other for full enzyme activity. studies of potential interactions between the enzymes have been initiated. so far, indication of a weak interaction has been detected in gelfiltration experiments and the two-hybrid system. to confirm these results, we are currently developing a method to study substrate channeling, as interfering with such interactions could lead to impaired growth and thus be used as inhibitory drugs. we have also cloned and sequenced the operons coding for the enzymes in the folate biosynthesis from different clinical isolates of streptococcus pyogenes . comparisons revealed some isolates with a mosaic structure in the operon, suggesting that horizontal transfer of genetic material has occurred. multi-resistance gene cluster on a plasmid in a clinical isolate of e. faecium pm werner g, hildebrandt b, klare i, witte w. department of nosocomial infections, robert koch institute, wernigerode branch, germany purpose: strain uw was isolated from an urine sample of a patient with a permanent catheter. the purpose of our study was to identify and localize the resistance determinants in this isolate. results: isolate uw was resistant to the following antibiotics: penicillin, ampicillin, gentamicin (high-level), streptomycin (highlevel), erythromycin, clindamycin, vancomycin, teicoplanin, ciprofloxacin, moxifloxacin, nourseothricin, rifampicin, and fusidic acid (lowlevel, mic / mg/l); but showed susceptibilities to oxytetracycline, phosphomycin, chloramphenicol, trimethoprim/sulfamethoxazol, linezolid, and quinupristin/dalfopristin. hybridization, pcr and sequencing experiments localized a cluster consisting of several resistance genes in a composite element on a plasmid. the cluster included genes and transposons tn (vana) Á/tn (ermb) Á/tn (aade Á/ sat Á/apha- ). the plasmid itself was not transferable in filter-matings into a fusidic acid high-level resistant enterococcus faecium recipient while selecting either for erythromycin or vancomycin resistances. however, after transposing a tn -related determinant into uw , determinants became mobilizable with the help of the conjugative transposon. transconjugants were, besides others, high-level resistant to fusidic acid, but susceptible to penicillin and ampicillin. pfge of transconjugants demonstrated a pattern almost identical to the recipient but clearly different from the donor. conclusion: resistance genes in e. faecium could be arranged in a cluster and are mobile via mobilizable/transferable plasmids. lilli d, rivanera d, barbacini ig, lozzi ma, mancini c. department of science and public health, university la sapienza, microbiology, rome, italy aim: hepatitis g virus (hgv), a new rna virus that is parenterally trasmitted has frequentley been found in patients with chronic hepatitis c infection but its role in chronic liver desease is unknown. the aim of this study was to determine the prevalence of hgv infection in patients infected with hcv. ninety-eight patients infected with hcv were evaluated for the presence of hgv rna. the hcv genotypes distribution was genotype b, genotype a, genotype a and four genotype c/ d. hcv rna and hgv rna were detected by rt-nested pcr. results: infection with hepatitis g virus was detected in ( . %) patients and ( . %) were hgv rna negative. none of our patients with genotypes a and c/ d results hgv rna positive. prevalence of hgv infection was % in patients infected with hcv genotype b and . % with genotype a. conclusions: infection with hgv occurred frequently ( . %) in this sample of patients with chronic hepatitis c. we observed a height prevalence of hcv/hgv coinfection in patients infected with hcv genotype a. this association with hcv genotype a was indipendent of the source of infection, infact some of our patients have not history of intravenous drug use. characterization of extended-spectrum beta-lactamase (esbl)mediated resistance in salmonella spp. from durban, south africa pm moodley p a , essack s b , gajee k a , sturm w a . a department of medical microbiology, nelson r. mandela school of medicine, school of infection, university of natal, durban, south africa , b school of pharmacy and pharmacology, university of durban westville, durban, south africa background: gastroenteritis is a common condition among the paediatric population presenting to king edward viii hospital in durban, south africa. from july , we noticed that the susceptibility of the salmonella spp. isolated from stool samples among these children were resistant to multiple antibiotics. aim: to characterize the phenotype of the resistance mechanisms involved. methods: minimum inhibitory concentrations (mics) of ampicillin, azithromycin, ciprofloxacin, cefepime, cefuroxime, cefotaxime, ceftazidime, ceftriaxone, cefoxitin, chloramphenicol, cotrimoxazole and gentamicin were determined by means of the agar dilution method. isolates were subjected to the e -test for extended-spectrum betalactamase (esbl) production. isoelectric focusing was performed as a preliminary step in enzyme characterization. results and conclusion: thirty isolates of multiresistant salmonella spp. were obtained. antibiogram typing revealed six different resistance phenotypes. all isolates depicted ceftazidime/ceftazidime Á/clavulanate ratio of !/ and were considered putative esbl-producers. isolates expressed Á/ beta-lactamases each with pi values ranging between and . indicative of tem-, shv-and/or ctx-m-related esbls. nine isolates expressed two beta-lactamases each and two isolates expressed three beta-lactamases each. there was evidence of the simultaneous expression of both tem-and shv-derived esbls as well as the simultaneous expression of multiple tem-or shv-derived esbls in single isolates, a phenomenon reported in esbl-positive klebsiella pneumoniae isolated at the same hospital. neutrophils exhibit reduced chemiluminescence response to serum opsonized klebsiella pneumoniae producing extended spectrum b-lactamases (esbl) pm objective: to investigate the ability of esbl and non-esblproducing klebsiella pneumoniae isolates treated with human serum to induce a chemiluminescence response in neutrophils. methods: oxidative burst induced by the interaction of esbl (n / ) and non-esbl-producing (n / ) klebsiella pneumoniae isolates with neutrophils from healthy individuals was monitored by measuring the chemiluminescence response (cl). pooled sera from healthy individuals served as source of complement for pretreatment of the bacteria. cl responses triggered by serum treated zymosan served as positive control. the serum opsonized klebsiella strains were arbitrarily graded as high (h) and low (l) inducers of cl when the cl response induced by the bacteria was cl b/ and !/ %, respectively, of that induced by opsonized zymosan. results: out of non-esbl-producing klebsiella isolates, . % induced high cl response in neutrophils whereas only % of esbl-producing klebsiella isolates did so (pb/ . ). conclusions: strains harboring the esbl plasmid were more virulent than non-esbl-producing strains by virtue of their higher tendency to escape serum-dependent recognition by neutrophils. osiris */an automated system for susceptibility testing in agar diffusion technique pm chegrani f, kolbert m, shah pm. universitätsklinik frankfurt, zentrum der inneren medizin med iii schwerpunkt infektiologie, frankfurt am main, germany objective: osiris measured zone sizes were compared to manually measured inhibition zones using round (rp) and mm mueller hinton square agarplates (sqp). variations of / mm in zone size measurements were defined as tolerable. 'very major errors' (vme) were defined as classification of a resistant organism as sensitive by osiris. thirty thousand two hundred and ninety-eight single measurements testing antibiotics on staphylococci and enterobacteriaceae were done according to the din recommendations. results: vancomycin, rifampicin, gentamicin gave the best results on rp with a concordance of , and %. vancomycin, rifampicin, teicoplanin performed best with , , % on sqp testing staphylococci . worst results on rp gave cefuroxim ( . %) and fosfomycin ( . %), on sqp fosfomycin ( . %), ofloxacin ( . %). for enterobacteriaceae amikacin ( %), gentamicin ( %), ciprofloxacin ( %) performed best on rp; worst nalidixinacid ( . %), piperacillin ( . %). concordance on sqp amikacin ( %), cefotaxim ( %), gentamicin ( %), nitrofurantoin ( . %), cotrimoxazol ( %). very major errors were seen in b/ % of all test performed. interpretation: osiris is a rapid and reliable system for susceptibility testing with round and square agarplates and has an excellent expert system. altindis m a , aktepe oc a , kocagoz t b . a kocatepe university school of medicine, microbiology, afyon, turkey , b diomed inc. tr, istanbul, turkey dio-bacit, in a two section plate, that contains % sheep blood agar on one side and sheep blood agar with bacitracin ( mg/ml) was compared for its efficiency in identification of group a beta hemolytic streptococcus (gabs) with other two different growth plates, one containing % sheep blood agar with bacitracin (b) and the other containing b-sxt. we used latex-agglutination for this comparision. throat specimens obtained from cases were inoculated to dio-bacit plates, first to one side with % sheep blood agar and to the other side with b. after an overnight inoculation at c, colonies with beta hemolysis an % sheep blood agar but no growth with b, were inoculated to % sheep blood agar again and antibiogram identification discs containing . u b and . and . mg sxt (oxoid, uk) were placed onto the plate and incubated overnight at c. after that, colonies with beta hemolysis were defined as b-sensitive while colonies resistant to sxt were defined to be gabs. all colonies are serologically classified by latex-agglutination (oxoid, uk). seventy-one ( . %) inoculations revealed growth of gabs at dio-bacit plates. after inoculating these colonies to % sheep blood agar, of them were found to be sensitive to b, while were found to be sensitive to b but resistant to sxt and of them were defined as gabs by latex test. when compared with latex-agglutination test, we found dio-bacit method's sensitivity and spesificity to be and . %, respectively. method: agar diffusion technique as recommended by din was used to determine izs (read using aura and manually) for staphylococci and enterobacteriaceae . variations in automated measured zone sizes of / mm to the manual readings were considered to be within acceptable range. results: six thousand and fifty-two zone sizes were determined for staphylococci and for enterobacteriaceae . mha displayed tendency to smaller zone sizes in automated readings than isa, as well in staphylococci and enterobacteriaceae . on the other side automated readings presented on isa more precise results than mha. overall less major discrepancies ( b/ mm) were found on isa. izs were generally smaller on mha. the tables below show differences in manually and automated measured zone sizes on different media and species. we discovered seven more cases of resistance in the case of metronidazole. we did not have such experience with clarithromycin. conclusion: our results show that e -test is comparable to ad for clarithromycin, but for metronidazole our findings confirm nccls recommendantion. classical ad is time consuming for every day use in the laboratory. the use of screening agar plate with mg/ml of metronidazole to detect possible resistance could be the solution. rokosz a a , sawicka-grzelak a a , meszaros j b , luczak m a . a department of medical microbiology, the university medical school, warsaw, poland , b department of bacteriology, state institute of hygiene, warsaw, poland purpose: to identify esbl-positive strains and to compare two methods applied for the detection of extended-spectrum beta-lactamases (esbls). methods: two hundred and sixty strains of gram-negative rods were cultured from clinical specimens from hospitalized patients. identification of strains was performed in the automatic atb system (biomerieux, france). these strains were identified as esbl-positive on the basis of the double-disc synergy test (ddst according to jarlier et al., ) results. all strains were also determined using a novel method of esbl detection (dd, diagnostic disc) according to appleton ( ) . two discs were applied in this test: cpd (cefpodoxime) and cd (cefpodoxime/clavulanic acid) (oxoid, england). results: consistent results of two methods (ddst and dd) were obtained in the case of from among of examined strains ( . %). consistent results concerned out of strains of enteric rods ( . %) and only five from among other strains (mostly nonfermenting rods). conclusions: the novel method of esbl-producers detection (dd) is more objective and easier for interpretation than the double-disc synergy test (ddst). diagnostic disc test should be used as the basic one or to confirm the results of ddst in difficult cases. assessment of e -test for determining penicillin resistance in pneumococci pm sener b, yeniþehirli g, ercis s, hasçelik g. department of clinical microbiology, hacettepe university medical faculty, ankara, turkey there is a greater need for susceptibility testing methods that distinguish between susceptible and resistant pneumococci. an alternative method could be the e -test, which is compared with the reference agar dilution method in this study. penicillin susceptibility of a total of pneumococci was determined by e -test and agar dilution methods. streptococcus pneumoniae atcc and enterococcus faecalis atcc were used as controls. the results were given in the effect of anoxic conditions on the minimum inhibitory concentration of metronidazole in helicobacter pylori pm de la obra sanz p, lomas e, roman jl, alarcon t, lopez-brea m. the objective of this study was to determine the effect of incubation under anoxic conditions on the metronidazole resistance of helicobacter pylori . methods: a total of clinical isolates were used in this study. mics were determined by an agar dilution method using mueller-hinton agar plus % lysed horse blood. three plates series contained twofold dilutions of metronidazole from to . mg/l were prepared. the first one was incubated under microaerophilic conditions (oxoid) for days; second and third series were incubated anaerobically (anaerobic system, oxoid) for and h, respectively, and were then transferred to the microaerophilic enviroment up to complete days of incubation. results: with microaerophilic incubation, of strains were resistant (mic and mic were and , respectively). with h anaerobic preincubation, four of strains were resistant (mic and mic were . and , respectively). with h anaerobic preincubation, of strains was resistant (mic and mic were . and , respectively). conclusions: anaerobic preincubations causes an increase in sensitivity to metronidazole, the extent of which was dependent on the length of the anaerobic period. methods: the susceptibility to antibiotics was performed by microdilution method according to nccls guidelines. the production of b-lactamase was tested by nitrocefin sticks (oxoid). results: the mics /mics (mg/ml) appeared, respectively: ampicillin / , amoxicillin/clavulanic . / . , cefaclor / , ceftriazone . / . , erythromycin . / . , azithromycin / . / . , clarithromycin . / . , ciprofloxacin . / . , imipenem / . / . , tetracycline / . / / . , trimethoprim/sulfamethoxazole . / . . b-lactamase was detected in . % of the strains. conclusions: ( ) m. catarrhalis isolates were uniformly susceptible to all tested antimicrobials except ampicillin. ( ) the production of blactamase was responsible for ampicillin resistance ( . %). ( ) m. catarrhalis strains had almost the same behavior 'in vitro' to the tested microlides (erythromycin, azithromycin, clarythromycin). rokosz a, sawicka-grzelak a, luczak m. department of medical microbiology, the university medical school, warsaw, poland purpose: to isolate, identify and determine the drug-susceptibility of fungal strains cultured from fecal samples routinely submitted for detection of clostridium difficile and its toxins in cases of antibioticassociated diarrhea (aad). methods: one hundred fecal samples from hospitalized patients were examined (may Á/october ). c. difficile toxins a/b were detected directly in stools with c. difficile tox a/b ii test (techlab † , usa). fecal specimens were inoculated on ccca and candida id (biomerieux, france) media. c. difficile and fungi were identified with standard microbiological procedures. susceptibility of fungal strains to anti-fungal agents was determined (atb fungus, biomerieux, france). results: c. difficile toxins were detected in and c. difficile strains were isolated from of examined specimens. sixty-two fungal strains of genera were cultured from stool samples ( c. albicans isolates). massive fungal growths were observed on primary plates in all cases. fifty-five fungal strains were susceptible to nystatin, -to fluorocytosine, -to amphotericin b, -to ketoconazole, -to miconazole and -to econazole. conclusions: in some cases of antibiotic-associated diarrhea fungal strains are responsible for symptoms of this disease. certain persons having aad should be treated with anti-fungal agents. results: a total of isolates ( . %) were penicillin nonsusceptible (intermediate and resistant) and ( . %) were erythromycin non-susceptible. non-susceptibility to both antibiotics was found in ( . %). conclusions: if penicillin administration eliminates all penicillin susceptible strains, the prevalence of penicillin non-susceptible strains will increase as well as the erythromycin non-susceptible ones. this means that the proportion of erythromycin non-susceptible strains should increase from . to . %. at the same time, if erythromycin eliminate all susceptible strains to this antibiotic, the prevalence of penicillin non-susceptible strains would increase from the initial . to . %. these data can explain the co-selection results observed in different surveillance studies. antimicrobial susceptibility and capsular types/groups of streptococcus pneumoniae isolates causing pneumococcal diseases in bulgaria pm setchanova l a , gergova r a , ioneva m b , sredkova v c . a department of microbiology, medical university, sofia, bulgaria , b department of microbiology, ii city hospital-sofia, sofia, bulgaria , c department of microbiology, faculty of medicine, pleven, bulgaria a prospective study of pneumococcal infections was performed in cooperation with five clinical microbiology laboratories in bulgaria. mics values to antimicrobials and serotype/serogroup distribution were determined for strains of streptococcus pneumoniae . pneumococci were isolated from patients with systemic or respiratory infections. the incidence of penicillin g-intermediate and penicillin gresistant isolate was . and . %, respectively. the rates of resistance to other antimicrobials were: cefotaxime/ceftriaxone */ . %; erythromycin */ . %; clindamycin */ . %; tetracycline */ %; chloramphenicol */ . %; trimethoprim/sulfamothoxazole */ %; ciprofloxacin */ . %; rifampin */ . %. the s. pneumoniae isolates belonged to capsular types/groups. the most common serotypes/serogroups in bulgaria are , , , , , , we aimed to determine the pneumococcal antibiotic resistance rates and the serotypes of those resistant isolates in our hospital. the mic values of isolates (year Á/ ) were determined by agar dilution method. serotyping was performed by using pooled antisera of the pneumo-test. the results were as follows (n / ). objectives: to asses the antimicrobial susceptibility of clinical isolates of pseudomonas aeruginosa obtained from to and to monitor trends in antimicrobial resistance. methods: mics were determined by microdilution testing according to nccls. the antibiotics tested were: ceftazidime (caz), aztreonam (atm), imipenem (imp), gentamicin (cn), tobramycin (tb), amikacin (ak) and ciprofloxacin (cip). results: a total of isolates were included. urine was the most common site of isolation for outpatients isolates ( . %) while for hospitalized patients respiratory samples were the most frequent ( . %). susceptibility to antibiotics was: % caz, . % atm, % imp, . % cn, . % tb, . % ak and . % cip. comparison of susceptibility data through Á/ showed that the increase in the resistance rate was significative for caz ( . vs . %), atm ( . vs %), cn ( vs %), tb ( . vs . %), ak ( . vs . %) and cip ( . vs %). significant differences were found under the following circumstances: isolates from intensive care units and from inpatients were significatively more resistant to caz, atm and imp. isolates from respiratory samples were more resistant to caz and atm and isolates from urine samples were more resistant to cip. conclusions: although the antimicrobial susceptibility level has been decreasing p. aeruginosa isolates still show good susceptibility percentages for all antibiotics tested. antimicrobial susceptibility testing of clinical isolates of bordetella pertussis : report on isolates from rouen, france pm lemee l a , nouvellon m a , caron f b , lemeland jf a . a chu rouen, bacteriologie, rouen, france , b chu rouen, maladies infectieuses et tropicales, rouen, france reports of an increased clinical incidence of pertussis and the development of resistance by bordetella pertussis to erythromycin prompted the collection and antimicrobial susceptibility testing of recent clinical isolates from patients, who were hospitalized in rouen between and . mics of nine antimicrobial agents (erythromycin, josamycin, spiramycin, roxithromycin, ketolide hmr , cotrimoxazole, ciprofloxacin, rifampicin and amoxicillin) were measured by agar dilution method on mueller-hinton agar containing % horse blood. mbcs of erythromycin and rifampicin were also determined against four isolates of b. pertussis . all isolates were fully susceptible to the nine antimicrobial agents tested. mics (mcg/ml) were . for erythromycin, ketolide hmr and ciprofloxacin, . for josamycin, . for spiramycin, roxithromycin and rifampicin, . / . for cotrimoxazole, and for amoxicillin. mbcs (mcg/ml) were . Á/ . for eyrthromycin and . Á/ for rifampicin. in conclusion, our isolates of b. pertussis remain extremely susceptible to all antimicrobial agents tested, especially macrolides. no resistance was detected. finally, if erythromycin remains the molecule of choice, other macrolides (c and c ) also confirm their good in-vitro activity. in addition, the good in-vitro potency of rifampicin, together with its great diffusion within the respiratory tract, suggests that rifampicin has potential clinical efficacy in pertussis too. the emergence of streptococcus pneumoniae (sp) with diminished susceptibility to penicillin g (psdp) suggests the use of other antibiotics such as newer fluoroquinolones (fq). the resistance phenotypes of consecutive pneumococcal strains isolated from patients of four hospitals (observatoire régional des pneumocoques du nord-pas de calais) were studied: strains were susceptible to penicillin g and were psdp. reference strains provided from the centre national de réfeacute;rence des pneumocoques were added to the study. the activity of pefloxacin, ciprofloxacin, norfloxacin, sparfloxacin, levofloxacin and moxifloxacin was studied. reserpine was used to detect the efflux phenotype. methods used were performed according to the recommendations of the comité de l'antibiogramme de la société française de microbiologie. for each strain, the resistance phenotype to fq was deduced by comparison of mics or diameters obtained with those obtained with the reference strains of known phenotypes. fq resistance phenotypes were not correlated to blactam agent susceptibilities. wild type phenotype was observed among . and . % of the susceptible and psdp strains, respectively. a 'wild efflux' mechanism, deduced by addition of reserpine to norfloxacin, represented the predominant phenotype. it was detected among sp susceptible to penicillin g ( . %) as well as among psdp ( . %). the aim of this study was to determine macrolide resistance phenotypes of sp isolated in three french departments (alpes maritimes, doubs, nord) from nasopharyngeal aspirates of children aged months to years attending a dcc. a random sample of children attending randomly selected dccs was obtained during three periods (spring, autumn and winter ) in each department ( children attending dccs and children sampled). analysis of macrolide susceptibility of sp strains was performed using the ca-sfm method. out of strains, . % had decreased susceptibility to penicillin (spdp) and . % were resistant to erythromycin. the triple disk diffusion method (erythromycin (e), clindamycin (cl) and spiramycin) was used to determine resistance phenotypes. macrolide resistance is a well known phenomenon in france and is confirmed by our study. these results show that the constitutive phenotype is predominant as in other parts of europe and the frequency of efflux mechanism is lower than that observed in the usa and canada. developing antibiotic resistance surveillance of helicobacter pylori in england and wales pm elviss nc, owen rj. central public health laboratory, laboratory of enteric pathogens, london, uk purpose: helicobacter pylori antibiotic resistance is a key contributing factor in Â/ % of infected patients failing drug treatment. our aim was to survey rates of primary in-vitro resistance at different locations, and links to disease severity. antral gastric biopsies/cultures were received from phls in chelmsford, mid-essex ( isolates Á/ ); london ( isolates Á/ ); and bangor, north wales ( isolates Á/ ). susceptibilities to metronidazole (mtz), clarithromycin (cla), tetracycline (tet) and amoxicillin (amx) were tested by disc diffusion and also by e -test for cla and mtz. results: overall resistance rates ( isolates) were % for mtz and % for cla. all were susceptible to amx and tet. dual resistance rate was %. breakdown by location showed some marked differences. mtz resistance was highest in london ( %) compared to % in chelmsford and % in bangor. by contrast cla rates were % for london, and about % for bangor and chelmsford. in london, the majority of mtz resistant isolates were from non-uk borne individuals ( % non-uk vs % uk). comparison of duodenal ulcer-associated isolates with those from non-ulcer patients indicated similar rates of mtz resistance ( %). conclusion: resistance rates may vary significantly between locations depending on the local population with non-uk birth being a key risk factor for primary resistance with a mtz resistant strain. local resistance rates should be taken into account in test and treat strategies. potrykus j, benetkiewicz m, wegrzyn g. department of molecular biology, university of gdansk, gdansk, poland purpose of the study : because of their ability to extrude a wide range of compounds, multidrug efflux pumps have recently become an important issue in combating bacterial infections. acrab-tolc is the major efflux system of escherichia coli . we investigated the effect of acra on plasmid-borne and intrinsic chloramphenicol, tetracycline and ampicillin resistance. results and conclusions: recently, we reported a chloramphenicol sensitivity of e. coli mutant expressing cat , the chloramphenicol resistance gene. the strain was shown to bear a nonsense mutation in the acra gene. our studies indicate that this mutation is, at least in part, responsible for the observed chloramphenicol sensitivity phenotype. the mutation seems also to influence the strain's susceptibility to ampicillin and teta (c )-mediated (plasmid-borne) tetracycline resistance. although the teta(c) protein retained its biological function, there was a considerable growth impairment of the mutant strain when cultured in tetracycline containing medium. deletion of the acrab locus prevented any growth in the presence of tetracycline. upon the addition of ampicillin, the mutant underwent lysis more rapidly than the control strain. such was also observed in acrab deletion derivatives of other e. coli strains. we are trying to elucidate the role of the acra gene product in the phenomena described above. existence of efflux pumps in wild type isolates of drug-resistance bacteria pm raja ray rr. medical microbiology and parasitology, calcutta university, kolkata, india efflux pumps possessed by the bacterial cells of different kinds of bacteria had presented as a newer mode of drug resistance in many organisms. the capacity of bacterial cells to cause outward flow of noxious agents was known, however, for a considerable time with respect to tetracycline. recently, interest in the efflux pump system has brought to light some previously ill-understood mechanisms of drug resistance, involving noxious agents, toxins or poisons. we have found high level of resistance in pseudomonads towards cetrimide and other germicides for which no definite chromosomal/plasmid-mediated genes/mechanisms could be identified. likewise, occurrence of nonantibiotic sensitive vibrios, staphylococci and pseudomonads in the background of their high level of resistance to most of the common antibiotics suggest a mechanism of interference with the efflux pump, which accounts for such sensitivity in such cases. involvement of multiple resistance of marine isolates of v. parahaemolyticus to numerous clinically used antibiotics to which they have never been exposed also suggests a possible role of efflux pumps in determining such resistance */that these can simultaneously develop against multiple marine toxins/poisons and other noxious agents. interaction between oxacillin and glycopeptides in a teicoplanin-resistant mutant of staphylococcus epidermidis with reduced susceptibility to vancomycin pm greco aa, ben hassen a. laboratory service of national bone-marrow transplant center, tunis, tunisia we selected a laboratory-generated mutant of staphylococcus epidermidis capable of growing in the presence of mg/l of teicoplanin ( b tm ), from a methicillin-resistant (mic!/ mg/l), teicoplanin-sensitive (mic mg/l) and vancomycin-sensitive (mic mg/l) clinical isolate of s. epidermidis ( b so). in a previous work, we studied the different phenotypic characteristics acquired by the teicoplanin-resistant mutant b tm ( th interdisciplinary meeting on anti-infectious chemotherapy, december , poster sessions, /p ). in this work, we examined the interaction between oxacillin and glycopeptides against this teicoplanin-resistant mutant of s. epidermidis with reduced susceptibility to vancomycin. to study the combined antibiotic activity of oxacillin and glycopeptides, we used different methods: a modified disk diffusion test, the e -test, time-kill assays and population analysis profiles. the synergistic activity of glycopeptides in combination with oxacillin against the teicoplaninresistant mutant b tm was demonstrated with a bactericidal effect. no synergy was seen against the parental strain b so. moreover, the synergy between glycopeptides and oxacillin occurred with suppression of the subpopulation with the highest level of glycopeptides resistance. we concluded that combination of glycopeptides and oxacillin may be a possible alternative in the treatment of infections caused by methicillin-resistant, teicoplanin-resistant s. epidermidis . compositional changes in microcosm biofilms induced by application of minocycline: a preliminary study pm the aim of the study was to observe the effect of application of minocycline upon microcosm dental plaques. the plaques were cultivated in a constant departmenth film fermentor (cdff), which produces biofilms under conditions mimicking those present in vivo. the composition of the biofilms was determined by viable counting on selective and non-selective media. the proportion of antibiotic resistant genera within the biofilm was determined by viable counts utilising media containing minocycline ( mg/ml). before commencing antibiotic pulsing, the biofilms had a total viable anaerobic count of . )/ cfu per biofilm, with negligible ( cfu/biofilm) minocycline-resistant bacteria. however, h after introduction of the antibiotic, the total count had been reduced to . )/ cfu/biofilm whilst the number of minocycline-resistant bacteria had risen to . )/ cfu/biofilm. at the final sampling time point ( h) the total viable anaerobic count was . )/ cfu/biofilm whilst the number of minocycline-resistant bacteria was . )/ cfu/biofilm. hence, there is a very low basal level of inherent resistance to minocycline within microcosm dental plaques, but this increases considerably once the biofilms are exposed to minocycline. mechanism of resistance to aminoglycosides (amg) e. coli isolated from children with community-acquired urinary tract infections (cau-tis) pm methods: during the Á/ years nine centers took part in the study. the mics of antimicrobials were determined by the agar dilution method as described in the nccls guidelines. results: a total of consecutive urine isolates from children aged month to years with cauti were collected. the most frequently isolated species from children with cauti was e. coli ( . %), followed by klebsiella spp. ( . %) and proteus spp. ( . %). results of the in vitro susceptibility testing of e. coli to amg are shown in table below. resistance of the strains was conditioned on production of amg-modifying enzymes. there has been found following phenotypes among resistance strains: gentamicin Á/ tobramycin Á/netilmicin ( . % */aac( )-v and . % */aac( )-iv enzymes) and gentamicin Á/tobramycin ( . %, due to ant( ƒ) enzyme). conclusion: amikacin is most active amg against e. coli . resistance to gentamicin and netilmicin was mainly determined by production of aac( )-v enzyme. effect of b-lactamase inhibitors (b-l-i) on the evolution of resistance (r) to b-lactams (b-l) in gram the b-lactams antimicrobials still are the most frequently used. among the bacteria responsible of high resistance to b-lactams are gramnegative rods; its most frequent mechanism is the production of b-lactamase. the use of b-l-i has reversed partially this mechanism of resistance. we expect changes in the frequencies of r using b-lƒci after more than years. since , the venezuelan group of bacterial resistance, with health institution in the country; analyse and publish data on bacterial resistance of isolates from patients with bacterial infection coming from hospitals. it was used diffusion disk, according nccls. the software program whonet (world health organization net) was used. we follow the trends of r of gram-negative rods to b-l alone and with b-l-i during the decade Á/ . statistical analyses were made by evaluating the differences among percentages of resistance between the two series (p / . ). results and discussion: the difference in r between b-l and b-l/b-l-i are: ( ) piperacillin, piperacillin/tazobactam: between and % of r for most isolated, except for escherichia coli ( %) and serratia spp. ( %); ( ) ampicilin, ampicilin/sulbactam: between and %; ( ) cefoperazone, cefoperazone/sulbactam: between and %. how is expected gramnegative rods resistance to b-lactams with a b-l-i is lower than the b-lactam alone; furthermore the difference between both series, grows higher with time. these results are relevant and they were not expected, since b-l-i have been shown to be b-lactamase inductors. trends in the resistance (r) to b-lactams and others antimicrobials in p. aeruginosa in venezuelan medical centres. nosocomial (nos) and communitarian resistance pm in order to approach the infection produced by resistant bacteria, it is convenient to consider the hospital and the community as two separate ecosystems. the hospital ecosystem has special relevance in the infection and r of gramnegative aerobic bacilli. today, they are the main responsible of nos infection, with special reference to pseudomonas aeruginosa . infection by resistant bacteria is a world wide problem, specially related to nos. since , the venezuelan group of bacterial resistance, with health institution in the country; identify, analyse and publish data on bacterial r to antimicrobials: b-lactams, quinolones and aminoglicosides of isolates from patients with bacterial infection coming from hospitals and the community. it was used diffusion disk, according nccls. the software program whonet (world health organiza-tion net) was used. statistical significance (p / . ) was determined by application of the x technique. we show significant differences in the r of p. aeruginosa nos and communitarian (highest differences: piperacilina / %, tobra / %). we also established significant differences between the r arising in public hospitals and private hospitals (highest differences: ceftazidime / %, amika / %). we show the tendency in decreasing of frequency of r since ; this is more evident in private hospitals (b-lactam and aminoglicosides). new antimicrobials and new mechanism of action, and in the future the new technology will solve today's problem. however, the most important tools we have today are prevention and antimicrobials, and we must make them suitable. susceptibility to antibiotics of enterobacter cloacae and citrobacter freundii from drinking water pm quintera sm, sousa jc, peixe l. department of microbiology, faculty of pharmacy, university of oporto, oporto, portugal the increased use of antimicrobials in farming, together with the practice of raw sewage discharge into receiving waters, has resulted in a significant increase in the number of antibiotic resistant bacteria present in aquatic environment. our objective was to determine the antimicrobial susceptibility, with focus on b-lactam resistance, among enterobacteriaceae strains isolated from raw drinking water samples. several isolates (n / ) of enterobacter cloacae and citrobacter freundii obtained from drinking waters were screened for antibiotic susceptibility patterns, using the agar diffusion technique, according to nccls's procedures. only % of e. cloacae strains, as well as % of c. freundii strains show resistance to amoxicillin and amoxicillin/ clavulanic acid. a reduced incidence of resistance to several others antibiotics was also observed. the obtained results suggest that strains isolated from raw drinking water have greater susceptibility to antimicrobial agents than pathogenic strains from hospital or outpatients infections. the 'natural' antimicrobial resistance phenotypes, usually described for c. freundii and e. cloacae , only seem to apply to strains isolated from human infections. notwithstanding the high susceptibility of the tested isolates to b-lactams, the role of environmental bacteria as a reservoir of resistance genes justify its periodical monitoring as a valid index for resistance spreading. a snapshot of the soil. using bacterial communities for tracing the evolution of metal-resistance pm quintera sm a , sousa jc a , peixe l a , monteiro nm b . a department of microbiology, faculty of pharmacy, university of oporto, oporto, portugal , b department of zoology and anthropology, faculty of sciences, university of oporto, oporto, portugal it is well known that pathogenic bacteria, specially those resistant to antimicrobial agents and heavy metals poses public health risks of great concern, and its detection, namely in soils is generally related to pollution. in this study, the heavy metal resistance patterns of the microflora isolated from polluted (dump area) and unpolluted soil environments were examined. the plate growth covering percentage in the soil samples was determined using mueller-hinton plates supplemented with different heavy metal (al '/, cd '/, cu '/, pb '/, hg '/ and zn '/) concentrations. parallelly, using icp-aes, it was possible to ascertain the real heavy metal concentration for each soil sample. we found that the percentage of plate growth covering from the used samples was closely linked to the level of chemical pollution measured for each location. moreover, using anova, we found significant differences between locations. the dump site showed the highest tolerance to all the tested metals (newman Á/keuls test). this pattern of results was consistent when using the data from the icp-aes. furthermore, it was possible to observe that pseudomonas spp., with a relatively high mic for the studied metals, might become a relevant model for both public health issues and eco-toxicological studies. biochemical characteristics of environmental isolates of listeria monocytogenes pm moshtaghi h a , garg sr b , mandokhot uv b . a shahrekord university, food hygiene, shahrekord, islamic republic of iran , b haryana agricultural university, food hygiene, hisar, india purpose: the investigations were carried out to study the biochemical reactions of listeria monocytogenes isolated from different sources in the environment. results: a total of isolates of l. monocytogenes were obtained from samples of agricultural soil, faecal matter of animals and sewage. all the isolates were gram-positive, small rods, catalase positive, oxidase negative, motile with tumbling motility in hanging drop at Á/ c, aerobic, facultative anaerobic, fermentative and produced acid from glucose. all the isolates of l. monocytogenes were beta haemolytic and positive for camp reaction with staphylococcus aureus . all the isolates were negative for phenyl alanine deaminase, ornithine decarboxylase, lysine decarboxylase, malonate utilization and beta galactosidase tests. these were also negative for acid production from arabinose, d-xylose, mannitol, soluble starch and sucrose but acid was produced in rhamnose, salicin, and trehalose. hydrogen sulfide production was recorded in tripticase soy broth with lead acetate paper strips but negative with triple sugar iron agar. all the isolates were found to hydrolyse aesculin. out of isolates of l. monocytogenes only two produced acid from lactose. in serotyping all the isolates were serotype b. conclusion: we can conclude that l. monocytogenes serotype b at least in fermentation of lactose shows different reactions. methods: the samples were pre-enriched in bhi broth with and without vancomycin ( mg/l) and then plated onto m-enterococcus agar with and without antibiotics: vancomycin ( mg/l), gentamicin ( mg/l), kanamycin ( mg/l), and streptomycin ( mg/l). representative colonies of each morphology were isolated and identified as enterococcus sp as previous described. pcr was used to identify e. faecium and e. faecalis and to characterise vancomycin resistant genotype. api strep was also used in the identification. susceptibility testing to antibiotics was performed by an agar dilution method (nccls). results: three hundred and fifty-three enterococci were isolated from of a total of faecal samples ( %, n / / ). the majority of enterococci were identified as e. faecium , e. faecalis and enterococcus sp. resistance to almost all antibiotics studied was observed: vancomycin */ . %; teicoplanin */ . ; ampicillin */ . %; tetracyclin */ . ; erythromycin */ . %; ciprofloxacin */ . %; chloramphenicol */ . %; gentamicin */ . %; streptomycin */ . %; kanamycin */ . %; linezolid */ %. the vancomycin resistant enterococci presented a vana genotype. conclusion: resistance to several common antibiotics used in therapy was observed among enterococci isolated from healthy human from community. many of these isolates presented multi-resistance. of concern is the presence of vana genotype among these populations that may constitute a reservoir of vancomycin resistant genes. antimicrobial resistance in tetracycline-resistant oral bacteria pm mercury release from dental amalgam may select for mercuryresistant oral bacteria. mercury resistance is often associated with multiple antibiotic resistances. the aims of this study were to determine whether tetracycline-resistant oral bacteria from children with and without amalgam fillings were also resistant to: (a) mercury; and (b) multiple antibiotics. tetracycline-resistant organisms were isolated on iso-sensitest/blood agar containing tetracycline ( mg/ml). the mic of hgcl and several antibiotics were determined using agar dilution (bsac). one hundred and three organisms were isolated from patients without amalgam. ninety-one were streptococcus species, seven neisseria species, three veillonella dispar and two rothia species. fifty-seven percent exhibited resistance to at least one antibiotic, % were mercury-resistant, % were penicillin-resistant, % were ampicillin-resistant and % erythromycin-resistant. fiftytwo organisms were isolated from patients with amalgam. forty-five were streptococcus species, five neisseria species, one v. dispar and one staphylococcus aureus . sixty-three percent exhibited resistance to at least one antibiotic, % were mercury-resistant, % penicillinresistant, % were ampicillin-resistant and % showed erythromycin-resistance. statistically, the results showed that in tetracyclineresistant organisms, the presence of dental amalgam did not affect the level of resistance to mercury or to the antibiotics tested. conway-wallace hl a , mullany p a , bedi r b , wilson m a . a eastman dental institute, university college london, microbiology, london, uk , b eastman dental institute, university college london, transcultural oral health, london, uk the purpose of this study: to determine the prevalence of antibioticresistant oral bacteria in children who had not received antibiotics during the months prior to sampling. plaque samples were obtained from children aged Á/ years and plated onto media containing: penicillin, ampicillin, tetracycline, erythromycin and vancomycin. resistant isolates were enumerated, sub-cultured and frozen for subsequent identification. the process was repeated and months later. the results obtained: bacteria resistant to each of the antibiotics were present in all of the children at each sampling time (except in the case of ampicillin and penicillin at months). the proportion of antibiotic-resistant bacteria in the oral microflora ranged from ]/ . (erythromycin) to / . % (ampicillin). the proportions of bacteria resistant to a particular antibiotic remained reasonably constant over the -month sampling period. in only two cases (penicillin and ampicillin) was there a statistically significant change in the proportions of resistant bacteria at different time periods. the conclusion reached: the results of the study have revealed that bacteria resistant to a wide range of antibiotics may be isolated from children who have not been administered these agents during the months prior to sampling. furthermore, in many cases the proportion of bacteria resistant to a particular antibiotic remains constant over a -month period. antimicrobial use in the intensive care unit: results of a pharmacoepidemiological study in italy pm periti p. e.i.f.t. srl, firenze, italy a retrospective survey of antimicrobial chemotherapy use in intensive care units in italy was carried out in using a computerized questionnaire under the auspices of the journal of chemotherapy. of the icus contacted, . % replied, being mainly general or post-surgical and pediatric units having a mean of beds, nine doctors and nurses. the antimicrobial agents used in these wards were almost always polychemotherapy with prevalent use of beta-lactams, aminoglycosides and glycopeptides or as empirical treatment during the first h after hospital admission. the continual use of medium-high dose combinational antimicrobial chemotherapy was justified by microbiological testing, which revealed that more than one-third of bacterial pathogens were resistant. approximately, % of gram-positive bacteria were methicillin-resistant, whereas about % of gram-negative strains were resistant to at least one of the tested antibiotics. forty percent of the responding icus furnished microbiological testing data, of which three quarters indicated the incidence of chemoresistance of the isolated strains. fungal infections were less frequent than bacterial, the most commonly isolated agent being candida spp. in conclusion, the sample of icus examined showed adequate and reasonable use of antimicrobial agents, with heavy reliance on medium-high dose combination therapy due to the elevated incidence of resistant isolates found. plasma concentrations (p), urinary excretion (u) and bactericidal activity of gatifloxacin (gat) mg versus ciprofloxacin (cip) mg in healthy volunteers after a single oral dose pm boy d a , kinzig-schippers m b , sö rgel f b , well naber kg a . a hospital st. elisabeth, urologic clinic, straubing, germany , b institute for biomedical and pharmaceutical research, ibmp, nürnberg-heroldsberg, germany twelve volunteers received a single oral dose of mg gat versus mg cip to assess p up to h, u (by hplc), and urinary bactericidal titers (ubt) in eight intervals up to h. the mean pmax of gat/cip was . / . mg. the ucum (mean) for gat/cip was . / . %. the ubts, i.e. the highest twofold dilution of urine still bactericidal, were determined for nine uropathogens and one reference strain */mics (mg/ml) (microdilution) for gat/cip: escherichia coli atcc ( . / . ); e. coli ( . / . ); klebsiella pneumoniae ( . / . ); proteus mirabilis ( . / . ); pseudomonas aeruginosa ( / . ); s. saprophyticus ( . / . ); two strains of s. aureus ( . / . ); two strains of e. faecalis ( . / and / ). the median ubts measured within the first h for gatifloxacin were between : and : for the five gram-negative strains (incl. p. aeruginosa ) and between : and : for the five gram-positive strains. the median ubts for ciprofloxacin were between : and : for the gramnegative strains (incl. p. aeruginosa ) and between : . and : for the five gram-positive strains. for the ubts up to h, gat was significantly superior to ciprofloxacin in all gram-positive strains, not different in the two e. coli strains, and inferior in the klebsiella , proteus and pseudomonas strains. for the ubts at Á/ h, gat was generally superior to cip, but showed no difference in the proteus and pseudomonas strains. gat showed overall comparable urinary bactericidal activity as cip. this is in agreement with a clinical study performed previously. malaria is one of the most prevalent endemic infectious disease affecting humans. in bichat hospital cases of malaria acute illness were reported during . among them, patients were hospitalised and intravenously treated by quinine. this retrospective study consisted of comparing the therapeutic drug monitoring (tdm) of quinine distinguishing, respectively and patients cured in infectious medical department (imd) and intensive care unit (icu) where a standardised quinine regimen was established ( and % malaria attacks, respectively). in icu, the treatment consisted of an infused loading dose mg/kg/ h of quinine diluted in % glucose followed by mg/kg/day. plasma quinine maximal concentrations were assessed after selective liquid Á/liquid extraction and spectrofluorometry detection. statistical analysis was performed using t -test. results showed that patients had comparable weight ( . / . and . / . kg) but quinine doses and plasma concentrations were significantly different in icu and imd, respectively ( . / . versus . / . mg/kg/day, pb/ . and . / . versus . / . mg/l, p b/ . ). in icu and imd, respectively: and % were in the therapeutic range ( Á/ mg/l) with and % below the requested therapeutic concentration ( mg/l) and and % above the limit of toxicity ( mg/l) conveying the importance of tdm in intravenous quinine treatment to avoid infra-therapeutic or toxic concentrations. simultaneous central nervous system distribution using microdialysis and pharmacokinetic Á/pharmacodynamic modelling of the electroencephalogram effect of norfloxacin in rats pm chenel m, marchand s, dupuis a, bouquet s, couet w. university of pharmacy, pharmacology, poitiers, france purpose: to investigate the epileptogenic activity of norfloxacin by a pharmacokinetic Á/pharmacodynamic (pk Á/pd) modelling approach and to assess the contribution of distributional processes across the blood Á/brain barrier (bbb) to the delayed effect. methods: rats (n / ) received an iv bolus dose of norfloxacin ( mg/kg). convulsant effect was quantified by electroencephalogram (eeg) recording during h post-dose. arterial blood samples were collected for drug assays in plasma. unbound norfloxacin concentrations were monitored in brain extracellular fluid (ecf) using microdialysis with in vivo calibration of the probes by retrodialysis with ciprofloxacin. results: the eeg effect reached its maximum between and min post-dose. a pk/pd effect compartment model was successfully fitted to these data. the relationship between effect and concentration at the effect site was best described by a spline function. norfloxacin concentrations in brain ecf were relatively low compared to plasma levels (ecf/plasma areas under curve (auc) ratio equal to . / . %), but central distribution was rapid. therefore, the effect versus brain ecf concentrations curves still exhibited a marked hysterisis. conclusion: the delay observed between plasma concentrations and norfloxacin convulsant effect cannot be explained by a slow distribution of norfloxacin across the bbb. pagoulatou a a , kanellakopoulou k b , vafiadou m a , kostakopoulos th c , kastriotis i b , giamarellou h c . a department of anesthesia, sismanoglio general hospital, greece , b th department of internal medicine, athens medical school, athens, greece , c department of urology, athens medical school, athens, greece csf kinetics of van and fu were studied in patients who underwent short urological surgery under spinal anesthesia. patients were excluded if they were already receiving an antibiotic or were suffering from renal and hepatic dysfunction. van was administered at g over h infusion. serum and csf samples were collected post-dose and the mean serum levels were as follows: min Á/ h: . mg/ml (five patients), Á/ h: . mg/ml (five patients), Á/ h: . mg/ml (six patients), Á/ h: . mg/ml (six patients) and Á/ h: . mg/ml (seven patients). fu was administered at mg dose over h infusion. serum and csf samples were taken post-dose and the mean serum concentrations were found as follows: Á/ min: . mg/ml (six patients), min Á/ h: . mg/ml (six patients), Á/ h: . mg/ml (five patients), Á/ h: . mg/ml (six patients), Á/ h: . mg/ml (five patients). in csf, both van and fu were undetectable. it is concluded that in the absence of meningeal inflammation van and fu do not penetrate (with the applied microbiological assay) the csf barrier. comparison of the pharmacology of intravenous and orally given moxifloxacin in an in-vitro model pm wiegand i, pfeil e, wiedemann b. university of bonn, pharmaceutical microbiology, bonn, germany purpose: the intravenous form (iv) of mg moxifloxacin (mox), one of the newer fluoroquinolones, has been recently approved by the fda. during the iv treatment higher peak serum concentrations are achieved in comparison to the oral administration (po) of the same dose. the antibacterial activity of fluoroquinolones is concentration dependent. we therefore simulated human pharmacokinetics of single po and iv dosages of mg mox in an in-vitro model using six different gram-negative and -positive pathogens to elucidate the different effect of these two dosing schedules. results: the comparison of the pharmacological parameter auc/ mic shows an increase ( table ) that could predict an enhanced antibacterial effect. however, the analysis of the killing curves with the following parameters, ka.max (maximal killing activity) and aac (area above the killing curve between and h), reveals no major difference between the po and iv dosage. conclusion: the serum concentration after oral administration is already sufficiently high to show the optimal bactericidal effect of mox that can only be slightly increased by higher peak concentrations and higher auc/mic ratios. thus the concentration dependence is not linear but ends already at concentrations achievable by oral dosing and documents that auc/mic calculations cannot easily be translated into dosing schedules. background : bacteria growing in vivo multiply much more slowly than in vitro. whether the bactericidal activity of quinolones may be affected by an increase in generation time (g) was studied in batch cultures. methods: by limiting the nutrient supply, generation times were lengthened from approximately . to . h up to . h. alternatively, the quinolones were added to the bacterial cultures during the lag-, exponential-and stationary phase. recent clinical isolates of escherichia coli were exposed to multiples of the mics of ciprofloxacin or norfloxacin. the 'killing rates' were calculated in analogy to the growth rate. results: the bactericidal activity of the quinolones tested against e. coli was minimally influenced by the reduced generation time. ciprofloxacin concentrations of ]/ )/mic eliminated the test strains within / h from the test system if added during the lag or exponential growth phase; four times higher concentrations were needed to reduce cfus by % within h, if added during the stationary phase. norfloxacin was significantly less active. conclusion: in contrast to norfloxacin, the bactericidal activity of ciprofloxacin is minimally affected by the generation time or growth phase of the bacteria. wiegand i, pfeil e, wiedemann b. university of bonn, pharmaceutical microbiology, bonn, germany purpose: moxifloxacin (mox) is one of the newer fluoroquinolones, now available for parenteral application. the pharmacology of an intravenous once-daily dose (od) of mg mox was determined with five gram-negative and -positive pathogens (streptococcus pyogenes , streptococcus pneumoniae , moraxella catarrhalis , escherichia coli , and klebsiella pneumoniae ). a twice-daily dose (bid) of mg mox was simulated with the gram-positive species in order to increase the bactericidal effect. results: to determine the efficacy, killing curves were analyzed, and following parameters were calculated: ka.max: maximal killing activity [log cfu]; ka. conclusion: an intravenous once-daily dose of mox is active against all tested pathogens. the gram-negative species are rapidly killed (ka. h similar to ka.max). there is no pronounced initial effect on the two gram-positive species but a general slow reduction in the viable cell count (ka.max is reached after h). the efficacy of mox (measured as aac and ka.max) on s. pyogenes and s. pneumoniae is to some extent increased after the second dose. however, the analysis of the killing curves reveals no major difference between od and bid. even the od nearly gives the maximal bacterical activity of mox against gram-positive pathogens. objectives: to evaluate the dose proportionality of amoxicillin and to compare the respective pk/pd parameters of two dosage regimens. methods: the dose proportionality of amoxicillin was evaluated using linear regression of mean auc -inf and c max data of different bioequivalence studies (n / volunteers) performed with formulations containing various amounts of amoxicillin alone or in the combination with clavulanic acid. the volunteers received a single oral dose in the range of Á/ mg. amoxicillin plasma concentrations were determined by hplc/uv or lc/ms/ms methods. time above mic (tmic) expressed in% of dosing interval was calculated with three target mic values ( . , . and . mg/l) for mg hourly and g -hourly dosage regimens. results: the absorption of amoxicillin (auc -inf ) showed a linear dependence with a correlation coefficient of . . the correlation coefficient of the linear regression for the cmax dependence on the actual dose was . . the respective tmic for both dosage regimens were very similar, with largely overlapping confidence intervals, supporting a pd breakpoint of mg/l for the g -hourly regimen (tmic ]/ mg/l: . %, % ci . , . %). conclusion: this analysis shows the dose proportionality of amoxicillin over the dosage range of Á/ mg and supports the pharmacodynamic rationale for a g bid dosage regimen. piperacillin/tazobactam concentration profile after high dose administration pattern in nosocomial pneumonias due to mecanical ventilation pm pedeboscq s a , gruson d b , bassoua v a , hilbert g b , pometan jp a . a st. andré hospital, pharmacy, bordeaux, france , b pellegrin hospital, reanimation, bordeaux, france the piperacillin (p)/tazobactam (t) antibacterial spectrum covers the largest part of bacteria responsible for pneumonias due to mechanical ventilation. but, due to important bacterial inoculum and pharmacokinetic parameter modifications in intensive care patients, high doses of beta-lactamines seem to be necessary to obtain antibiotic concentrations above suspected bacteria's mic (minimal inhibitory concentration) . this led us to compare, in patients with pneumonia due to mechanical ventilation, two intermittent administration patterns: g three times a day (usual pattern) versus g four times a day (high dose pattern). this study is carried out in collaboration with intensive care unit, bacteriological department and pharmacy where antibiotic concentrations are determined. twenty-three takings of blood are executed within a h period, in addition to two bronchial secretion samples. concerning p seric concentrations, the high dose pattern seems to be more adapted because of relatively high residual concentrations ( !/ mg/ml). three hours after each injection, t seric concentrations are lower than the mg/ml activity threshold. first and second day residual bronchial concentrations of p seem to be sufficient although t concentrations are below activity threshold. these results are to be correlated with the mic determined by the bacteriological department, and only this correlation will make us able to conclude the better efficacy of the high dose pattern in intensive care patients. anti-inflammatory drugs interference in absorption and tissue penetration of amoxycillin pm del fiol fs a , menon sz b , caramez th b , celotto tf b , lopes ras b . a university of sorocaba, pharmacology, sorocaba, brazil , b school of pharmacy, university of sorocaba, sorocaba, brazil antibiotics and anti-inflammatories are frequently associated in clinical practice. there is some concern about the quantity of antibiotic that reaches the infection sites, which may be reduced in the presence of an anti-inflammatory drug. the purpose of the present study was to analyse how steroids (dexamethasone (dexa)) and aines (celecoxib (cele)) influence on the penetration of amoxicillin to inflamed tissues. thirty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their backs to form granulomatous tissue. one week later the animals were divided into three groups. one group received only amox ( mg/kg), another received amox ( mg/kg) plus cele ( . mg/kg) and the last received amox ( mg/kg) plus dexa ( . mg/kg). one hour later the animals were sacrificed and the concentration of amoxicillin in the serum and tissue investigated. there was no difference among the groups in the quantity absorbed (amox / . / . mg/ml; amox'/cele / . / . mg/ml and amox'/dexa / . / . mg/ml). there was a reduction in the tissue concentration of amoxicillin (p b/ . tukey-kramer) for the group that received the drug with dexamethasone. for the other groups, there was no difference in the tissue concentration of amoxicillin. the results indicated that in inflamed tissue, a significant reduction of antibiotic penetration was induced by sinultaneous dexamethasone therapy. prediction of the optimal amoxicillin dose regimen based on coupling of pharmacokinetic data and bactericidal activity pm background: given its short half-life, amoxicillin (amx) should be administered at least three times a day to patients with acute exacerbations of chronic bronchitis, in order to achieve serum concentrations well above the mic of the responsible pathogen. however, several authors have recommended twice-daily administration of a higher dose for a shorter period. we assessed the relationship between amx sputum concentrations and antibacterial activity following two treatment schedules in healthy volunteers. subjects and methods: twelve healthy volunteers were randomized to receive amx for days at a dose of either g bd or mg bd. serum and sputum were collected every day, h after the morning administration, and again days after the last dose. amx concentrations were determined by hplc with fluorometric detection. sputum killing activity was determined against haemophilus influenzae , streptococcus pneumoniae and moraxella catarrhalis . results: mean serum concentrations measured h after the morning administration were . ( mg bd) and . mg/l ( g bd), and were above the mics of the three microrganisms. in contrast, sputum concentrations were always below . mg/l. in terms of sputum killing activity, g bd was more effective than mg bd against s. pneumoniae and m. catarrhalis , whereas no sputum samples were active on h. influenzae . conclusion: the optimal amoxicillin treatment schedule cannot be established on the basis of serum pharmacokinetics only. galmiche h, louchahi k, tod m, drugeon h, giroud jp, rouveix b. service de pharmacologie clinique, hopital cochin, paris, crepit , hopital avicenne, bobigny, service de microbiologie, hopital laennec, nantes, france background: cysteine-based mucolytics are commonly used in combination with antibiotics to treat patients with acute exacerbations of chronic bronchitis (aecb). they are also used to allow in vitro mic determination in sputum specimens. we conducted an in vitro and ex-vivo compatibility study designed to detect a possible interaction between mucolytics and antibiotics. methods: serial samples of bronchial secretions were collected from aecb patients and from healthy volunteers who received g of amoxicillin twice a day for days. two mucolytics were used to fluidify sputum specimens: , -dihydroxy- , -dithiolbutan (digest-eur † ) and acetylcysteine ( % solution). amoxicillin was assayed using a chromatographic system with fluorometric detection. each sample was also tested in a microbiological assay. results: amoxicillin could not be detected in the presence of the mucolytic agents. conclusions: this mucolytic Á/amoxicillin interaction may be explained by amoxicillin fixation to fluidified mucoproteins, and should be taken into account when assessing antibiotic efficacy in vivo. del fiol fs a , ferro c b , albuquerques et b . a university of sorocaba, pharmacology, sorocaba, brazil , b uniso, school of pharmacology, sorocaba, brazil physicians frequently recommend that macrolides should be administered with milk to decrease the discomfort they cause. thus the objective of this study was to verify the interference of milk in the absorption and distribution of erythromycin (eryt); clarithromycin (clar); roxithromycin (roxi) and azithromycin (azit). forty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their backs for granulomatous tissue formation. one week later the animals were divided into groups that received the drugs eryt, clar, roxi and azit with and without milk ( . ml/kg [ca'/'/] / . mg/ml). the animals were sacrificed and the serum and tissue concentration of the drugs was investigated. there was no reduction (pb/ . tukey-kramer) in the serum and tissue concentration in the presence of milk for azit and clar. there was a % reduction for roxi in the serum concentration in the presence of milk ( . / . and . / . mg/ml), but no alteration in the tissue concentration. there was a % reduction for eryt (p b/ . ), in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and a % reduction in the tissue concentration. the milk decreased the effectiveness of treatments with erythromycin and roxithromycin and the bioavailabilities of this macrolides were affected by co-administration with milk. del fiol fs a , souza gp b , duzzi mr b . a university of sorocaba, pharmacology, sorocaba, brazil , b uniso, school of pharmacology, sorocaba, brazil the degree to which tetracyclines are absorbed differs greatly. this absorption is impaired by the concurrent ingestion of divalent and trivalent cations. thus the objective of this study was to investigate the interference of milk in the absorption and distribution of tetracycline (tetr), oxytetracycline (oxyt), minocycline (mino) and doxycycline (doxy). forty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their back for granulomatous tissue formation. one week later the animals were divided into groups that received the drugs: tetr; oxyt; mino; and doxy with and without milk ( . ml/kg [ca'/'/] / . mg/ml). the animals were sacrificed and the drug concentrations in the serum and tissue were determined. there was no reduction (pb/ . tukey-kramer) in the serum and tissue concentrations in the presence of milk for mino. there was a % reduction (p b/ . ) for doxy in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and % in the tissue concentration. for oxyt, there was a reduction of % (pb/ . ) in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and % in the tissue concentration. the tetr results show a . % reduction (p b/ . ) in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and % in the tissue concentration. milk decreased tetracycline bioavailability and effectiveness. isotopic studies with oxine labelled platelet. platelet kinetics in thrombocytopenic malaria patients pm introduction : thrombocytopenia is a common feature in human malaria ( ). excessive splenic platelet pooling has been suggested to play a role in uncomplicated cases of malaria, but a moderately shortened platelet life span during the period with decreasing parasitaemia seems the most plausible cause of the frequently observed thrombocytopenia ( , ). consumption coagulopathy, eventually manifested as disseminated intravascular coagulation, has been described in malaria ( ). in uncomplicated malaria, however disseminated intravascular coagulation is rarely found ( ). results: in malaria patients the sequestration was not different to normal. platelet half-life was reduced in patients with p. falciparum malaria to Á/ h (normal Â/ days). in one patient with p. vivax malaria platelet half live was . h. conclusion: no significant differences in the sequestration of platelets when compared to healthy individuals could be detected by in-labelled platelet scintigraphy. especially, no enhanced splenic sequestration, as previously expected, was the cause of the thrombocytopenia. therefore, other mechanisms than sequestration are responsible for the dramatically reduced life span of the platelets during acute malaria. zaharanka ag, rozhdestvensky da. vitebsk state medical university, vitebsk, belarus aim: the studying of chromatingeterogenic test (ct) results in sperm of subjects taking doxycycline (d) and some macrolides (erythromycine (e), jozamycine (j), and azytromycine (a)) in moderate therapeutic doses. methods: forty healthy volunteers ( Á/ years) were studied. daily dose of d was . ; e was administered in dose . four times per day days; j */ . before meals twice daily days; a */ . before meals once daily days. ct for evaluation of dna condition in human spermatozoids was performed before treatment (twice), on the th and th days of treatment, as well as after and months after treatment course completing. results: ct data analysis revealed that the mean amount of defective spermato-zoids before treatment was . '/ . %. by the th day of d treatment the index of de-natured dna was . '/ . % (pb/ . ), by the th day */ . '/ . % (pb/ . ). one and months after the d treatment course the amount of generative cells with denatured dna was . '/ . and . '/ . %, respectively (p b/ . in both case). under e treatment the amount of defective spermatozoids changed as . '/ . ( th day), . '/ . ( th day), . '/ . (after month), and . '/ . % (after months) (pb/ . in any case). under a using the ct results at the same control points were . '/ . , . '/ . , . '/ . , . '/ . % (pb/ . in any case); and under j treatment */ . '/ . , . '/ . , . '/ . , . '/ . %, respectively (p !/ . in any case). conclusions: the data obtained permit to conclude that d demonstrates the high level of toxicity to male generative cells. this effect preserves during months after the course of d treatment. objective: to study the effect of aggressive isolation and decontamination measures to control an outbreak of multi-resistant acinetobacter baumanii (mr-ab) in an icu. the outbreak: the index case was transferred from a mediterranean hospital, directly into an open-plan -bedded icu, with severe injuries to his head and thorax. he died shortly after admission. sputum, bronchoalveolar lavage fluid, blood cultures and a chest drain swab grew mr-ab, resistant to ampicillin, co-amoxiclav, aztreonam, amikacin, ceftazidime, cefotaxime, cefuroxime, ciprofloxacin, gentamicin, meropenem, piptazobactam, tobramycin and sensitive only to colistin. within days, mr-ab was isolated from two further icu patients. all isolates demonstrated identical antimicrobial susceptibility profiles. the icu was closed to admissions and thoroughly cleaned. all patients were isolated and their contacts screened. the icu was reopened, however, mr-ab was isolated from a fourth patient. this patient was isolated, the icu closed, for a second time, thoroughly cleaned, and all contacts isolated until discharge. all subsequent patients screened were negative for mr-ab conclusion: this illustrates the importance of aggressive isolation measures and thorough supervised cleaning in control of an outbreak, and the need to screen patients for resistant bacteria before admission to the intensive care unit in a general hospital. extended spectrum beta-lactamase-positive bacteria isolated in neonatal intensive care unit pm sandorcinova z a , siegfried l a , kmetova m a , viragova s b . a institute of medical microbiology, faculty of medicine, university of p.j. safarik, kosice, slovakia , b hospital, kosice-saca, neonatal intensive care unit, kosice, slovakia extended-spectrum beta-lactamases hydrolyse all penicilins, cephalosporins, including third-generation cephalosporins and aztreonam. esbl are predominantly produced by klebsiella spp. but may be presented in other enterobacteriaceae, too. the aim of present study was to investigate the occurrence of esbl-producing bacteria isolated from patients hospitalized at the neonatal intensive care units (icu). fifty escherichia coli and klebsiella spp. were isolated from rectum of patients hospitalized at the neonatal icu. the mics of antimicrobial agents were determined by the standard agar plate dilution method according to the nccls guidelines. for screening of esbl production we investigated strains showing reduced susceptibility (mic equal and/or more than ml/l) to at least one of third generation cephalosporins. esbl production was detected by double disk synergy test (ddst), e -test for esbl, and pcr employing specific primers for the presence of blashv and blatem genes. by using ddst and e -test, among e. coli isolates, an expression of esbl was detected in the three by the former method, while among klebsiella spp. isolates, a production of esbl was found in the two by the latter method. in esbl-positive e. coli strains the presence of blatem genes fragments was detected while in esbl-positive klebsiella spp. genes encoding for shv-type beta-lactamases were found. isolation of staphylococci from wound swabs and their susceptibility to antibiotics pm markov mij, shopovski e, despotovski v, angelevski a, nikolovski b. military hospital, microbiology, skopje, the former yugoslav republic, macedonia purpose: to determine percent of staphylococci from wound swabs and to establish their susceptibility to antibiotics. material and method: the wound swabs have been evaluated with standards microbiological techniques. bacteria have been identified with strips from the 'atb expression' system. the susceptibility testing has been performed with strips with dilution technique, read by the same system. results: during the last years ( Á/ ), a total of wound swabs have been evaluated in the military hospital in skopje. positive bacterial finding have been determined in ( %) swabs with isolated bacterial species, from which ( . ) were staphylococci: staphylococcus aureus ( . %) isolations ( methycillin resistant s. aureus ); s. epidermidis ( . %); s. haemolyticus ( . %); s. hominis ( . %); s. chromogenes and s. lugdunensis nine ( . %); s. intermedius , s. lentus , s. sciuri and s. warneri all with seven ( . %) isolations. the susceptibility of s. aureus was to: penicillin %, ampicillin %, amoksicillin/clavulonic acid %, ceftazidime %, gentamicin %, tetracycline's %, erythromycin %, lincomycin %, ciprofloksacin %, cotrimoxazole %, vancomycin %, fusidic acid %, cefixime %, and azitromicin %. conclusion: in our study most frequently isolated bacteria from the wound swabs were staphylococci, especially s. aureus . susceptibility, except for the penicillin ( %), was high to other antibiotics. the study went on from january to december . at maribor teaching hospital, staphylococcus aureus isolates were collected. in , ( %) and in , ( . %) were mrsa. mrsa were recovered from routine clinical material and from surveillance swabs (nose, throat, skin). for isolation, conventional culture media were used and for surveillance swabs mrsa-screening plate (manitol salt agar with % oxacillin) and trypticase soy broth with . % nacl were added. s. aureus was identified by catalase, dna-se and tube-coagulase test. antibiotic susceptibility was determined by the disk-diffusion method according to ncci guidelines. all mrsa isolates were tested for sensitivity to the following antimicrobials: gentamicin, netilmicin, ciprofloxacin, erythromycin, chloramphenicol, tetracycline, cotrimoxazol, vancomycin and clindamycin. it was found that all mrsa isolates were sensitive to vancomycin and partially or totally resistant to the rest. there were no important differences between the years and . our mrsa isolates were completely ( %) susceptible to vancomycin, but resistant to the other antimicrobials in use to some extent. although the monitoring of mrsa susceptibility to antimicrobials once a year did not show any important change in antimicrobial resistance, the periodical monitoring of mrsa susceptibility to antimicrobials and revaluation of current treatment regimens of mrsa infections is necessary. staphylococcus aureus strains with reduced susceptibility to vancomycin among clinical isolates in university hospital in warsaw pm the visa and especially h-visa are very difficult to be found in the routine laboratory. in our investigations we examined of s. aureus strains isolated and stored in our laboratory for several last years ( Á/ ) . most strains were isolated in , and some in . for all staphylococcal strains mrsa as well as mssa the mics of vancomycin were performed by the standard dilution method. among strains isolated in the last year three strains were recognized as visa (mic values were mg/l). the frequency of visa was . %. in the aim of founding the h-visa strains the population analysis was used. for this analysis all strains growing on the concentration mg/l of vancomycin from the inoculum were chosen. it was strains, but only of them were recognized as h-visa. the frequency of h-visa among investigated strains was about %. most but not all of the h-visa and all visa strains were methicillin resistant. in vitro activity of vancomycin, teicoplanin and oxacillin against staphylococci isolated from patients of surgical intensive care unit pm kucukates e, karayel n, kansiz e. institute of cardiology, university of istanbul, istanbul, turkey objectives: oxacillin-resistant staphylococci have emerged as a major infection control problem in our hospital. the aim of this study was to evaluate the in vitro activity of vancomycin, teicoplanin and oxacillin against staphylococci. material and methods: this study was performed between january and december , at university of istanbul, institute of cardiology. the antimicrobial susceptibilities of staphylococci isolates for vancomycin, teicoplanin and oxacillin have been investigated by e -test according to nccls guidelines. results: fifty-five ( . %) of clinical isolates were staphylococcus aureus . one hundred and twenty-four ( . %) of clinical isolates were coagulase negative staphylococci (cns). none of staphylococci isolates were resistant to vancomycin. but three of cns isolates were intermediate and six of cns isolates were resistant to teicoplanin. twenty-eight ( . %) of s. aureus were resistant to oxacillin. ninety ( . %) of cns isolates were also resistant to methicillin. conclusions: nosocomial staphylococcal infections, especially in intensive care units increase day by day. staphylococcal infections are a major problem in many hospitals. according to our experiences the rate of oxacillin resistant staphylococci isolates in our hospital has also increased. methicillin-resistant staphylococcus aureus (mrsa) is a clinically significant pathogen because mrsa is resistant to many kinds of antibiotics and causes nosocomial infections around the world. the antiseptics are used for prevention of infections by mrsa. antisepticresistant mrsa strains have been isolated from clinical specimens. antiseptic resistance genes confer resistance to many kinds of drugs structurally and the resistance mechanism is the energy-dependent drug efflux system. in addition, the fluoroquinolone (fq)-resistance gene, nora , confers also resistance to many kinds of antiseptics. we studied the relation of the susceptibility to antiseptics and fqs of mrsa strains isolated in japan. a total of strains of mrsa were isolated from hospitals in japan from to . acriflavin (af), acrinol, benzethonium chloride, benzalkonium chloride and chlorhexidine digluconate were used as the antiseptics. norfloxacin and sparfloxacin were also used in this experiment. the mic was determined by agar double-dilution method as recommended by the nccls. about % of mrsa showed resistance to af (mic: !/ ug/ml). no strain was resistant to a specific antiseptic. fq-susceptible strains were susceptible to all antiseptics. this study showed that antiseptic-resistant mrsa are widely spread at hospitals in japan. the drug of choice in treatment of serious infections caused by mrsa was still vancomycin, however sometimes failures were observed, especially in monotherapy. some conflicting are present in literature about an effect of combined action of vancomycin and betalactams. in the present work, the common effect of vancomycin and methicillin against chosen staphylococcus aureus strains was examined. the investigated strains were characterized as visa, h-visa and clones obtained from h-visa in population analysis. two methods were performed: e -tests with methicillin and vancomycin placed on the media supplemented with the second antibiotic and the chessboard micro-analysis with increasing concentrations of both antibiotics. the fic indexes were calculated for different combinations of concentrations. on the basis of the fic indexes it was shown that the simultaneous action of vancomycin and methicillin was synergistic in all examined strains visa and h-visa, but only in appropriate concentrations. in different combinations the observed effect was addition or indifference. antagonism was never observed. the synergistic effect was not observed in the case of standard s. aureus strain sensitive to methicillin. supplementation of media with % of nacl substantially decreased the observed effect. incidence of antibiotic resistance in staphylococcus aureus strains in hungary with special reference to mrsa pm ghidán Á , maró di c, csukás z, kamotsay k, szabó d, ostorházi e, rozgonyi f. institute of medical microbiology, semmelweis university, budapest, hungary between january and december , a total of staphylococcus aureus strains isolated from patients admitted to the clinics of the semmelweis university were examined for antibiotic sensitivity with the disc diffusion test. resistance to individual antimicrobials were as follows: penicillin %, oxacillin %, erythromycin %, ciprofloxacin %, amikacin %, netilmicin %, tobramycin %, gentamicin %, clindamycin %, mupirocin %, tetracyclines %, chloramphenicol %, teicoplanin % and vancomycin %. all mrsa were b-lactamase producer. they showed coresistance to erythromycin ( %), ciproflxacin ( %), amikacin ( %), netilmicin ( %) and mupirocin ( %). multiple resistant mrsa strains to mupirocin'/tetracyclines'/chloramphenicol amounted to . %. triple resistance to oxacillin'/ciprofloxacin'/netilmicin was %. the detection of meca gene by pcr in randomly chosen mrsa qualified with mg oxacillin disc resulted in only % meca positivity indicating that the traditional disc diffusion test overestimates the frequency of mrsa strains particularly in such an environment where the usage of penicillins and cephalosporins is so liberal as in hungary. consequently, the selective pressure for blactam-resistance and b-lactamase induction exists everywhere. this conclusion is coherent with the relatively low frequency of multiple resistant mrsa strains and urge the need of a routinely available genetic method to apply for mrsa detection. objectives: the main objectives of this study were to monitor antibiotic resistance, identify new/emerging resistance mechanisms at an early stage, prevent their dissemination, early detection and prevent the outbreaks. methods: our laboratory used antibiotic disc sensitivity testing methodology (nccls ) . zone sizes were measured objectively using a biomic automated radius zone reader. results: throughout years (january till december ) we have surveyed organisms collected from outpatient departments ( , . %), radio-oncology department ( , . %), medical department ( , . %), obg department ( , . %), surgical-oncology non icu department ( , . %), icu department ( , . %). from ( %) strains of enterobacteriaceae ( . %) were resistant to th generation fluoroquinolone and ( . %) strains were esbl positive. from ( %) strains of staphylococcus aureus were only five ( . %) strains resistant to methicilin (mrsa). we collected ( %) strains of enterococci, whereabout only two ( . %) were resistant to glycopeptides (vre). from ( %) strains of pesudomonas aeruginosa , ( . %) were resistant to aminoglycosides. conclusions: national restrictive antibiotic policy hand in hand with local hospital antibiotic policy and regular rotation of antibiotics used in prevention and treatment on all departments is leading in our case in positive situation in antibiotic resistance in comparing with other slovakian and european centers. antimicrobial resistance of nosocomial strains of staphylococcus aureus pm dekhnich av, stratchounski ls, edelstain ia, narezkina ad. institute of antimicrobial chemotherapy, smolensk, russian federation purpose: to determine the antimicrobial resistance of staphylococcus aureus causing nosocomial infections in smolensk regional hospital. results: a total of s. aureus strains isolated during Á/ were studied. antimicrobials tested included oxacillin (oxa), erythromycin (ery), clindamycin (cli), gentamicin (gen), vancomycin (van), linezolid (lnz), tetracycline (tet), chloramphenicol (chl), rifampicin (rif), fusidic acid (fus), trimethoprim/sulfamethoxazole (ts), ciprofloxacin (cip), mupirocin (mup), quinupristin/dalfopristin (qd). susceptibility testing and its interpretation were performed by agar dilution according to nccls guidelines where applicable. results are presented in the conclusions: the most active antimicrobials were vancomycin, linezolid, quinupristin/dalfopristin, fusidic acid, mupirocin, followed by co-trimoxazole, rifampicin. beta-lactams, macrolides, lincosamydes, tetracyclines and chloramphenicol should not be used for the treatment of nosocomial s. aureus infections. we investigated all staphylococcal infections within years among neonates hospitalized for infection in the neonatal icu in a tertiary neonatal referral center. univariate analysis, to assess risk factors for neonates infected with staphylococcus aureus ( ) vs. without s. aureus ( ) was performed. from the total number of cases, in cases s. aureus was isolated from various samples; in cases from blood cultures, in cases from urine, in cases from eye swabs and in cases from gastric content (no significant differences in comparison with control group). colonization with s. aureus , was a predictor of infection: nasal swabs, throat swabs, ear swabs, skin swabs and umbilical swabs were significantly more commonly observed in neonates infected with s. aureus , than with other infections. etiological analysis showed that co-pathogens escherichia coli and viridans streptococci were significantly more frequently associated with neonatal infection caused by s. aureus , in comparison to other organisms. according to localization of infection site, conjunctivitis and thrush stomatitis was the commonest s. aureus neonatal infections. outcome was similar to other infections and without any significant differences between both groups. mortality was similar to other infections, probably because: initial therapy in our centre contains an antistaphylococcaly active agent (cefuroxime or cefotaxime plus aminoglycosides). morozova ot, semina na. laboratory of hospital infections, central research institute of epidemiology, moscow, russian federation purpose is to study the role of enterococcus spp. in the aetiology of nosocomial infections among the patients of the childrens clinical hospital and susceptibility of these strains to antibiotics. methods: the strains of enterococci were isolated from patients with hospital infections in Á/ . results: the aetiological structure of enterococcus -infections showed the predominance of skin and soft tissue infections ( . %), urinary tract infections ( . %), bloodstream infection ( . %), pneumoniae ( . %), infection of central nervous system, gastrointestinal tract, eye, surgical wound infections were of rare incidence ( Á/ . %). various nosological forms of infections were caused more often by e. faecalis than e. faecium ( . , . %). the antibiotic resistance to ampicillin and other beta-lactams occured in % of e. faecium isolates, but all strains of e. faecalis were susceptible to these drugs. high-level gentamicin resistance demonstrated e. faecalis isolates */ . %, e. faecium */ %; and high-level streptomycin resistance showed e. faecalis */ . %, e. faecium */ . %. all the e. faecalis were active against fluoroquinolones, but e. faecium were resistant in . %. there were no vancomycin resistant enterococci. conclusion: e. faecalis predominated in the aetiological structure of nosocomial infections due to enterococcus spp. antibiotic resistance patterns for two species of enterococci were different, all the strans were susceptible to vancomycin. evaluation of antimicrobial resistance of enterococcus spp. experience of years ( Á/ ) pm lopez-barba j, jesus de la calle i, solino-ocañ a i, rodríguez-iglesias m, perez-ramos s. microbiology laboratory, puerto real university hospital, cádiz, spain objective: determination of quantitative changes in antimicrobial resistance of enterococcus isolated from clinically significant not urinary samples of patients remitted to the laboratory of microbiology during a years period ( Á/ ) . material: the period of the study was comprised between and . the samples has been processed for the isolation of enterococcus following conventional methods. were isolated enterococci strains. the identification and susceptibility to antibiotics have been performed in automated system microscan(c) dade-behring(c) through panels combo cgp. the data were processed by the statistical system statgraphics plus . . results: of the enterococcus , have been identified e. faecalis and e. faecium . the resistance is shown in table . conclusions: the resistance to va and tei of e. faecalis remains through last years ( Á/ %). the high resistance to erythromycin and tetracilin ( !/ %) and the resistance ( Á/ %) to quinolones, antibiotics all of them used in community-acquired infections justify the susceptibility testing to the clinical strains isolated of this group of microorganism. in e. faecium the antimicrobial resistances was high and increasingly to imipenem, meropenem, erythromycin and quinolones. characteristics of strains e. faecium colonizing the neutropenic patients pm abbassi ms, achour w, gréco a, ben hassen a. laboratory of bone marrow transplant center, tunis, tunisia digestive colonization by enterococcus faecium in the neutropenic patients under gut decontamination is important. seventeen multiresistant strains of e. faecium isolated from stools of seven neutropenic patients were the target of an epidemiological analysis through the determination of the mics of amoxicillin, gentamicin, vancomycin, the transferability gentamicin resistance to the recipient strain e. faecalis jh - by filter-mating assay, analysis of plasmid profiles of e. faecium -strain and of transconjugants and the amplification by pcr of the gene aac( ?)-aph( ??) coding for the bifunctional enzyme by using primer m who gives a fragment of kb. among the seventeen strains, eleven had the same antibiotype a , had a gentamicin mic!/ mg/l. the mic of the amoxicillin was of mg/l. all the strains were sensitive to vancomycin. ten strains harbored a plasmid of kb transfered at a frequency of . Á/ , also found in gentamicin-resistant transconjugants. however, strains belong to nine distinguished plasmids profiles. all high-level gentamicin resistant-strains had a positive pcr amplification of the aac ( ?)-aph ( ƒ) gene. the features of the studied strains establish their endogen origin, specific for every patient, sharing only high-level resistance to gentamicin. gut decontamination treatment with gentamicin enhance either the spread and the preservation of easy-transferable plasmid carrying genetic transposable element. frequency and antibiotic resistance of bacteria isolated from patients suffering infectious complications following the implantation of prosthetic devices pm kristó f k, rozgonyi f. institute of medical microbiology, semmelweis university, budapest, hungary for patients with indwelling joint prosthesis, early recognition and prompt therapy for infection in any location may be critical to reduce the risk of seeding the joint implant heamatogenously. a year period ( ) a total of swabs of aspiration from patients with infectious complications following the implantation of prosthetic devices were cultured. cultivation and identification of the strains were performed by conventional methods and by vitek system (biomȇrieux) and susceptibility testing by disc diffusion. potentional pathogens were recovered in cases ( . %). gram positive cocci, in particular staphylococcus spp. proved to be the most commonly isolated bacteria. coagulase-negative staphylococcus (cns) was isolated more frequently ( %), followed by s. aureus ( %), enterococcus faecalis and e. faecium ( %), gram-negatives ( %), anaerob isolates ( . %). resistance to individual antimicrobials of s. aureus and cns were as follows: methicillin and %, clindamycin . and . %, fluoroquinolones and %. mupirocin resistant strains of s. aureus were not found, while . % were among the cns strains. our results could be essential for the rational selection of treatment at our orthopedic wards. results: in the analysed period the percentage of isolated enterococcus sp. strains among all non-repetitive clinical isolates in , and was . , . and %, respectively. cultured strains were identified as e. faecalis, e. faecium, e. gallinarum and e. avium . the most prevalent was e. faecium strains, isolated with a frequency of , and %, respectively. vancomycin-resistant strains were all identified as e. faecium and in they comprised % of all isolates of this species. conclusions: ( ) the frequency of isolation of enterococcus sp. in blood cultures of haematological patients remained relatively stable in Á/ . ( ) the predominant enterococcal species isolated from these patients was e. faecium . ( ) in we recorded for the first time an emergence of vancomycin-resistant e. faecium , which comprised % of all isolates of this species. kalai s, ben hassen a, achour w, greco a. laboratory of microbiology, national bone marrow transplantation center, tunis, tunisia from april to june , non-repeated strains of pseudomonas aeruginosa were isolated from immunocompromised patients. thirty-six percent of strains were isolated from abscess, % from blood culture and % from urine. susceptibility to antibiotic was studied by the routine disk diffusion method (ca-sfm). mics were determined using agar dilution to antibiotics ( b-lactams, four aminosides and two fluoroquinolones). serotyping of the different trains was performed using antisera to the international antigenic typing systems serotypes. the study showed % of resistance to c cochin port royal hospital, service de gynecologie-obstetrique site st vincent-de-paul, paris, france , d cochin port royal hospital, cclin, paris, france aims: to determinate risk markers of an outbreak of postpartum endometritis due to group a streptococcus . design: a case-control study using data collected with a structured form. setting: the cases of postpartum endometritis were diagnosed in the department of obstetric of the paris hospital network during days (december Á/january ). the group of controls consisted of women delivered in the same department during the same period. participants: cases (n / ) and controls (n / ). findings: cases had smoked more often during pregnancy ( vs. %; p / . ) and received more often immunosuppressive treatment than controls ( vs. %; p / . ). instrumental delivery has been needed more often for cases than controls ( vs. %; p / . ). cases had been hospitalized after delivery in a ward z of the department more often than controls ( vs. %; p / . ). they had been examined after delivery more often by a midwife x ( vs. %; p / . ) and a nurse y has provided care to cases and not to controls ( vs. %; p / . ). conclusion: smoking, receiving immunosuppressive treatment during pregnancy, and instrumental delivery were significantly associated with postpartum endometritis (pb/ %). a midwife and a nurse might be involved in the transmission of the infection. petoukhova i a , sokolova e a , dmitrieva n a , nummaev b b . a laboratory microbiology, cancer research center of russia, moscow, russian federation , b department of oncogynecology, cancer research centre, moscow, russian federation the aim of the study was to assess efficacy of perioperative ap. total pts were included. two hundred and one pts with cervical cancer (cc) undergone extensive hysterectomy, pts with cancer of vulva (cv)-extensive vulvoectomy, pts with ovarian cancer (oc) Á/ extensive/combined operations. fifty-one pts (group ) received ap with amoxicillin/clavulanate (am/cl) . g iv min prior to operation, then . g iv thrice per day for Á/ days. fifty pts (group ) received cefotaxime (ctx) g iv min prior to operation, then g four times per day for Á/ days'/metronidazole (mtz) mg two times per day for the same period. one hundred and forty pts were retrospective control (they received ii Á/iii generation cephalosporins or linkosamides only after operation). the rate of swi in pts with cc, cv and oc was . , , %, respectively; dwi */ . , and %, respectively; uti */ , , . %, respectively. the ap with am/cl was more effective compared to ctx'/mtz (swi */ vs %, respectively, pb/ . , dwi */ . vs %, respectively, p b/ . ). the rate of postoperative uti was equivalent in two groups ( vs %, p /n.s.). thus, ap with am/cl is preferrable option in extensive operations in og pts. fifty-eight patients were randomized into two groups. group , a treatment consisting of patients and group , consisting of patients as a control. the patients were at the age of / . and had had different surgical interventions with general anaesthesia from to h and accompanying copd ( %). artificial pulmonary ventilation was used in cases. in the early post-surgery period the patients of group were administered inhalation therapy, including ipratopium of bromide in the combination with fenoterol (atrovent, berodual) and ambrocsol (lazolvan) through a nebulizer. the inhalation therapy was not administered to the patients of group . under the influence of the inhalation therapy pulmonary ventilation and respiratory metabolism was restored faster in all the resuscitation patients (in group */by the end of the first h, in group */on the rd Á/ th day). the percent rate of pef was . '/ . and . '/ . , respectively. artificial pulmonary ventilation ended in . and . h, respectively. the time of the patients' stay in the resuscitation department was . days in group and . days in group . by the end of the st week pneumonia developed in one patient from group and in eight patients in group . aerosol therapy application accelerates medication delivery to the respiratory tracts, increases the local activity and effects good prophylaxis for surgical hospital pneumonia. variation in ethiology of early and late onset ventilator associated pneumonia pm nikolopoulos j, daganou m, michailidou m, karabela e, kavada k, retsou s, antoniadou a, rasidakis a. department of respiratory abstracts s failure and icu, sotiria general and chest disease hospital, athens, greece purpose: to compare the distribution of causative microorganisms, their susceptibility to antibiotics and outcome of 'early' and 'late' vap in a greek icu. methods and results: retrospective study of mechanical ventilated (mv) patients (pts) with early and late vaps during a -month period. diagnosis of vap was made by clinical, radiographic criteria and quantitative cultures of bronchial secretions. vap was diagnosed in pts ( %) out of consecutive admissions in icu. all pts before the development of vap received antibiotics. three episodes of vap ( . %) were developed before the th day of mv (early vap) and were caused: ( ) by multi-resistant acinetobacter; and ( ) by antibiotic-susceptible pseudomonas aeroginosa . in this group one pt died from septic shock related to vap and two pts survived. fourteen pts ( . %) developed vap after the th day of mv (late vap). five cases were caused by multi-resistant p. aeroginosa , two cases by mrsa, two cases by multi-resistant acinetobacter, two cases by susceptible to antibiotics klebsiella pneumoniae , and three were polymicrobial and caused by multi-resistant microorganisms (mrsa and gnb). four pts died ( . %) from septic shock related to vap, five pts ( %) died because of another cause and five pts ( %) survived. conclusions: early and late onset episodes of vap were caused by 'potentially drug-resistant bacteria'. p. aeroginosa as a cause of early vap was susceptible. mortality attributed to early and late vap was similar. antibiotic prophylaxis in oncological and major reconstructive orthopaedic surgery pm de biase p, ciampalini l, astone a, capanna r. azienda ospedaliera careggi, oncological and reconstructive centre, aoc, florence, italy during last year patients scheduled for oncological surgery or major reconstructive procedures were randomised to either vancomycin or teicoplanin prophylaxis. prophylaxis was performed with either vancomycin g i.v. twice daily or teicoplanin once daily i.v. two hundred patients were included. four patients did not agree the study protocol and were excluded. we treated patients with teicoplanin and patients with vancomycin. out of the patients were operated for oncological disease, while the remaining underwent major orthopaedic procedures. we experienced cases of red man syndrome, and five cases of moderate hypotension. five patients had postoperative complications: two deep venous thrombosis, one pulmonary embolism, two postoperative haematoma. in five patients we observed a wound dehiscence; two of these patients showed clinical sign of ssi and microbiological examinations were positive for mrsa. one patient recovered from infection with medical therapy, while the other patient showed a local tumour recurrence and was amputated at the thigh. at last surgery infection was still present clinical and at microbiological examination. in conclusion we had an infection rate of . % which is comparable to the infection rate of a 'clean' surgery in patients with normal risk of infection. teicoplanin showed lower toxicity, has a longer half-life and has a simpler way of infusion and it is our current choice in high risk surgery. injuries with contaminated sharp articles in health care workers in general hospital celje, slovenia pm lesnicar g, sibanc b. department of infectious diseases, medical center celje, celje, slovenia in a prospective study carried out from january till june , we registered subcutaneous injuries with sharp objects, mostly in nurses and cleaning service workers. in % of cases the incident occurred outside the hospital, in persons who were not medical workers. in cases the injury causing object was a needle that had been used in known patients, of which were hepatitis b positive. fifty-five ( . %) of the injured health workers had been previously vaccinated against hepatitis b; the protective antibodies to hepatitis b in the blood were found in / ( . %) health workers only, while the tests for antibodies to hepatitis c and hiv were negative in all cases. following the incident, the majority of the injured persons, i.e. , were vaccinated against hepatitis b, while persons ( . %) also received passive prophylaxis with human immunoglobulin against hepatitis b. none of the injured persons have developed the disease or showed evidence of sero-conversion. in the year the general as well as specific preventive measures practised in our hospital became more rigorous. thus, approximately % of our health workers at risk have already been vaccinated against hepatitis b. to improve measures preventing dissemination of multidrug-resistant bacteria (mrb), a cross-sectional survey ( ) was conducted to analyse healthcare workers' (hcws) isolation precaution knowledge for mrb infection at investigation and outpatient departments excluding four declaring not to be involved in care to mrb patients (emergency, obstetrics, nuclear medicine, and bacteriology). two hundred and eight hcws answered ( % of the paramedical staff, % of the physicians). thirty-three percent of them reported they do not know frequently or always the patient mrb status. they ( %) wish mrb status to be mentioned on the test form or on the advice request letter. mrb patient visit or test was appropriately timed in % answers. gowns ( %) or masks ( %) use were not systematically reported. other hcws ( %) reported better isolation precaution knowledge than physicians ( %) and nurses ( %) than investigation assistants ( %). physicians declared lower compliance with use of gowns, gloves or draw-sheets than other hcws. they had also lower education in isolation precautions and were less interested in education programs. this study suggests the necessity to improve mbr infection information. physicians and investigation assistants seem to be insufficiently aware of hospital infection control. therefore, education strategies targeted at physicians and investigation assistants working at outpatient and investigation departments should be developed. outbreak of clostridium difficile -associated diarrhoea in infectious disease department: risk factors and hygiene measures assessment pm henoun loukili n, martin m, remy v, hansmann y, christmann d. hôpitaux universitaires de strasbourg, maladies infectieuses et tropicales, strasbourg, france (shock)'/ * (bedridden status)'/ * (age !/ years)'/ * (previous antibiotic treatment) and points (women) / * (shock)'/ * (bedridden status)'/ * (age !/ years)'/ * (immunosuppression). the vast majority of patients ( and % of males and females, respectively) could be classified in the subgroups with lower scores (six points or less) which had a very limited risk of death ( Á/ . and Á/ . % for men and women, respectively), whereas for patients in the highest score subgroup ( points or more), the risk was % for men and % for women. conclusion: risk stratification of patients with ap is possible from simple clinical variables available on admission. procalcitonin (pct) and c-reactive protein (crp) for differentiation of systemic inflammatory response syndrome (sirs), sepsis and severe sepsis pm lupse m a , ursu l b , slavcovici a a , carstina d a . a clinical department, teaching hospital of infectious diseases, cluj-napoca, romania , b laboratory department, teaching hospital of infectious diseases, cluj-napoca, romania objectives: to evaluate the value of pct in the differentiation of patients with sirs, sepsis, severe sepsis and bacteremia in comparison to crp. design: prospective study including patients who meet criteria for sirs, sepsis or severe sepsis (consensus conference of the accp/ sccm) admitted over -month period. patients and method: a total of patients were included: eight with sirs, with sepsis and with severe sepsis. sixteen from patients had bacteremia. pct and crp were evaluated in the first h after admission: pct by brahms pct-q test and crp by turbidimetric assay. the sensitivity, specificity, predictive value of different cutoff points for crp and pct were determined. results: with a cut off point of . ng/ml for pct and . mg/dl for crp sensitivity and specificity for sepsis were %, respectively % (ppv . , npv . ) and %, respectively % (ppv . , npv ). a cutoff point of ng/ml for pct accurately predict severe sepsis (sensitivity %, specificity %, ppv ). a pct level of at least ng/ml was a good predictor for bacteremia (sensitivity %, specificity %, ppv . and npv ). conclusion: pct is a good discriminating marker to characterize the level of inflammation caused by infection and can predict bacteremia . giamarellou h a , aoun a b , klastersky j b , anagnostopoulos n c , galani l c , grecka p c , panaretou e c , papageorgiou e c , repoussis p c , syrseloudis pct has been considered as a useful diagnostic marker in neutropenic patients with bacteremia and/or severe sepsis (giamarellos-bourboulis ej et al. clin infect dis ; : ) . in an attempt to define its value in the diagnosis of localized infections in neutropenic hosts, daily determinations of pct and of c-reactive protein (crp) were performed before and after the onset of fever in subjects male and female aged . / . years with various haematologic malignancies (aml , nhl , mds , all ) developing neutropenia ( b/ pmns/mm ) after chemotherapy. thirty-three patients were presented with fever of unknown origin (fuo) and with localized bacterial infections (lbi; pneumonia , acute pyelonephritis , soft tissue infections , acute pharyngitis ). pct was determined by an immunoluminometric assay and crp by nephelometry. it is concluded that febrile neutropenia followed by a localized bacterial infections is accompanied by significantly higher levels of pct than in case of fuo ( . / . vs . / . ng/ml). similar differences are not observed with crp, which lacks the appropriate specificity. our study included patients who were categorized as having proven ( ), probable ( ), or possible ( ) systemic fungosis according to eortc criteria; showed no sign of infection, and were used as controls. blood samples were received on the st, rd, and th day from the onset of signs of a fungal infection, and then twice a week. pct levels were determined by an immunochemioluminent assay, and candida and aspergillus antigen levels by elisa. in only five patients pct indicated early signs of infection, albeit at barely detectable limits. six patients, however, showed significantly increasing titres preceding time of death. positive antigens titres were observed only in patients who had proven or probable systemic fungosis. only half of the control group had negative antigen titres; a high rate of false negatives was also observed. both pct and antigens titres increased in parallel in / patients with unfavorable outcome. pct and antigens titres cannot reliably indicate early diagnosis of systemic fungal infections although may be used as a prognostic tool of severity. lactulose, a factor that decreases endotoxaemia, in obstructive jaundice? pm koutelidakis im a , papaziogas v a , makris i a , giamarellos-bourboulis ej b , giamarellou h b , papaziogas t a . a thessaloniki med school, nd surgical clinic, thessaloniki, greece , b th department internal medicine, athens medical school, athens, greece bacterial translocation is a process implicated in the pathogenesis of spontaneous peritonitis. in order to evaluate the impact of lactulose administration on systemic endotoxaemia, obstructive jaundice was induced in rabbits by common bile duct ligation. animals were divided into two groups, group a of five rabbits not receiving lactulose and group b of six rabbits, which received . ml/kg of lactulose orally by an oral catheter. blood was collected daily, before and after operation for a total duration of four days. samples were applied for culture and for determination of endotoxins (lps) by the lal qcl- assay. concentrations of lps (mean /sd) of group a were . / . , . / . , . / . and . / . eu/ml on the st, nd, rd and th day, respectively. respective concentrations of lps (mean /sd) of group b were . / . , . / . , . / . and . / . . all blood cultures were sterile in both groups. differences activity of linezolid against nosocomial strains of staphylococcus aureus in russia: results of multicentre study pm were included in the study. antimicrobial susceptibility testing was performed by agar dilution method in accordance with the nccls recommendations. all tested strains including mrsa strains ( . % of all strains) were found to be susceptible to linezolid with the mic ranged from . to mg/l. both mic and mic were mg/l. conclusions: linezolid had excellent in vitro activity that was not affected by resistance to other classes of antimicrobials susceptibility to antiseptics of mrsa isolated in japan during Á % to piperacillin, % to ceftazidime, % to cefepime, % to imipenem, % to amikacin and % to ciprofloxacin. fiftythree percent of p. aeruginosa strains were multiresistant ( strains) and were isolated in patients. wild phenotype to b-lactams was observed in % of strains. the most frequent b-lactams resistance phenotypes were: cephalosporinase over production ( %) and penicillinase ( %). imipenem, ceftazidime and piperacillin-tazobactam were the most active b-lactams (mic of . and mg/l, respectively) these results showed high rates of antibiotic resistance and predominance of o serotype in multiresistant strains compared to the o serotype in europe. infectious complications sustained by stenotrophomonas (xanthomonas ) maltophilia in hiv at present, very limited informations are available about s. maltophilia infections in the setting of hiv disease. patients and methods: a retrospective survey of clinical and microbiological records of hiv-infected patients referring to out tertiary care centre between and was performed, in order to identify all episodes of s. maltophilia infections, and analyze its epidemiological, clinical, and microbiological variables. results: sixty-one episodes of s. maltophilia infection were observed in patients: sepsis/bacteraemia in cases ( . %), lower airways infection in five, urinary tract infection in four, pharyngitis in two, lymphadenitis and liver abscess in one case each. forty-seven out of episodes of s. maltophilia infections ( %) occurred as nosocomial disease, generally in association with advanced immunodeficiency, neutropenia, instrumentation, and prior antimicrobial therapy. bacterial isolates showed an elevated resistance profile against many betalactam compounds, aztreonam, imipenem, and aminoglycosides. conclusion: s. maltophilia represents an emerging opportunistic pathogen in hiv-infected patients extended spectrum beta-lactamases producing germs in intensive care units pm university of medicine and pharmacy 'victor babes ', microbiology, timisoara, romania injury and complications lasted months, demanded for surgical procedures and total cost was comparison of different methods for detection of extended spectrum beta lactamases (esbls) and their genetic relatedness among enterobacteriaceae clinical isolates in a research medical institute pm methods: one hundred out of isolates that were screened positive for esbls were tested with double disk synergy test (ddst), three dimensional test (tdt), e -test-esbl and vitek-esbl test. pulsed field gel electrophoresis (pfge) analysis was applied to esbls; five klebsiella pneumoniae and eight escherichia coli . results: revealed the prevalence of esbls in . % of clinical isolates. the sensitivities of the ddst, tdt, e -test and vitek were , , . and . %, respectively. in the ddst, aztreonam was the most sensitive indicator ( . %). pfge demonstrated that % of k. pneumoniae were derived from a single clone whereas . % of e. coli isolates were derived from two different clones. non-clonal origin was demonstrated in % of k. pneumoniae and . % of e. coli . conclusion: there is an increased prevalence of esbls. the ddst is the most sensitive, practical and cost effective diagnostic method reliable for routine use in our laboratory. both clonal spread and plasmid dissemination contributed to the concurrent nosocomial outbreaks caused by esbl-producing k severe nosocomial infections due to stenotrophomonas maltophilia pm the teaching hospital of infectious diseases total og pts after extensive hysterectomy ( pts), extensive vulvoectomy ( pts) and extensive/combined operations for ovarian cancer ( pts) were analysed. ic developed in pts. one hundred and sixtyseven pts had no ic. twenty-eight of rf analysed were independent rf of ic. most important included: age !/ years (p / . ), grade Á/ obesity (p / . ), diabetes mellitus (p / . ), diagnosis of cervical cancer (p / . ), history of pre-cancer of vulva postpartum endometritis due to group a streptococcus : a case-control study pm unite operationnelle d 'hygiene all isolates were sensitive to vancomycin. among gram-negative bacteria klebsiella spp. was isolated in . % of cases, acinetobacter baumanii in . %, enterobacter spp. in . %, providencia spp. in . %. of the klebsiella spp. isolates % were resistant to amikacin, % to cephalosporins, % to piperacillin/ tazobactam. all were sensitive to imipenem. of the a. baumanii isolates % were resistant to amikacin, aztreonam, cefoperazone, cefotaxime, ceftriaxone, piperacillin; % to ampicillin-sulbactam, % to ceftazidime, and % sensitivity to imipenem antimicrobial activity of selected pharmacopoeial antiseptics analysed according to european standards pm european committee for standardisation approved several european standards (en), describing test methods establishing, whether an antiseptic has or does not have a bactericidal or fungicidal activity under the laboratory conditions defined by en. the aim of the study was to investigate, if some chemical compounds in concentrations recommended by polish pharmacopoeia for skin disinfection, comply european standards requirements. methods: basic bactericidal (en ) and fungicidal (en ) activity were investigated as well as bactericidal activity of products for hygienic and surgical handrub and hand wash used in human medicine (pren ). all methods and used neutralizers were validated. standard strains: staphylococcus aureus , pseudomonas aeruginosa , escherichia coli , e. hirae , candida albicans and a. niger were used, when en standards were evaluated. results: ethanol, izopropanol and n -propanol caused viable microbial count reduction required by ens in pharmacopoeial concentrations the purpose of the study: we collected bacteriological samples from adult and neonate patients who were admitted in intensive care units (icu) . the aim was to observe the colonization status with microbes that may have a nosocomial potential and to establish circulating phenotypes in icus. the results obtained from a total of samples strains of gram negative bacteria (enterobacteriaceae family) were isolated. fourteen strains showed extended spectrum beta-lactamases (esbl) phenotype (eight strains of klebsiella pneumoniae , three of escherichia coli , two of klebsiella ornithynolitica , one of klebsiella oxytoca ). we used both disc diffusion test (extended antibiotic susceptibility test and synergy test to visualize 'champagne stopper' pattern) and mini api † system.the conclusion reached: we put in evidence a massive colonization with germs that may have a nosocomial potential especially microbes that produce esbl ( . % from all enterobacteriaceae isolated) which implies a rational policy in prescribing antibiotics in hospitals from western part of romania.carbapenem activity against nosocomial gram-negative rods pm sawicka-grzelak a a , rokosz a a , meszaros j b , luczak m a . a department of medical microbiology, university medical school, warsaw, poland , b department of general and transplantation surgery, university medical school, warsaw, poland purpose: to determine a susceptibility of nosocomial gramnegative rods to carbapenems.methods: two hundred strains of gram-negative rods were cultured from clinical specimens from hospitalized patients (july Á/november ). identification of strains was performed in the automatic atb system (biomerieux, france). susceptibility of strains to carbapenems: imipenem and meropenem was determined with disc diffusion method according to nccls recommendations. esbl-producing strains were detected with double-disc synergy test (ddst according to jarlier et al., ) or a novel method of esbl detection (dd, diagnostic disc) according to appleton ( ) . two discs were applied in this test: with cefpodoxime (cpd) and with cefpodoxime/clavulanic acid (cd ) (oxoid, england) .results: one hundred and ten strains of enteric rods and strains of non-fermenting rods were cultured. twenty eight ( %) esblpositive strains were detected. carbapenems were active against % of enteric rods. the percentage of non-fermenting rods susceptible to imipenem was and to meropenem */ .conclusions: carbapenems: imipenem and meropenem demonstrated high activity against clinical strains of enteric rods. however, the antibiotics were less active against nosocomial strains of nonfermenting rods.inhaled antibiotics against multiresistant bacteria in bronchial secretions of icu patients: a preliminary report pm horianopoulou m a , kanellopoulou m b , paraskevopoulos i a , valakis k a , kyriakidis a a , lambropoulos s a . a intensive care unit, sismanoglio general hospital, athens, greece , b department of microbiology, sismanoglio general hospital, athens, greece purpose: the aim of this study was to assess the effectiveness of aerosolized ampicillin/sulbactam, ceftazidime and colistin, in icu patients with multiresistant acinetobacter baumannii or pseudomonas aeruginosa colonization of the respiratory tract.methods: fifty-three intubated, mechanically ventilated patients participated in the study. multiresistant a. baumannii , sensitive only to ampicillin/sulbactam, or p. aeruginosa , sensitive to ceftazidime or colistin, were isolated from the bronchial secretions ( Á/ cfu/ ml). all patients were subsequently treated with intravenous ampicillin/sulbactam, ceftazidime or colistin, whereas of them were also given the same antibiotic in aerosolized form.results: a decrease in the number of colonies by Á/ cfu/ml was observed, following Á/ days of combined treatment with both intravenous and inhaled antibiotic. none of the patients developed vap. in the patients who only received the antibiotic intravenously, the decrease ranged from zero to cfu/ml, after days of treatment. two of patients developed vap.conclusions: our results suggest that the administration of aerosolized antibiotics represents an effective means of preventing ventilator-associated pneumonia caused by a. baumanni and p. aeruginosa . introduction: pseudomonas aeruginosa is an important nosocomial pathogen. resistance to certain beta-lactam antimicrobial agents among p. aeruginosa is increasing. despite the development of new antibiotics multiresistant strains of p. aeruginosa represent an important therapeutic problem. the aim of this study was to investigate the activity of imipenem, amikacin, piperacillin, ciprofloxacin, ceftazidime, against clinical isolates of p. aeruginosa . methods: a total of isolates by tracheal aspiration from hospitalized patients, admitted to intensive care units were identified as p. aeruginosa using an algorithm that included: gram stain, pigment, oxidaze ('/ ,) and gram negative identifications microscan walkaway- (dade behring) were used according to the manufactures instructions. minium inhibitory concentrations were determined using walkaway, interpretation based on ncclsm -s , january ' .results: the respiratory tract was the single site of isolation for this study. the best activity was showed by imipenem %, followed by amikacin %, piperacillin, pip/tazobactam, ciprofloxacin had the same sensitivity %.conclusion: a high level resistance to antibiotics was observed to p. aeruginosa isolated from tracheal aspiration. carbapenems seem to be the most active against p. aeruginosa in this study. materials and methods: clinical samples were collected from patients admitted to this hospital. only one isolate per patient was included. antimicrobial susceptibility testing was performed as recommended by the nccls. all bacterial isolates were tested by dd and ad to provide a comparison of both test results. very major error was considered when the strains were resistant (r) by ad and susceptible (s) by dd and major error when s by ad and r by dd. categories of s and r were stablised using the breakpoints suggested by mensura ( ) . colistin r strains was typed by rep-pcr.results: among the strains included in this study ( . %) were s to colistin and five ( . %) were colstin r by ad, of this five colistin r strains four ( . %) were s to colistin by dd and one was r by both methods. all r isolates were similar by rep-pcr.conclusions: most of the ad colistin resistant strains were s when tested by dd indicating that this method is not useful to determine the resistance to colistin. rep-pcr patterns show that the spread of a colistin r clone seems to be involved. methods: gnb were isolated from per-operative biopsies (pob) and/ or from articular punction (ap). patients (pts) received cfp, g bid'/ ofl, mg tid or cip, mg bid intravenously for days, followed by a prolonged oral fq monotherapy. cure was defined as: resolution of all clinical signs of infection, normalization of the biological inflammatory profile at the end of treatment (eot) and absence of infection at the same site during the post-treatment followup period (ptfu).results: all of the studied patients [mean age / years] had hospital acquired bji. seventeen/ had an infected orthopedic device (prosthetic joints / , other orthopedic prosthetic devices / ). culture of pob and ap yielded to pseudomonas sp. ( ), enterobacter cloacae ( ), others ( ). vancomycin was added for six pts co-infected by gnb-mrsa. nineteen/ pts underwent a surgical intervention (debridement / , removal-replacement / , amputation / ). after ptfu period of months (range Á/ ), the overall success rate was / ( . %) without serious adverse events.conclusion: cfp Á/fq combination was safe and efficient in the treatment of hypercase gnb, bji.treatment of posttraumatic mrsa osteomyelitis of the femur with longterm cotrimoxazole */a case report pm the authors report a case of posttraumatic osteomyelitis of the femur caused by methicillin-resistant staphylococcus aureus (mrsa), following the shot injury. relapses of the infection occured in months interval and were treated by revision, debridement, lavage and vancomycin. because of laboratory signs of renal insufficiency vancomycin became contraindicated for treatment of the third relapse of infection and the different approach was employed: classic open treatment of bone infection sec. orr was combined with a long-term administration of high-dose cotrimoxazole. the patient was given cotrimoxazole mg daily divided in four doses ( mg/kg/ h) for months, then for gastrointestinal complaints with lowered dose of g daily for next months. the wound completely healed. during months after the final surgery there was no relaps of infection, but the atrophic pseudoarthrosis of the femur resulted. the patient can walk with a rigid orthesis and two crutches. the whole treatment of the objective: to present a variety of severe nosocomial infections due to stenotrophomonas maltophilia in patients hospitalized in tertiary medical units from cluj.results: during the last year nine strains of s. maltophilia obtained from patients with severe infections and hospitalized in different wards were isolated. all but one were considered nosocomial infections: four cases of pneumonia, one urinary tract infection, three cases of surgical wound infections and one case of endocarditis under surgical treatment. the cases of pneumonia were either primary occurring in a granulocytopenic patient with leukemia or secondary in patients that underwent surgical treatment. in the case of endocarditis the ethiology was established after surgery from the damaged valve in a negative hemoculture patient with a poor outcome under medical treatment. in all cases of surgical wound infection bacteremia occurred diagnosed on clinical basis in the presence of severe sepsis or hematogenous dissemination in the lung. the urinary tract infection occurred in a patient after urinary surgery and having a catheter in place. the immediate evolution was favorable in all cases but treatment was difficult due to the highly resistant strains and to underling diseases.conclusions: s. maltophilia should be considered in nosocomial severe infections and prophylaxis by interrupting environmental transmission has to be promoted. sawicka-grzelak a, rokosz a, luczak m. department of medical microbiology, university medical school, warsaw, poland purpose: to identify and determine the drug-susceptibility of esblpositive strains isolated from urine samples.methods: seven hundred and twelve strains of gram-negative rods were cultured from urine samples from hospitalized patients during months (july Á/november ). identification and susceptibility were performed in the automatic atb system (biomerieux, france) using id e, id gn and atb ur strips. esbl-activity was detected with double-disc synergy test (ddst according to jarlier et al., ) or using a novel method of esbl detection (dd, diagnostic disc) according to appleton ( ) . two discs were applied in this test: with cefpodoxime (cpd) and with cefpodoxime/clavulanic acid (cd ) (oxoid, england).results: five hundred and ninety-five strains ( . %) belonging to enterobacteriaceae family, strains ( . %) of non-fermenting rods and two strains ( . %) of other gram-negative rods were isolated. eighty-two esbl-producing strains ( . % of all strains) were detected. fifty-nine esbl-positive strains were susceptible to nitrofurantoin, -to norfloxacin and ciprofloxacin and -to fosfomycin.conclusions: esbl-positive strains were detected most frequently among enteric rods ( strains). nitrofurantoin and quinolones were the most active in vitro antibacterial agents against examined esblpositive uropathogens. results: the mechanisms of resistance were evaluated phenotypically using different aminoglycosides. a total of aminoglycoside resistant gram-negative strains were studied. one hundred fifty-eight strains were collected in Á/ , in and in . the resistant profiles were determined: enterobacteriaceae */ ; pseudomonas aeruginosa */ ; acinetobacter spp. */ . the most frequently phenotypes were gt (gentamicin, tobramycin) */ % and gtnet (gentamicin, tobramycin, netilmicin) */ %. the gt phenotype due to production ant( ƒ)-i enzyme, the gtnet Á/aac( )-v ( strains), aac( )-iv */one strain and aac( ƒ)-i */one strain. the resistance to amikacin in % strains was due to production aac( ?)-i ( %) and aph( ?)-vi ( %). the most of examined strains were simultaneously resistant to kanamycin and neomycin caused by production of aph( ?)-i ( %). only seven strains were resistant to all aminoglycosides due to impermeability of outer membrane. no substantial differences were observed between years.conclusions: the main mechanism of aminoglycoside resistance is fermentative modification. the high rate to gentamicin and tobramycin was due to production of ant( ƒ)-i and aac( )-v. amikacin and isepamicin were the most active aminoglycosides against gramnegative nosocomial isolates.the incidence of clostridium difficile associated diarrhoea (cdad) increased in our department from january to june .objective: to confirm the out break of cdad, to identify the risk factors and assess the effectiveness of the measures implemented for controlling this outbreak.methods: cdc definitions were used to identify the cases. the scope of the outbreak was defined. cdad incidences during the outbreak period and during the same period in were compared. risk factors (reduced mobility, antibiotic treatments . . .) were studied for patients whom length of hospital stay (lhs) was more than days. contact precautions and environmental cleaning with clona implemented were assessed.results: seventeen episodes of cdad were identified. sex ratio: . , mean age / . , mean lhs / days, mean delay for cdad occurring / days. one hundred and fifty-two patients involved in the study of risk factors. relative risk (r.r.) evaluated were: blactams (r.r. / . , ic %: . Á/ . ), reduced mobility (r.r. / . , ic %: . Á/ . ). incidence of cdad was less than two cases per days of hospitalisation after june .conclusion: we confirmed the outbreak of cdad in our department b-lactams and reduced mobility were identified as risk factors for cdad. measures implemented to control the outbreak were effectiveness.positive heart transport fluid cultures associated with severe infections in heart transplant recipients pm at the mount sinai hospital in new york city heart transplants were performed between and june . cultures were routinely performed on all heart transplant transport fluids. culture data was available for of these patients. in total / ( . %) were positive for bacteria, fungi or both. the organisms isolated included coagulase negative staphylococci ( ), pseudomonas aeruginosa ( ), staphylococcus aureus ( ), acinetobacter baumanii ( ), serratia mercescens ( ), enterobacter cloacae ( ), escherichia coli ( ), proteus mirabilus ( ), enterococcus faecalis ( ), viridans streptococci ( ), and fungi (aspergillus fumigatus ( ), penicillium species ( ), and rhodotorula rubra ( ). two heart transplant recipients had two organisms isolated from the transport fluid. isolation of resistant gram-negative bacilli in the transport fluid was associated with significant infection in / patients ( %) with the same organism. the observed infections were pneumonia secondary to e. cloacae , sternal wound infection secondary to p. aeruginosa , and bacteremia secondary to p. aeruginosa . it appears prudent to provide prophylaxis against resistant gram negative bacilli to prevent infections. zacharof ak, flevaris c, petrogianopoulos c, karachalios g, vroulis j, chartzoulakis g, drakogiorgos g, loizidou a, svoukas g. nd department of internal medicine, hellenic red cross hospital, athens, greece objective: we studied the trends of nosocomial bloodstream infection and calculated the population-attributable risk for death among hospitalized patients. methods: we perform a -year retrospective study for all patients (n / ), admitted to our department between and .results: between and , a total of patients developed episodes of nosocomial bloodstream infection. the crude infection rates increased linearly from . to . per discharges ( . Á/ . episodes per patient-days) during the -year study period. increases in the infection rates were due to gram-positive cocci, yeasts and essentially explained by infections caused by coagulasenegative staphylococci, staphylococcus aureus , enterococci, and candida species, respectively. although the crude mortality in patients with nosocomial bloodstream infections decreased from % in to % in , the in-hospital population-attributable mortality among infected patients increased from . deaths per discharges in to . per discharges in . the etiologic fraction or the proportion of deaths in patients with bloodstream infection to all deaths occurring in the hospital increased from . % in to . % in .conclusions: the incidence and the population-attributable risk for death among patients experiencing nosocomial bloodstream infections increased progressively during the last years in our department.ventilator-associated pneumonia before and after intensive care unit temporary closure pm results: we compared the incidence, causative organisms and mortality of vap in two different time periods. period june to december . period june to december . between those two periods the icu remained closed for months because of reconstruction works.period : sixty-seven consecutive patients (pts) were studied with bronchial secretions cultures at least days after mechanical ventilation (mv) initiation. the vap was diagnosed by clinical, radiological and microbiological criteria in pts ( %). causative organisms included: pseudomonas aeruginosa , acinetobacter , staphylococcus aureus , klebsiella pneum. , enterobacter . in three cases vap was proved polymicrobial. fourteen ( ) episodes of vap ( %) were developed after days mv (late vap) and were attributed to multiresistant microorganisms. mortality of vap was %.period : among consecutive pts, vap was diagnosed in ( %). causative organisms included: acinetobacter , p. aeruginosa , s. aureus , k. pneumoniae , escherichia coli . five ( ) cases were polymicrobial and cases were 'late vap' ( %). causative microorganisms had similar patterns of sensitivity to antibiotics (compared to period one). mortality of vap was %.conclusion: temporary icu closure had no significant influence on the incidence, distribution of causative organisms, their sensitivities to antibiotics and mortality of the vap. efstathiou sp a , pefanis av a , tsioulos di a , tsiakou ag a , zacharos id a , kanavaki s b , mountokalakis td a . a third university department of medicine, sotiria general hospital, athens, greece , b microbiology laboratory, sotiria general hospital, athens, greecepurpose: the aim of this study was to derive a scoring system for the prediction of outcome in adult patients with acute pyelonephritis (ap) severe enough to need hospitalization. therefore, the charts of patients ( men, median age years) were reviewed.results: logistic regression analysis identified in both sexes four independent correlates of in-hospital mortality, the coefficients of which divided by . and rounded to the nearest interval, resulted in the following integer-based scoring system: points (men) / * between concentrations of lps of the two groups were statistically significant on the nd and the th day (p b/ . ). it is concluded that the administration of lactulose may decrease systemic endotoxaemia in the field of obstructive jaundice.nosocomial infections: a prevalence study in the island of crete pm doukakis s a , tzimis l b , perogambrakis g a , kalloniatou m a , christodoulakis n a , evaggelopoulos a a , koutsoumba d a , kastanakis s a . a first medical department, 'saint george ' general hospital, chania, greece , b pharmacy department, 'saint george ' general hospital, chania, greeceprevalence surveillance is a rapid and inexpensive mode to estimate the problem of hospital-acquired infections (hais). to study the problem of nosocomial infections in our hospital, a prevalence study was made from our team in . the study included patients (the total number of hospitalized patients at the time of the study). from these patients were males ( . %) and females ( . %). one hundred and ninety-one patients ( . %) belonged in the groups of age between and years. fifty-seven patients had a urine catheter ( . %). one hundred and fifty-five/ patients ( %) received antibiotics and from these patients received one antibiotic and the remaining patients two or more. a nosocomial infection was found in patients and consequently the prevalence of hais was . %. among these, urinary tract infections were six ( . %), lower respiratory tract infections were three ( . %), surgical site infections were three ( . %), and bloodstream infections was one ( . %). the incidence of multiresistant bacteria was primarily enterococcus spp and secondary, pseudomonas aeruginosa , enterobacter spp, klebsiella pneumoniae , escherirchia coli , staphylococcus aureus , enterobacter cloacae . unfortunately prophylactic chemotherapy of long duration was found despite the suggestions of the infection control committee. regarding age the highest incidence of hais occurred in the third age group. bagirova ns, dmitrieva n. laboratory of microbiology, cancer research center of russia, moscow, russian federation objectives: to determine the pathogens and susceptibility to antimicrobials.methods: blood samples were collected from adult pts ( Á/ ). the bacteraemic episodes were classified according to the definitions of the cdc. laboratory detection of bacteraemia and fungaemia was performed according to cumitech b (blood cultures iii, ). susceptibility testing was performed by disk diffusion method (nccls).results: the total number of blood samples */ , -positive ( . % episodes of significant bacteraemias). bsi was confirmed microbiologically in of febrile pts ( . %). the most frequent pathogens were gram('/) cocci ( . %) (p b/ . ), gram((/) bacilli */ . %, fungi */ . %. coagulase-negative staphylococci (cns) represented . %, staphylococcus aureus . %, streptococcus spp. . %, enterococcus spp. . %, enterobacteriaceae . %, pseudomonas aeruginosa . %, other non-fermenting */ . %, yeast . %, mould . %, anaerobes . %. one hundred percent cns were resistant to penicillin, . % to oxacillin, . % to clindamycin, . % to cefazolin, . % to ceftazidime, . % to ciprofloxacin, . % to gentamicin, and no isolate was resistant to vancomycin. the predominant pathogens in all types of hm were gram('/) cocci (mainly cns). all gram('/) microorganisms were sensitive to vancomycin. katashinsky o a , opriatova t b , tchuev p c . a state clinical hospital, anaesteziology, odessa, ukraine , b state clinical hospital, bacteriology, odessa, ukraine , c medical university, anaesteziology, odessa, ukrainethis investigation was carried out in odessa state clinical hospital during Á/ . of the patients with post-operation complications, % of gram-negative cultures were sensitive to ceftazidime and % to amikacin. sixty-nine percent of gram-positive cultures were resistant to penicillin g, but they were sensitive to vancomycin and nitrofurantoin in and % of cases, respectively. sixty percent of isolates of pseudomonas aeruginosa were sensitive to ceftazidime and % to amikacin. rates of resistance to carbenicillin and gentamicin were and %, respectively. one hundred and fortythree isolates of escherichia coli were studied and % of them were resistant to ampicillin % to cephalothin and % to tetracycline. of the isolates tested against ciprofloxacin, all were sensitive. one hundred and eight isolates of staphylococcus aureus were studied and they were resistant only to penicillin g ( %). staphylococcus aureus was sensitive to erythromycin ( %), tetracycline ( %), oxacillin ( %) and vancomycin ( %). all isolates enterococcus faecalis were sensitive to nitrofurantoin and % to ciprofloxacin. the majority of s. aureus and e. faecalis isolates were susceptible to most other antibiotics, but the majority of e. coli isolates were resistant to the studied antibiotics expect ciprofloxacin.campylobacter foetus bacteraemia in an immunocompromised patient: a case report pm monno r a , ierardi e b , rendina m b , ceci g a , luzzi i c , de vito d a , rizzo g a , francavilla a b . a department of internal medicine and public health, university of bari, bari, italy , b department of emergency and organ transplantation, university of bari, bari, italy , c istituto superiore di sanità, rome, italy a -year-old woman was admitted for recurrent fever. the patient underwent a liver transplantation and splenectomy in . she had followed immunosuppressive therapy until when tacrolimus was added for chronic rejection. in non-hodgkin lymphoma was diagnosed and chemotherapy was started. six months later because of the presence of two metastatic encephalic foci affecting the optic chiasm, a new chemotherapy course was started with the regression of lesions. in january she was treated with steroid recycle and cyclosporine Á/azathioprine Á/prednisone reintroduction. fever occurred after months and cytomegalovirus (cmv) infection was diagnosed. treatment with ganciclovir was started with clinical remission. in november cmv infection recurred and blood cultures were positive for a bacterium that was identified as campylobacter fetus . the patient was successfully treated with intravenous ciprofloxacin. bacteremia frequently occurs in cancer patients. bacteremia due to c. fetus are rare, occurring mainly in immunocompromised patients. c. fetus expresses a proteinaceous surface layer that confers serum resistance. in our patients steroid and immunosuppression may have contributed to the development of lymphoma. all of these factors and chemotherapy have contributed to cmv infection and all have made the patient susceptible to bacteremia with this infrequently found bacterium. the clinical microbiologist should be aware of this infection in immunocompromised host. minenko sv, dmitrieva nv, sokolova en, ptushkin vv. bone marrow transplantation department, n.n. blokhin cancer research center, moscow, russian federation c'/a is the standard regimen as empirical therapy for febrile neutropenia (fn). activity of c against g'/ bacteria and g(/ bacteria, producing chromosomally-mediated b-lactamases (e.g. ampc) is suboptimal. cep is active against a broad range of g'/ and g(/, including ampc producing bacteria. the purpose of the study was to compare the efficacy of two regimens in the treatment of fn.methods: patients with fn received either cep ( g/ h) or c ( g/ h) plus amikacin ( mg/kg/day) or netilmicin ( mg/kg/day). data were collected prospectively.results: a total of pts with episodes ( / ) of fn were included. fifteen/ in cep group and / in c'/a group. the median duration of neutropenia grade , distribution of age, sex and underlying disease were comparable in both arms. mdi was in and %, cdi in and % fuo in and % in fep and c'/a groups correspondingly. response to the initial empirical regimen according to ihs criteria was in % of cep and . % of c'/a groups (p / . ). modification of therapy with a change of cep or c to carbapenem took place in and % of cep and c'/a groups (p / . ). no patient in either treatment group died due to the presenting infection. tolerability of cep was good and no laboratory abnormalities took place. transient elevation of serum creatinin level was observed in two patients c'/a group.conclusions: cep monotherapy is as effective as c'/a combination in the treatment of patients with fever and granulocytopenia purpose of the study: retrospectively, we analysed patients with intraabdominal infection for aetiology, risk factors, and outcome from hospitals in slovak republic within year (march Á/march ). results: in this group significantly more frequent were older patients ( !/ years) with cancer ( vs. %, p b/ . ). acinetobacter spp. ( vs. %, p b/ . ) and enterobacteriaceae ( vs. %, pb/ . ) were predictive for monoinfection. pre-treated patients with other antibiotics had inferior prognosis and more risk factors: permanent urinary catheter ( vs. %, pb/ . ), ventilation or intubation ( vs. %, pb/ . ) and polymicrobial infection ( vs. %, pb/ . ). the risk factors with poor prognosis were enterobacteriaceae ( vs. %, pb/ . ), diabetes mellitus as underlying disease ( vs. %, p b/ . ) and uraemia ( vs. %, p b/ . ). surprisingly, negative prognostic factor was also non-effective previous antibiotic therapy. failed patients died significantly more frequently patients due to underlying disease. cefoperazone/sulbactam was shown to be useful, effective and well tolerated also in one group of patients with % efficacy of treatment, and it belongs to a group of antibiotics suitable for treatment of nosocomial infections.